Multi-chain chimeric polypeptides and uses thereof

MULTI-CHAIN CHIMERIC POLYPEPTIDES AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to: U.S. Patent Application No. 62/724,969, filed August 30, 2018; U.S. Patent Application No. 62/817,230, filed March 12, 2019; U.S. Patent Application Serial No. 62/725,043, filed August 30, 2018; U.S. Patent Application Serial No. 62/725,010, filed August 30, 2018; U.S. Patent Application Serial No. 62/749,007, filed October 22, 2018; U.S. Patent Application Serial No. 62/746,832, filed October 17, 2018; U.S. Patent Application Serial No. 62/749,506, filed October 23, 2018; U.S. Patent Application Serial No. 62/817,241, filed March 12, 2019; U.S. Patent Application Serial No. 62/816,683, filed March 11, 2019; and U.S. Patent Application Serial No. 62/881,088, filed July 31, 2019, each of which is herein incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to the field of biotechnology, and more specifically, to antigen-binding molecules.

BACKGROUND

Tissue factor (TF), a 263 amino acid integral membrane glycoprotein with a molecular weight of ~46 kDa and the trigger protein of the extrinsic blood coagulation pathway, is the primary initiator of coagulation in vivo. Tissue factor, normally not in contact with circulating blood, initiates the coagulation cascade upon exposure to the circulating coagulation serine protease factors. Vascular damage exposes sub-endothelial cells expressing tissue factor, resulting in the formation of a calcium-dependent, highaffinity complex with pre-existing plasma factor Vila (FVIIa). Binding of the serine protease FVIIa to tissue factor promotes rapid cleavage of FX to FXa and FIX to FIXa. The proteolytic activity of the resulting FXa and an active membrane surface then inefficiently converts a small amount of prothrombin to thrombin. The thrombin generated by FXa initiates platelet activation and activates minute amounts of the procofactors factor V (FV) and factor VIII (FVIII) to become active cofactors, factor Va (FVa) and factor Villa (FVIIIa). FIXa complexes with FVIIIa on the platelet surface forming the intrinsic tenase complex, which results in rapid generation of FXa. FXa complexes with FVa to form the pro-thrombinase complex on the activated platelet surface which results in rapid cleavage of prothrombin to thrombin.

In addition to the tissue factor-FVIIa complex, a recent study showed that the tissue factor-FVIIa-FXa complex can activate FVIII, which would provide additional levels of FVIIIa during the initiation phase. The extrinsic pathway is paramount in initiating coagulation via the activation of limited amounts of thrombin, whereas the intrinsic pathway maintains coagulation by dramatic amplification of the initial signal.

Much of the tissue factor expressed on a cell surface is “encrypted,” which must be “decrypted” for full participation in coagulation. The mechanism of “decryption” of cell-surface tissue factor is still unclear at this time, however, exposure of anionic phospholipids plays a major role in this process. Healthy cells actively sequester anionic phospholipids such as phosphatidyl serine (PS) to the inner leaflet of the plasma membrane. Following cellular damage, activation, or increased levels of cytosolic Ca2+, this bilayer asymmetry is lost, resulting in increased PS exposure on the outer leaflet, which increases the specific activity of cell-surface tissue factor-FVIIa complexes. PS exposure is known to decrease the apparent Km for activation of FIX and FX by tissue factor-FVIIa complexes, but additional mechanisms could include conformational rearrangement of tissue factor or tissue factor-FVIIa and subsequent exposure of substrate binding sites.

SUMMARY

The present invention is based on the discovery that soluble tissue factor can be used as a scaffold for chimeric polypeptides including an antigen-binding domain. Based on this discovery provided herein are multi-chain chimeric polypeptides that include: (a) a first chimeric polypeptide including: (i) a first target-binding domain; (ii) a soluble tissue factor domain; and (iii) a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide including: (i) a second domain of a pair of affinity domains; and (ii) a second target-binding domain, where the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains. Also provided herein are compositions that include any of the multi-chain chimeric polypeptides described herein, nucleic acids that encode any of the multi-chain chimeric polypeptides described herein, and cells that include any of the nucleic acids that encode any of the multi-chain chimeric polypeptides described herein. Also provided herein are methods of stimulating an immune cell and methods of treating a subject in need thereof that include the use of any of the multi-chain chimeric polypeptides described herein. Also provided herein are methods of producing any of the multi-chain chimeric polypeptides described herein.

Accordingly, provided herein is a multi-chain chimeric polypeptide comprising:

(a) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and (iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and (ii) a second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains. In some embodiments, the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide. In some embodiments, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide. In some embodiments, the first target-binding domain and the second target-binding domain bind specifically to the same antigen. In some embodiments, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments, the first target-binding domain and the second target-binding domain comprise the same amino acid sequence. In some embodiments, the first target-binding domain and the second target-binding domain bind specifically to different antigens. In some embodiments, one or both of the first target-binding domain and the second target-binding domain is an antigen-binding domain. In some embodiments, the first target-binding domain and the second target-binding domain are each antigen-binding domains. In some embodiments, the antigen-binding domain comprises a scFv or a single domain antibody. In some embodiments, one or both of the first target-binding domain and the second target-binding domain bind specifically to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, IGF1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-β receptor II (TGF3־RII), a ligand of TGF3־RIII, a ligand of DNAM-1, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor for IL-2, a receptor for IL3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD 155, a receptor for CD 122, and a receptor for CD28. In some embodiments, one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin, a soluble cytokine protein, or a ligand protein. In some embodiments, the soluble interleukin, soluble cytokine, or ligand protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L. In some embodiments, one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin, a cytokine receptor, or a soluble cell surface receptor. In some embodiments, the soluble receptor is a soluble TGF-β receptor II (TGF-β RII), a soluble ΤΟΡ-βΚΙΙΙ, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-1, a scMHCI, a scMHCII, a scTCR, a soluble CD155, or a soluble CD28. In some embodiments, the first chimeric polypeptide further comprises one or more additional target-binding domain(s), where at least one of the one or more additional antigen-binding domain(s) is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the at least one of the one or more additional antigen-binding domain(s), and/or a linker sequence between the at least one of the one or more additional antigen-binding domain(s) and the first domain of the pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal and/or C-terminal end of the first chimeric polypeptide. In some embodiments, at least one of the one or more additional target-binding domains directly abuts the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first domain of the pair of affinity domains. In some embodiments, the at least one of the one or more additional target-binding domains directly abuts the first target-binding domain in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first target-binding domain. In some embodiments, at least one of the one or more additional target-binding domains is disposed at the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target-binding domains is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the N-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the at least one additional target-binding domain of the one or more additional targetbinding domains disposed at the C-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first targetbinding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the at least one of the one or more additional targetbinding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, directly abuts the soluble tissue factor domain and/or the first domain of the pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises a linker sequence disposed (i) between the soluble tissue factor domain and the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, and/or (ii) between the first domain of the pair of affinity domains and the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains. In some embodiments, the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide. In some embodiments, at least one of the one or more additional target-binding domains directly abuts the second domain of the pair of affinity domains in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second domain of the pair of affinity domains in the second chimeric polypeptide. In some embodiments, at least one of the one or more additional target-binding domains directly abuts the second target-binding domain in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second target-binding domain in the second chimeric polypeptide. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence. In some embodiments, the first target-binding domain, the second targetbinding domain, and the one or more additional target-binding domains each bind specifically to the same antigen. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same epitope. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each comprise the same amino acid sequence. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to different antigens. In some embodiments, one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains is an antigen-binding domain. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains are each an antigen-binding domain. In some embodiments, antigen-binding domain comprises a scFv. In some embodiments, one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains bind specifically to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, Pcadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-β receptor II (TGF-βRII), a ligand of TGFβRIII, a ligand of DNAM-1, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD 155, a receptor for CD 122, and a receptor for CD3, and a receptor for CD28. In some embodiments, one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin, a soluble cytokine protein, or a ligand protein. In some embodiments, the soluble interleukin, soluble cytokine, or ligand protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L. In some embodiments, one or more of the first targetbinding domain, the second target-binding domain, and the one or more additional targetbinding domains is a soluble interleukin or cytokine receptor. In some embodiments, the soluble receptor is a soluble TGF-β receptor II (TGF-β RII), a soluble ΤΟΡ-βΚΙΙΙ, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-1, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a soluble CD3, or a soluble CD28. In some embodiments, the first chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the first chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the second chimeric polypeptide. In some embodiments, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain does not comprise one or more of: a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein; a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein; a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein. In some embodiments, the soluble human tissue factor domain does not comprise any of: a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein; a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein; a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein. In some embodiments, the soluble tissue factor domain is not capable of binding to Factor Vila. In some embodiments, the soluble tissue factor domain does not convert inactive Factor X into Factor Xa. In some embodiments, the multi-chain chimeric polypeptide does not stimulate blood coagulation in a mammal. In some embodiments of any of the single-chain chimeric polypeptides provided herein, the human soluble tissue factor domain does not initiate blood coagulation. In some embodiments of any of the single-chain chimeric polypeptides provided herein, the soluble tissue factor domain comprises or consists of a soluble wildtype human tissue factor.

In some embodiments, the pair of affinity domains is a sushi domain from an alpha chain of human IL-15 receptor (IL15Ra) and a soluble IL-15. In some embodiments, the soluble IL15 has a D8N or D8A amino acid substitution. In some embodiments, the human IL15Ra is a mature full-length IL15Ra. In some embodiments, the pair of affinity domains is selected from the group consisting of barnase and barnstar, a PKA and an AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. In some embodiments, the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its Nterminal end.

In some embodiments, the multi-chain chimeric polypeptide comprises a composition. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises at least one dose of the multi-chain chimeric polypeptide. In some embodiments, a kit comprises at least one dose of the composition.

In some embodiments, provided herein is a method of stimulating an immune cell, the method comprising: contacting an immune cell with an effective amount of any of the multi-chain chimeric polypeptides or compositions described above. In some embodiments, the method comprises contacting the immune cell in vitro. In some embodiments, the method comprises obtaining the immune cell from a subject. In some embodiments, the method comprises obtaining the immune cell from the subject prior to the contacting step. In some embodiments, the method comprises contacting the immune cell in vivo. In some embodiments, the method comprises selecting the immune cell from the group consisting of: an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, a Th 17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, and a natural killer cell. In some embodiments, the method comprises genetically modifying the immune cell to express a chimeric antigen receptor or a recombinant T-cell receptor. In some embodiments, the method comprises introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor after the contacting step. In some embodiments, the method comprises administering the immune cell to a subject in need thereof. In some embodiments, the method comprises identifying or diagnosing the subject as having an age-related disease or condition. In some embodiments, the age-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. In some embodiments, the method comprises identifying or diagnosing the subject as having a cancer. In some embodiments, the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the method comprises diagnosing or identifying the subject as having an infectious disease. In some embodiments, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Also provided herein is a method of inducing or increasing proliferation of an immune cell, the method comprising: contacting an immune cell with an effective amount of any of the multi-chain chimeric polypeptides or compositions described above. In some embodiments, the method comprises contacting the immune cell in vitro. In some embodiments, the method comprises obtaining the immune cell from a subject. In some embodiments, the method comprises obtaining the immune cell from the subject prior to the contacting step. In some embodiments, the method comprises contacting the immune cell in vivo. In some embodiments, the method comprises selecting the immune cell from the group consisting of: an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, a Thl7 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, and a natural killer cell. In some embodiments, the method comprises modifying the immune cell to express a chimeric antigen receptor or a recombinant T-cell receptor. In some embodiments, the method further comprises introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor after the contacting step. In some embodiments, the method further comprises administering the immune cell to a subject in need thereof. In some embodiments, the method comprises identifying or diagnosing the subject as having an age-related disease or condition. In some embodiments, the age-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. In some embodiments, the method comprises identifying or diagnosing the subject as having a cancer. In some embodiments, the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the method comprises diagnosing or identifying the subject as having an infectious disease. In some embodiments, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Also provided herein is a method of inducing differentiation of an immune cell into a memory or memory-like immune cell, the method comprising: contacting an immune cell with an effective amount of any of the multi-chain chimeric polypeptides or compositions discussed above. In some embodiments, the method comprises contacting the immune cell in vitro. In some embodiments, the method comprises obtaining the immune cell from a subject. In some embodiments, the method further comprises obtaining the immune cell from the subject prior to the contacting step. In some embodiments, the method comprises contacting the immune cell in vivo. In some embodiments, the immune cell is selected from the group consisting of: an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a Th 17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, and a natural killer cell. In some embodiments, the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor. In some embodiments, the method further comprises introducing into the immune cell a nucleic acid encoding a chimeric antigenreceptor or a recombinant T-cell receptor after the contacting step. In some embodiments, the method further comprises administering the immune cell to a subject in need thereof. In some embodiments, the method comprises identifying or diagnosing the subject as having an age-related disease or condition. In some embodiments, the agerelated disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. In some embodiments, the method comprises identifying or diagnosing the subject as having a cancer. In some embodiments, the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, nonsmall cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the method comprises diagnosing or identifying the subject as having an infectious disease. In some embodiments, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Also provided herein is a method of killing a cancer cell, an infected cell, or a senescent cell in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the multi-chain chimeric polypeptides or compositions discussed above. In some embodiments, the method comprises identifying or diagnosing the subject as having a cancer. In some embodiments, the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the method comprises identifying or diagnosing the subject as having an aging-related disease or condition. In some embodiments, the aging-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of any of the multi-chain chimeric polypeptides or compositions discussed above. In some embodiments, the method comprises identifying or diagnosing the subject as having a cancer. In some embodiments, the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the method comprises identifying or diagnosing the subject as having an aging-related disease or condition. In some embodiments, the aging-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. In some embodiments, the method comprises diagnosing or identifying the subject as having an infectious disease. In some embodiments, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Also provided herein is a nucleic acid encoding any of the multi-chain chimeric polypeptides discussed above. Some embodiments comprise vector containing any of the nucleic acids discussed above. In some embodiments, the vector is an expression vector. Some embodiments comprise a cell containing any of the nucleic acids discussed above.

Also provided herein is a method of producing a multi-chain chimeric polypeptide, the method comprising: culturing the cell discussed above in a culture medium under conditions sufficient to result in the production of the multi-chain chimeric polypeptide; and recovering the multi-chain chimeric polypeptide from the cell and/or the culture medium. In some embodiments, the method comprises producing a multi-chain chimeric polypeptide by the methods discussed above.

In some embodiments, the mutant soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 3. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 3. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 3. In some embodiments, the multi-chain chimeric polypeptide of claim 140, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO: 3. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 4. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 4. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO:4. In some embodiments, the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO: 4.

As used herein, the term “chimeric” refers to a polypeptide that includes amino acid sequences (e.g., domains) originally derived from two different sources (e.g., two different naturally-occurring proteins, e.g., from the same or different species). For example, a chimeric polypeptide can include domains from at least two different naturally occurring human proteins. In some examples, a chimeric polypeptide can include a domain that is a synthetic sequence (e.g., an scFv) and a domain that is derived from a naturally-occurring protein (e.g., a naturally-occurring human protein). In some embodiments, a chimeric polypeptide can include at least two different domains that are synthetic sequences (e.g., two different scFvs).

An “antigen-binding domain” is one or more protein domain(s) (e.g., formed from amino acids from a single polypeptide or formed from amino acids from two or more polypeptides (e.g., the same or different polypeptides) that is capable of specifically binding to one or more different antigen(s). In some examples, an antigen-binding domain can bind to an antigen or epitope with specificity and affinity similar to that of naturally-occurring antibodies. In some embodiments, the antigen-binding domain can be an antibody or a fragment thereof. In some embodiments, an antigen-binding domain can include an alternative scaffold. Non-limiting examples of antigen-binding domains are described herein. Additional examples of antigen-binding domains are known in the art.

A “soluble tissue factor domain” refers to a polypeptide having at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical) to a segment of a wildtype mammalian tissue factor protein (e.g., a wildtype human tissue factor protein) that lacks the transmembrane domain and the intracellular domain. Non-limiting examples of soluble tissue factor domains are described herein.

The term “soluble interleukin protein” is used herein to refer to a mature and secreted interleukin protein or a biologically active fragment thereof. In some examples, a soluble interleukin protein can include a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to a wildtype mature and secreted mammalian interleukin protein (e.g., a wildtype human interleukin protein) and retains its biological activity. Non-limiting examples of soluble interleukin proteins are described herein.

The term “soluble cytokine protein” is used herein to refer to a mature and secreted cytokine protein or a biologically active fragment thereof. In some examples, a soluble cytokine protein can include a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to a wildtype mature and secreted mammalian interleukin protein (e.g., a wildtype human interleukin protein) and retains its biological activity. Non-limiting examples of soluble cytokine proteins are described herein.

The term “soluble interleukin receptor” is used herein in the broadest sense to refer to a polypeptide that lacks a transmembrane domain (and optionally an intracellular domain) that is capable of binding one or more of its natural ligands (e.g., under physiological conditions, e.g., in phosphate buffered saline at room temperature). For example, a soluble interleukin receptor can include a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to an extracellular domain of wildtype interleukin receptor and retains its ability to specifically bind to one or more of its natural ligands, but lacks its transmembrane domain (and optionally, further lacks its intracellular domain). Non-limiting examples of soluble interleukin receptors are described herein.

The term “soluble cytokine receptor” is used herein in the broadest sense to refer to a polypeptide that lacks a transmembrane domain (and optionally an intracellular domain) that is capable of binding one or more of its natural ligands (e.g., under physiological conditions, e.g., in phosphate buffered saline at room temperature). For example, a soluble cytokine receptor can include a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to an extracellular domain of wildtype cytokine receptor and retains its ability to specifically bind to one or more of its natural ligands, but lacks its transmembrane domain (and optionally, further lacks its intracellular domain). Non-limiting examples of soluble cytokine receptors are described herein.

The term “antibody” is used herein in its broadest sense and includes certain types of immunoglobulin molecules that include one or more antigen-binding domains that specifically bind to an antigen or epitope. An antibody specifically includes, e.g., intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multi-specific antibodies. One example of an antigen-binding domain is an antigen-binding domain formed by a VH -VL dimer. Additional examples of an antibody are described herein. Additional examples of an antibody are known in the art.

“Affinity” refers to the strength of the sum total of non-covalent interactions between an antigen-binding site and its binding partner (e.g., an antigen or epitope). Unless indicated otherwise, as used herein, “affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of an antigen-binding domain and an antigen or epitope. The affinity of a molecule X for its partner Y can be represented by the dissociation equilibrium constant (Kd). The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®). Additional methods for determining the affinity for an antigen-binding domain and its corresponding antigen or epitope are known in the art.

A “multi-chain polypeptide” as used herein to refers to a polypeptide comprising two or more (e.g., three, four, five, six, seven, eight, nine, or ten) protein chains (e.g., at least a first chimeric polypeptide and a second polypeptide), where the two or more proteins chains associate through non-covalent bonds to form a quaternary structure.

The term “pair of affinity domains” is two different protein domain(s) that bind specifically to each other with a Kd of less than of less than 1 x IO'7 M (e.g., less than 1 x IO'8 M, less than 1 x IO'9 M, less than 1 x 10'10 M, or less than 1 x 10'11 M). In some examples, a pair of affinity domains can be a pair of naturally-occurring proteins. In some embodiments, a pair of affinity domains can be a pair of synthetic proteins. Nonlimiting examples of pairs of affinity domains are described herein.

The term “epitope” means a portion of an antigen that specifically binds to an antigen-binding domain. Epitopes can, e.g., consist of surface-accessible amino acid residues and/or sugar side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter may be lost in the presence of denaturing solvents. An epitope may comprise amino acid residues that are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding. Methods for identifying an epitope to which an antigen-binding domain binds are known in the art.

An “immune effector cell” refers to a cell of the immune system of a mammal that is capable, directly or indirectly, of recognizing and/or causing cytostasis or cell death of a pathogenic cell (e.g., a cancer cell) in the mammal. Non-limiting examples of immune effector cells include macrophages, T-lymphocytes (e.g., cytotoxic Tlymphocytes and T-helper cells), natural killer cells, neutrophils, monocytes, and eosinophils. Additional examples of immune effector cells are known in the art.

The term “treatment” means to ameliorate at least one symptom of a disorder. In some examples, the disorder being treated is cancer and to ameliorate at least one symptom of cancer includes reducing aberrant proliferation, gene expression, signaling, translation, and/or secretion of factors. Generally, the methods of treatment include administering a therapeutically effective amount of composition that reduces at least one symptom of a disorder to a subject who is in need of, or who has been determined to be in need of such treatment.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows exemplary diagrams for a multi-chain chimeric polypeptide: (i) a first chimeric polypeptide including a first target-binding domain (A), a soluble tissue factor domain, a first domain of an affinity pair of domains (soluble interleukin IL-15), and an additional target-binding domain (B); and (ii) second chimeric polypeptide including a second domain of an affinity pair of domains (IL-15 receptor alpha sushi domain), a second target-binding domain (C), and an additional antigen-binding domain (D). The top cartoon diagram depicts the association of the first and the second chimeric polypeptides through the pair of affinity domains. The bottom schematic diagrams show the order of the domains in the first and second chimeric polypeptides.

Figure 2 shows exemplary diagrams for a multi-chain chimeric polypeptide: (i) a first chimeric polypeptide including a first target-binding domain (A), a soluble tissue factor domain including five amino acid substitutions in order to remove binding of the soluble tissue factor domain to FVIIa, a first domain of an affinity pair of domains (soluble interleukin IL-15 including a D8N or D8A amino acid substitution), and an additional target-binding domain (B); and (ii) second chimeric polypeptide including a second domain of an affinity pair of domains (IL-15 receptor alpha sushi domain), a second target-binding domain (C), and an additional antigen-binding domain (D). The top cartoon diagram depicts the association of the first and the second chimeric polypeptides through the pair of affinity domains. The bottom schematic diagrams show the order of the domains in the first and second chimeric polypeptides. In other embodiments of any of the multi-chain chimeric polypeptides described herein the soluble tissue factor domain can comprise or consists of a soluble wildtype human tissue factor domain (comprising or consisting of a contiguous sequence within wildtype human tissue factor).

Figure 3 shows a schematic diagram of an exemplary IL-12/IL-15RaSu DNA construct.

Figure 4 shows a schematic diagram of an exemplary IL-18/TF/IL-15 DNA construct.

Figure 5 shows a schematic diagram of the interaction between the exemplary IL12/IL-15RaSu and IL-18/TF/IL-15 DNA constructs.

Figure 6 shows a schematic diagram of the interaction between the exemplary IL12/IL-15RaSu and IL-18/TF/IL-15 fusion proteins resulting in IL- 18/TF/IL-15 :IL-12/IL15RaSu complex (18tl5-12s).

Figure 7 shows a chromatograph of 18tl5-12s purification elution from an antiTF antibody affinity column.

Figure 8 shows an exemplary chromatographic profile of anti-TF Ab / SECpurified 18tl5-12s protein following elution on an analytical size exclusion column, demonstrating separation of monomeric multiprotein 18tl5-12s complexes from protein aggregates.

Figure 9 shows an example of a 4-12% SDS-PAGE of the 18tl5-12s complex following disulfide bond reduction. Lane 1: SeeBlue Plus2 marker; Lane 2: an anti-tissue factor antibody affinity column-purified 18tl5-12s (0.5 pg); Lane 3: an anti-tissue factor antibody affinity column-purified 18tl5-12s (1 pg).

Figure 10 shows SDS PAGE analysis of deglycosylated and non-deglycosylated 18tl5-12s. Lane 1: an anti-tissue factor antibody affinity column-purified 18tl5-12s (0.5 pg), non-deglycosylated; Lane 2: anti-TF Ab-purified 18tl5-12s (1 pg), nondeglycosylated; Lane 3: 18tl5-12s (1 pg), deglycosylated, Lane 4: Markl2 unstained maker.

Figure 11 shows a sandwich ELISA for the 18tl5-12s complex, comprising an anti-human tissue factor capture antibody and a biotinylated anti-human IL-12 detection antibody (BAF 219).

Figure 12 shows a sandwich ELISA for the 18tl5-12s complex, comprising an anti-human tissue factor capture antibody and a biotinylated anti-human IL-15 detection antibody (BAM 247).

Figure 13 shows a sandwich ELISA for the 18tl5-12s complex, comprising an anti-human tissue factor capture antibody and a biotinylated anti-human IL-18 detection antibody (D045-6).

Figure 14 shows a sandwich ELISA for the 18tl5-12s complex, comprising an anti-human tissue factor (143) capture antibody and an anti-human tissue factor detection antibody.

Figure 15 shows proliferation of IL dependent 32ϋβ cells mediated by the 18tl5-12s complex (open squares) and recombinant IL-15 (black squares).

Figure 16 shows biological activity of IL-18 within the 18tl5-12s complex (open squares), where recombinant IL-18 (black squares) and recombinant IL-12 (black circles) serve as positive and negative controls, respectively.

Figure 17 shows biological activity of IL-12 within the 18tl5-12s complex (open squares), where recombinant IL-12 (black circles) and recombinant IL-18 (open squares) serve as positive and negative controls, respectively.

Figures 18A and 18B show cell-surface expression of CD25 on NK cells induced by the 18tl5-12s complex and cell-surface CD69 expression of NK cells induced by the 18tl5-12s complex.

Figure 19 shows a flow cytometry graph of intracellular interferon gamma expression of NK cells induced by the 18tl5-12s complex.

Figure 20 shows cytotoxicity of 18tl5-12s induced human NK cells against K562 cells.

Figure 21 shows a schematic diagram of an exemplary IL-12/IL-15RaSu/aCD16 DNA construct.

Figure 22 shows a schematic diagram of an exemplary IL-18/TF/IL-15 DNA construct.

Figure 23 shows a schematic diagram of the interaction between the exemplary IL-12/IL-15RaSu/aCD16scFv and IL-18/TF/IL-15 DNA constructs.

Figure 24 shows a schematic diagram of an exemplary 18tl5-12s/aCD16 protein complex.

Figure 25 shows a sandwich ELISA for the 18tl5-12sl6 complex, comprising an anti-human tissue factor capture antibody and a biotinylated anti-human IL-12 or IL-18 detection antibody.

Figure 26 shows a schematic diagram of an exemplary TGF3RIEIL-15RaSu DNA construct.

Figure 27 shows a schematic diagram of an exemplary IL-21/TF/IL-15 construct.

Figure 28 shows a schematic diagram of the interaction between the exemplary IL- IL-21/TF/IL-15 and TGFpRII/IL-lSRaSu constructs.

Figure 29 shows a schematic diagram of the interaction between the exemplary TGF3RIEIL-15RaSu and IL-21/TF/IL-15 fusion proteins, resulting in an IL-21/TF/IL15/TGF3RIEIL-15RaSu complex (21tl5-TGFRs).

Figure 30 shows a chromatograph of 21tl5-TGFRs purification elution from an anti-TF antibody affinity column.

Figure 31 shows an exemplary 21tl5-TGFRs size exclusion chromatograph showing a main protein peak and a high molecular weight peak

Figure 32 shows an example of a 4-12% SDS-PAGE of the 21tl5-TGFRs complex following disulfide bond reduction. Lane 1: Markl2 unstained marker (numbers on the left side indicate molecular weights in kDa); Lane 2: 21tl5-TGFRs (0.5 pg); Lane 3: 21tl5-TGFRs (1 pg); Lane 4: 21tl5-TGFRs, deglycosylated (1 pg), wherein the MW was the expected size of 53kDa and 39.08 kDa.

Figure 33 shows a sandwich ELISA for the 21tl5-TGFRs complex, comprising an anti-human tissue factor capture antibody and a biotinylated anti-human IL-21 detection antibody (13 81, BioLegend).

Figure 34 shows a sandwich ELISA for the 21tl5-TGFRs complex, comprising an anti-human tissue factor capture antibody and a biotinylated anti-human IL-15 detection antibody (BAM 247, R&D Systems).

Figure 35 shows a sandwich ELISA for the 21tl5-TGFRs complex, comprising an anti-human tissue factor capture antibody and a biotinylated anti-human TGF3RII detection antibody (BAF241, R&D Systems).

Figure 36 shows a sandwich ELISA for the 21tl5-TGFRs complex, comprising an anti-human tissue factor (143) capture antibody and an anti-human tissue factor detection antibody.

Figure 37 shows IL dependent proliferation of 32D3 cells mediated by the 21tl5-TGFRs complex (open squares) compared to IL-15 (black squares).

Figure 38 shows biological activity of the TGFpRII domain within the 21tl5TGFRs complex (open squares). TGF3RII/Fc (black squares) served as a positive control.

Figure 39 A shows a flow cytometry graph of cell-surface CD25 expression of NK cells induced by the 21tl5-TGFRs complex.

Figure 40 shows a flow cytometry graph of cell-surface CD69 expression of NK cells induced by the 21tl5-TGFRs complex.

Figure 41 shows a flow cytometry graph of intracellular interferon gamma expression of NK cells induced by the 21tl5-TGFRs complex.

Figure 42 shows cytotoxicity of 21tl5-TGFRs-induced human NK cells against K562 cells.

Figure 43 shows a schematic diagram of an exemplary IL-7/IL-15RaSu DNA construct.

Figure 44 shows a schematic diagram of an exemplary IL-21/TF/IL-15 DNA construct.

Figure 45 shows a schematic diagram of the interaction between the exemplary IL-7/IL-15RaSu and IL-21/TF/IL-15 DNA constructs.

Figure 46 shows a schematic diagram of the interaction between the exemplary IL-7/IL-15RaSu and IL-21/TF/IL-15 fusion proteins resulting in an IL-21/TF/IL-15:IL7/IL-15RaSu complex (21tl5-7s).

Figure 47 shows the expansion of primary natural killer (NK) cells by stimulation with 21tl5-7s + anti-TF IgGl antibody.

Figure 48 shows activation of expanded primary NK cells, using CD25 MFI and CD69 MFI as markers of NK cell activation.

Figure 49 shows cytotoxic activity of expanded NK cells against K562 human tumor cells, wherein NK cells stimulated with 21tl5-7s + anti-TF IgGl antibody demonstrate greater specific lysis of K562 cells than NK cells not stimulated with 21tl57s + anti-TF IgGl antibody.

Figure 50 shows a schematic diagram of an exemplary IL-21/IL-15RaSu DNA construct.

Figure 51 shows a schematic diagram of an exemplary IL-7/TF/IL-15 DNA construct.

Figure 52 shows a schematic diagram of the interaction between the exemplary IL-21/IL-15RaSu and IL-7/TF/IL-15 DNA constructs.

Figure 53 shows a schematic diagram of the interaction between the exemplary IL-21/IL-15RaSu and IL-7/TF/IL-15 fusion proteins resulting in an IL-7/TF/IL-15:IL21/IL-15RaSU complex (7tl5-21s).

Figure 54 shows in diagrammatic form the activation and expansion of primary natural killer (NK) cells by stimulation with 2Itl 5-TGFRs + anti-TF IgGl antibody.

Figure 55 shows size exclusion chromatography (SEC) profiles of anti-TF IgGl antibody, 7tl5-21s and the complex containing equal amounts of anti-TF IgGl antibody and 7tl5-21s.

Figure 56 shows the oxygen consumption rate (OCR) in pmoles/min for human NK cells isolated from blood (2 x 106 cells/mL) of two different donors.

Figure 57 shows the extracellular acidification rate (ECAR) in mpH/minute for human NK cells isolated from blood (2 x 106 cells/mL) of two different donors.

Figure 58 shows a schematic of the 7tl5-16s21 construct.

Figure 59 shows an additional schematic of the 7tl5-16s21 construct.

Figures 60A and 60B show binding of 7tl5-16s21 to CHO cells expressing human CD 16b as compared to a control protein.

Figures 61A-61C are results from ELISA experiments using antibodies against IL-15, IL-21, and IL-7 in detecting 7tl5-16s21.

Figure 62 shows results of the 32D3 cell proliferation assay with 7tl5-16s21 or recombinant IL-15.

Figure 63 shows the chromatographic profile of 7tl5-16s21 protein containing cell culture supernatant following binding and elution on anti-TF antibody resin.

Figure 64 shows the analytical SEC Profile of 7tl5-16s21.

Figure 65 shows a schematic of the TGFRtl5-16s21 construct.

Figure 66 shows an additional schematic of the TGFRtl5-16s21 construct.

Figures 67A and 67B show binding affinity of TGFRtl5-16S21 and 7tl5-21s with CHO cells expressing human CD16b. Figure 67A shows binding affinity of TGFRtl516S21 with CHO cells expressing human CD16b. Figure 67B shows binding affinity of 7tl5-21s with CHO cells expressing human CD16b.

Figure 68 shows results of TGFpi inhibition by TGFRtl5-16s21 and TGFR-Fc.

Figure 69 shows results of 32D3 cell proliferation assay with TGFRtl5-16s21 or recombinant IL-15.

Figures 70A-70C show results of detecting IL-15, IL-21, and TGFPRII in TGFRtl5-16s21 with corresponding antibodies using ELISA.

Figure 71 shows the chromatographic profile of TGFRtl5-16s21 protein containing cell culture supernatant following binding and elution on anti-TF antibody resin.

Figure 72 shows results of a reduced SDS-PAGE analysis of TGFRtl5-16s21.

Figure 73 shows a schematic of the 7tl5-7s construct.

Figure 74 shows an additional schematic of the 7tl5-7s construct.

Figure 75 shows the chromatographic profile of 7tl5-7s protein containing cell culture supernatant following binding and elution on anti-TF antibody resin.

Figure 76 shows detection of TF, IL-15 and IL-7 in 7tl5-7s using ELISA.

Figure 77 shows a schematic of the TGFRtl5-TGFRs construct.

Figure 78 shows an additional schematic of the TGFRtl5-TGFRs construct.

Figure 79 shows results of TGFpi inhibition by TGFRtl5-TGFRs and TGFR-Fc.

Figure 80 shows results of 32D3 cell proliferation assay with TGFRtl5-TGFRs or recombinant IL-15

Figures 81A and 8 IB show results of detecting IL-15 and TGF3RII in TGFRtl5TGFRs with corresponding antibodies using ELISA.

Figure 82 is a line graph showing the chromatographic profile of TGFRtl5TGFRs protein containing cell culture supernatant following binding and elution on antiTF antibody resin.

Figure 83 shows the analytical SEC profile of TGFRtl5-TGFRs.

Figure 84 shows TGFRtl5-TGFRs before and after deglycosylation as analyzed by reduced SDS-PAGE.

Figures 85 A and 85B show spleen weight and the percentages of immune cell types in TGFRtl5-TGFRs-treated and control-treated mice. Figure 85A shows spleen weight in mice treated with TGFRtl5-TGFRs as compared to PBS control. Figure 85B shows the percentage of CD4+ T cells, CD8+ T cells, and NK cells in mice treated with TGFRt 15-TGFRs as compared to PBS control.

Figure 86A and 86B show the spleen weight and immunostimulation over 92 hours in mice treated with TGFRtl5-TGFRs. Figure 86A shows spleen weight of mice treated with TGFRtl5-TGFRs at 16, 24, 48, 72, and 92 hours after treatment. Figure 86B shows the percentages of immune cells in mice treated with TGFRtl5-TGFRs at 16, 24, 48, 72, and 92 hours after treatment.

Figure 87A and 87B show Ki67 and Granzyme B expression in mice treated with TGFRtl5-TGFRs overtime.

Figure 88 shows enhancement of cytotoxicity of splenocytes by TGFRtl5-TGFRs in C57BL/6 Mice.

Figure 89 shows changes in tumor size in response to PBS treatment, chemotherapy alone, TGFRtl5-TGFRs alone, or chemotherapy and TGFRtl5-TGFRs combination, in a pancreatic cancer mouse model.

Figure 90 shows the cytotoxicity of NK cells isolated from mice treated with TGFRtl5-TGFRs.

Figure 91 shows a schematic of the 7tl5-21sl37L (long version) construct.

Figure 92 shows an additional schematic of the 7tl5-21sl37L (long version) construct.

Figure 93 is a line graph showing the chromatographic profile of 7tl5-21sl37L (long version) protein containing cell culture supernatant following binding and elution on anti-TF antibody resin.

Figure 94 shows the analytical SEC profile of 7tl5-21sl37L (long version).

Figure 95 shows binding of 7tl5-21sl37L (short version) to CD137L (4.1BBL) Figures 96A-96C show detection of IL15, IL21, and IL7 in 7tl5-21sl37L (short version) with ELISA. Figure 96A shows detection of IL15 in 7tl5-21sl37L (short version) with ELISA. Figure 96B shows detection of IL21 in 7tl5-21sl37L (short version) with ELISA. Figure 96C shows detection of IL7 in 7tl5-21sl37L (short version) with ELISA.

Figure 97 shows results from a CTLL-2 cell proliferation assay.

Figure 98 shows the activity of 7tl5-lsl37L (short version) in promoting IL21R containing B9 cell proliferation.

Figure 99 shows a schematic of the 7tl5-TGFRs construct.

Figure 100 shows an additional schematic of the 7tl5-TGFRs construct.

Figure 101 shows results of TGFpi inhibition by 7tl5-TGFRs and TGFR-Fc.

Figures 102A-102C show detection of IL-15, TGFpRII, and IL-7 in 7tl5-TGFRs with ELISA.

Figure 103 shows results of a 32D3 cell proliferation assay with 7tl5-TGFRs or recombinant IL-15.

Figure 104 is a line graph showing the chromatographic profile of 7tl5-TGFRs protein containing cell culture supernatant following binding and elution on anti-TF antibody affinity column.

Figure 105 shows 7tl5-TGFRs before and after deglycosylation as analyzed using reduced SDS-PAGE.

Figure 106 shows ELISA detection of IL-7, IL-15 and TGFPRII in the 7tl5TGFRs protein.

Figures 107A and 107B show spleen weight and the percentages of immune cell types in 7tl5-TGFRs-treated and control-treated mice. Figure 107A shows spleen weight in mice treated with 7tl5-TGFRs at various dosages, as compared to PBS control. Figure 107B shows the percentage of CD4+ T cells, CD8+ T cells, and NK cells in mice treated with 7tl5-TGFRs at various dosages, as compared to PBS control.

Figures 108Aand 108B show upregulation of CD44 expression of CD4+ and CD8+ T cells by 7tl5-TGFRs in C57BL/6 mice.

Figures 109A and 109B show upregulation of Ki67 expression and Granzyme B expression of CD8+ T cells and NK Cells by 7tl5-TGFRs in C57BL/6 mice.

Figure 110 shows enhancement of cytotoxicity of splenocytes by 7tl5-TGFRs in C57BL/6 mice.

Figure 111 shows a schematic of the TGFRtl5-21sl37L construct.

Figure 112 shows an additional schematic of the TGFRtl5-21sl37L construct.

Figure 113 is a line graph showing the chromatographic profile of TGFRtl5-21sl37L protein containing cell culture supernatant following binding and elution on anti-TF antibody affinity column.

Figure 114 shows a schematic of the TGFRtl5-TGFRs21 construct.

Figure 115 shows an additional schematic of the TGFRtl5-TGFRs21 construct.

Figure 116 is a line graph showing the chromatographic profile of TGFRtl5TGFRs21 protein containing cell culture supernatant following binding and elution on anti-TF antibody affinity column.

Figure 117 shows TGFRtl5-TGFRs21 before and after deglycosylation as analyzed by reduced SDS-PAGE.

Figures 118A and 118B show detection of components of TGFRtl5-TGFRs21 using ELISA.

Figures 119A and 119B show the percentages and proliferation of CD4+ T cells, CD8+ T cells, and natural killer (NK) cells present in the spleen of control-treated and TGFRtl5-TGFRs21-treated mice.

Figure 120 shows upregulation of Granzyme B expression of splenocytes in mice treated with TGFRtl5-TGFRs21.

Figure 121 shows enhancement of cytotoxicity of splenocytes by TGFRtl5-TGFRs21 in C57BL/6 Mice.

Figure 122 shows a schematic of the TGFRtl5-TGFRsl6 construct.

Figure 123 shows an additional schematic of the TGFRtl5-TGFRsl6 construct.

Figure 124 shows a schematic of the TGFRtl5-TGFRsl37L construct.

Figure 125 shows an additional schematic of the TGFRtl5-TGFRsl37L construct.

Figure 126 shows changes in the surface phenotype of lymphocyte populations after stimulation with 18tl5-12s, 18tl5-12sl6, and 7tl5-21s.

Figure 127 shows an increase in phospho-STAT4 and phospho-STAT5 levels in NK cells after stimulation with 18tl5-12s.

Figures 128A-128C show in vivo stimulation of Tregs, NK cells, and CD8+ T cells in ApoE_/ mice fed with a Western diet and treated with TGFRtl5-TGFRs.

Figures 129A-129C show immunostimulation in C57BL/6 mice following treatment with TGFRtl5-TGFRs.

Figures 130A and 130B show in vivo induction of proliferation of NK cells and CD8+ T cells in ApoE_/ mice fed with a Western diet and treated with TGFRtl5-TGFRs.

Figures 131A and 13 IB show enhancement of cytotoxicity of NK cells following treatment of NK cells with TGFRtl5-TGFRs.

Figures 132A and 132B show enhancement of ADCC activity of NK cells following treatment of NK cells with TGFRtl5-TGFRs.

Figures 133A-133H show antitumor activity of TGFRtl5-TGFRs plus anti-TRPl antibody (TA99) in combination with chemotherapy in a melanoma mouse model.

Figures 134A-134C show amelioration of the Western diet-induced hyperglycemia in ApoE_/ mice by TGFRtl5-TGFRs.

Figure 135 shows cell surface staining summarizing the differentiation of NK cells into cytokine-induced memory like NK Cells (CIML-NK Cells) after stimulation with 18tl5-12s and cultured in rhIL15.

Figure 136 shows upregulation shows upregulation of CD44hi memory T cells upon treatment with TGFRtl5-TGFRs.

Figure 137 shows a graph of Factor X (FX) activation following treatment with single-chain or multi-chain chimeric polypeptides.

Figure 138 shows clotting time for a buffer with varying concentrations of Innovin in a prothrombin time (PT) test.

Figure 139 shows clotting time for multi-chain chimeric polypeptides in a PT Assay.

Figure 140 shows clotting time of the multi-chain chimeric polypeptides in a PT assay when mixed with 32DB cells.

Figure 141 shows clotting time of multi-chain chimeric polypeptides in a PT assay when mixed with human PBMC.

Figure 142 shows binding of 7tl5-21sl37L (long version) and 7tl5-21sl37L (short version) to CD137 (4. IBB).

Figure 143A-143D show detection of IL7, IL21, IL15, and 4.1BBL in 7tl521sl37L (long version) by the respective antibodies using ELISA.

Figure 144 shows IL-15 activity of 7tl5-21sl37L (long version) and 7tl521sl37L (short version) as evaluated by a IL2Ra3y-containing CTLL2 cell proliferation assay.

DETAILED DESCRIPTION

Provided herein are multi-chain chimeric polypeptides that include: (a) a first chimeric polypeptide including: (i) a first target-binding domain; (ii) a soluble tissue factor domain; and (iii) a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide including: (i) a second domain of a pair of affinity domains; and (ii) a second target-binding domain, where the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains. Also provided herein are compositions that include any of the multi-chain chimeric polypeptides described herein, nucleic acids that encode any of the multi-chain chimeric polypeptides described herein, and cells that include any of the nucleic acids that encode any of the multi-chain chimeric polypeptides described herein. Also provided herein are methods of stimulating an immune cell and methods of treating a subject in need thereof that include the use of any of the multi-chain chimeric polypeptides described herein. Also provided herein are methods of producing any of the multi-chain chimeric polypeptides described herein.

In some examples of any of the multi-chain chimeric polypeptides described herein the total length of first chimeric polypeptide and/or the second chimeric polypeptide can each independently be about 50 amino acids to about 3000 amino acids, about 50 amino acids to about 2500 amino acids, about 50 amino acids to about 2000 amino acids, about 50 amino acids to about 1500 amino acids, about 50 amino acids to about 1000 amino acids, about 50 amino acids to about 950 amino acids, about 50 amino acids to about 900 amino acids, about 50 amino acids to about 850 amino acids, about 50 amino acids to about 800 amino acids, about 50 amino acids to about 750 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 480 amino acids, about 50 amino acids to about 460 amino acids, about 50 amino acids to about 440 amino acids, about 50 amino acids to about 420 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 380 amino acids, about 50 amino acids to about 360 amino acids, about 50 amino acids to about 340 amino acids, about 50 amino acids to about 320 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 280 amino acids, about 50 amino acids to about 260 amino acids, about 50 amino acids to about 240 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 100 amino acids, about 100 amino acids to about 3000 amino acids, about 100 amino acids to about 2500 amino acids, about 100 amino acids to about 2000 amino acids, about 100 amino acids to about 1500 amino acids, about 100 amino acids to about 1000 amino acids, about 100 amino acids to about 950 amino acids, about 100 amino acids to about 900 amino acids, about 100 amino acids to about 850 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 480 amino acids, about 100 amino acids to about 460 amino acids, about 100 amino acids to about 440 amino acids, about 100 amino acids to about 420 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 380 amino acids, about 100 amino acids to about 360 amino acids, about 100 amino acids to about 340 amino acids, about 100 amino acids to about 320 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 280 amino acids, about 100 amino acids to about 260 amino acids, about 100 amino acids to about 240 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 150 amino acids, about 150 amino acids to about 3000 amino acids, about 150 amino acids to about 2500 amino acids, about 150 amino acids to about 2000 amino acids, about 150 amino acids to about 1500 amino acids, about 150 amino acids to about 1000 amino acids, about 150 amino acids to about 950 amino acids, about 150 amino acids to about 900 amino acids, about 150 amino acids to about 850 amino acids, about 150 amino acids to about 800 amino acids, about 150 amino acids to about 750 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 480 amino acids, about 150 amino acids to about 460 amino acids, about 150 amino acids to about 440 amino acids, about 150 amino acids to about 420 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 380 amino acids, about 150 amino acids to about 360 amino acids, about 150 amino acids to about 340 amino acids, about 150 amino acids to about 320 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 280 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 240 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 200 amino acids, about 200 amino acids to about 3000 amino acids, about 200 amino acids to about 2500 amino acids, about 200 amino acids to about 2000 amino acids, about 200 amino acids to about 1500 amino acids, about 200 amino acids to about 1000 amino acids, about 200 amino acids to about 950 amino acids, about 200 amino acids to about 900 amino acids, about 200 amino acids to about 850 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 480 amino acids, about 200 amino acids to about 460 amino acids, about 200 amino acids to about 440 amino acids, about 200 amino acids to about 420 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 380 amino acids, about 200 amino acids to about 360 amino acids, about 200 amino acids to about 340 amino acids, about 200 amino acids to about 320 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 280 amino acids, about 200 amino acids to about 260 amino acids, about 200 amino acids to about 240 amino acids, about 200 amino acids to about 220 amino acids, about 220 amino acids to about 3000 amino acids, about 220 amino acids to about 2500 amino acids, about 220 amino acids to about 2000 amino acids, about 220 amino acids to about 1500 amino acids, about 220 amino acids to about 1000 amino acids, about 220 amino acids to about 950 amino acids, about 220 amino acids to about 900 amino acids, about 220 amino acids to about 850 amino acids, about 220 amino acids to about 800 amino acids, about 220 amino acids to about 750 amino acids, about 220 amino acids to about 700 amino acids, about 220 amino acids to about 650 amino acids, about 220 amino acids to about 600 amino acids, about 220 amino acids to about 550 amino acids, about 220 amino acids to about 500 amino acids, about 220 amino acids to about 480 amino acids, about 220 amino acids to about 460 amino acids, about 220 amino acids to about 440 amino acids, about 220 amino acids to about 420 amino acids, about 220 amino acids to about 400 amino acids, about 220 amino acids to about 380 amino acids, about 220 amino acids to about 360 amino acids, about 220 amino acids to about 340 amino acids, about 220 amino acids to about 320 amino acids, about 220 amino acids to about 300 amino acids, about 220 amino acids to about 280 amino acids, about 220 amino acids to about 260 amino acids, about 220 amino acids to about 240 amino acids, about 240 amino acids to about 3000 amino acids, about 240 amino acids to about 2500 amino acids, about 240 amino acids to about 2000 amino acids, about 240 amino acids to about 1500 amino acids, about 240 amino acids to about 1000 amino acids, about 240 amino acids to about 950 amino acids, about 240 amino acids to about 900 amino acids, about 240 amino acids to about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 650 amino acids, about 240 amino acids to about 600 amino acids, about 240 amino acids to about 550 amino acids, about 240 amino acids to about 500 amino acids, about 240 amino acids to about 480 amino acids, about 240 amino acids to about 460 amino acids, about 240 amino acids to about 440 amino acids, about 240 amino acids to about 420 amino acids, about 240 amino acids to about 400 amino acids, about 240 amino acids to about 380 amino acids, about 240 amino acids to about 360 amino acids, about 240 amino acids to about 340 amino acids, about 240 amino acids to about 320 amino acids, about 240 amino acids to about 300 amino acids, about 240 amino acids to about 280 amino acids, about 240 amino acids to about 260 amino acids, about 260 amino acids to about 3000 amino acids, about 260 amino acids to about 2500 amino acids, about 260 amino acids to about 2000 amino acids, about 260 amino acids to about 1500 amino acids, about 260 amino acids to about 1000 amino acids, about 260 amino acids to about 950 amino acids, about 260 amino acids to about 900 amino acids, about 260 amino acids to about 850 amino acids, about 260 amino acids to about 800 amino acids, about 260 amino acids to about 750 amino acids, about 260 amino acids to about

700 amino acids, about 260 amino acids to about 650 amino acids, about 260 amino acids to about 600 amino acids, about 260 amino acids to about 550 amino acids, about 260 amino acids to about 500 amino acids, about 260 amino acids to about 480 amino acids, about 260 amino acids to about 460 amino acids, about 260 amino acids to about 440 amino acids, about 260 amino acids to about 420 amino acids, about 260 amino acids to about 400 amino acids, about 260 amino acids to about 380 amino acids, about 260 amino acids to about 360 amino acids, about 260 amino acids to about 340 amino acids, about 260 amino acids to about 320 amino acids, about 260 amino acids to about 300 amino acids, about 260 amino acids to about 280 amino acids, about 280 amino acids to about 3000 amino acids, about 280 amino acids to about 2500 amino acids, about 280 amino acids to about 2000 amino acids, about 280 amino acids to about 1500 amino acids, about 280 amino acids to about 1000 amino acids, about 280 amino acids to about 950 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids to about 850 amino acids, about 280 amino acids to about 800 amino acids, about 280 amino acids to about 750 amino acids, about 280 amino acids to about 700 amino acids, about 280 amino acids to about 650 amino acids, about 280 amino acids to about 600 amino acids, about 280 amino acids to about 550 amino acids, about 280 amino acids to about 500 amino acids, about 280 amino acids to about 480 amino acids, about 280 amino acids to about 460 amino acids, about 280 amino acids to about 440 amino acids, about 280 amino acids to about 420 amino acids, about 280 amino acids to about 400 amino acids, about 280 amino acids to about 380 amino acids, about 280 amino acids to about 360 amino acids, about 280 amino acids to about 340 amino acids, about 280 amino acids to about 320 amino acids, about 280 amino acids to about 300 amino acids, about 300 amino acids to about 3000 amino acids, about 300 amino acids to about 2500 amino acids, about 300 amino acids to about 2000 amino acids, about 300 amino acids to about 1500 amino acids, about 300 amino acids to about 1000 amino acids, about 300 amino acids to about 950 amino acids, about 300 amino acids to about 900 amino acids, about 300 amino acids to about 850 amino acids, about 300 amino acids to about 800 amino acids, about 300 amino acids to about 750 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 480 amino acids, about 300 amino acids to about 460 amino acids, about 300 amino acids to about 440 amino acids, about 300 amino acids to about 420 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 380 amino acids, about 300 amino acids to about 360 amino acids, about 300 amino acids to about 340 amino acids, about 300 amino acids to about 320 amino acids, about 320 amino acids to about 3000 amino acids, about 320 amino acids to about 2500 amino acids, about 320 amino acids to about 2000 amino acids, about 320 amino acids to about 1500 amino acids, about 320 amino acids to about 1000 amino acids, about 320 amino acids to about 950 amino acids, about 320 amino acids to about 900 amino acids, about 320 amino acids to about 850 amino acids, about 320 amino acids to about 800 amino acids, about 320 amino acids to about 750 amino acids, about 320 amino acids to about 700 amino acids, about 320 amino acids to about 650 amino acids, about 320 amino acids to about 600 amino acids, about 320 amino acids to about 550 amino acids, about 320 amino acids to about 500 amino acids, about 320 amino acids to about 480 amino acids, about 320 amino acids to about 460 amino acids, about 320 amino acids to about 440 amino acids, about 320 amino acids to about 420 amino acids, about 320 amino acids to about 400 amino acids, about 320 amino acids to about 380 amino acids, about 320 amino acids to about 360 amino acids, about 320 amino acids to about 340 amino acids, about 340 amino acids to about 3000 amino acids, about 340 amino acids to about 2500 amino acids, about 340 amino acids to about 2000 amino acids, about 340 amino acids to about 1500 amino acids, about 340 amino acids to about 1000 amino acids, about 340 amino acids to about 950 amino acids, about 340 amino acids to about 900 amino acids, about 340 amino acids to about 850 amino acids, about 340 amino acids to about 800 amino acids, about 340 amino acids to about 750 amino acids, about 340 amino acids to about 700 amino acids, about 340 amino acids to about 650 amino acids, about 340 amino acids to about 600 amino acids, about 340 amino acids to about 550 amino acids, about 340 amino acids to about 500 amino acids, about 340 amino acids to about 480 amino acids, about 340 amino acids to about 460 amino acids, about 340 amino acids to about 440 amino acids, about 340 amino acids to about 420 amino acids, about 340 amino acids to about 400 amino acids, about 340 amino acids to about 380 amino acids, about 340 amino acids to about 360 amino acids, about 360 amino acids to about 3000 amino acids, about 360 amino acids to about 2500 amino acids, about 360 amino acids to about 2000 amino acids, about 360 amino acids to about 1500 amino acids, about 360 amino acids to about 1000 amino acids, about 360 amino acids to about 950 amino acids, about 360 amino acids to about 900 amino acids, about 360 amino acids to about 850 amino acids, about 360 amino acids to about 800 amino acids, about 360 amino acids to about 750 amino acids, about 360 amino acids to about 700 amino acids, about 360 amino acids to about 650 amino acids, about 360 amino acids to about 600 amino acids, about 360 amino acids to about 550 amino acids, about 360 amino acids to about 500 amino acids, about 360 amino acids to about 480 amino acids, about 360 amino acids to about 460 amino acids, about 360 amino acids to about 440 amino acids, about 360 amino acids to about 420 amino acids, about 360 amino acids to about 400 amino acids, about 360 amino acids to about 380 amino acids, about 380 amino acids to about 3000 amino acids, about 380 amino acids to about 2500 amino acids, about 380 amino acids to about 2000 amino acids, about 380 amino acids to about 1500 amino acids, about 380 amino acids to about 1000 amino acids, about 380 amino acids to about 950 amino acids, about 380 amino acids to about 900 amino acids, about 380 amino acids to about 850 amino acids, about 380 amino acids to about 800 amino acids, about 380 amino acids to about 750 amino acids, about 380 amino acids to about 700 amino acids, about 380 amino acids to about 650 amino acids, about 380 amino acids to about 600 amino acids, about 380 amino acids to about 550 amino acids, about 380 amino acids to about 500 amino acids, about 380 amino acids to about 480 amino acids, about 380 amino acids to about 460 amino acids, about 380 amino acids to about 440 amino acids, about 380 amino acids to about 420 amino acids, about 380 amino acids to about 400 amino acids, about 400 amino acids to about 3000 amino acids, about 400 amino acids to about 2500 amino acids, about 400 amino acids to about 2000 amino acids, about 400 amino acids to about 1500 amino acids, about 400 amino acids to about 1000 amino acids, about 400 amino acids to about 950 amino acids, about 400 amino acids to about 900 amino acids, about 400 amino acids to about 850 amino acids, about 400 amino acids to about 800 amino acids, about 400 amino acids to about 750 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 480 amino acids, about 400 amino acids to about 460 amino acids, about 400 amino acids to about 440 amino acids, about 400 amino acids to about 420 amino acids, about 420 amino acids to about 3000 amino acids, about 420 amino acids to about 2500 amino acids, about 420 amino acids to about 2000 amino acids, about 420 amino acids to about 1500 amino acids, about 420 amino acids to about 1000 amino acids, about 420 amino acids to about 950 amino acids, about 420 amino acids to about 900 amino acids, about 420 amino acids to about 850 amino acids, about 420 amino acids to about 800 amino acids, about 420 amino acids to about 750 amino acids, about 420 amino acids to about 700 amino acids, about 420 amino acids to about 650 amino acids, about 420 amino acids to about 600 amino acids, about 420 amino acids to about 550 amino acids, about 420 amino acids to about 500 amino acids, about 420 amino acids to about 480 amino acids, about 420 amino acids to about 460 amino acids, about 420 amino acids to about 440 amino acids, about 440 amino acids to about 3000 amino acids, about 440 amino acids to about 2500 amino acids, about 440 amino acids to about 2000 amino acids, about 440 amino acids to about 1500 amino acids, about 440 amino acids to about 1000 amino acids, about 440 amino acids to about 950 amino acids, about 440 amino acids to about 900 amino acids, about 440 amino acids to about 850 amino acids, about 440 amino acids to about 800 amino acids, about 440 amino acids to about 750 amino acids, about 440 amino acids to about 700 amino acids, about 440 amino acids to about 650 amino acids, about 440 amino acids to about 600 amino acids, about 440 amino acids to about 550 amino acids, about 440 amino acids to about 500 amino acids, about 440 amino acids to about 480 amino acids, about 440 amino acids to about 460 amino acids, about 460 amino acids to about 3000 amino acids, about 460 amino acids to about 2500 amino acids, about 460 amino acids to about 2000 amino acids, about 460 amino acids to about 1500 amino acids, about 460 amino acids to about 1000 amino acids, about 460 amino acids to about 950 amino acids, about 460 amino acids to about 900 amino acids, about 460 amino acids to about 850 amino acids, about 460 amino acids to about 800 amino acids, about 460 amino acids to about 750 amino acids, about 460 amino acids to about 700 amino acids, about 460 amino acids to about 650 amino acids, about 460 amino acids to about 600 amino acids, about 460 amino acids to about 550 amino acids, about 460 amino acids to about 500 amino acids, about 460 amino acids to about 480 amino acids, about 480 amino acids to about 3000 amino acids, about 480 amino acids to about 2500 amino acids, about 480 amino acids to about 2000 amino acids, about 480 amino acids to about 1500 amino acids, about 480 amino acids to about 1000 amino acids, about 480 amino acids to about 950 amino acids, about 480 amino acids to about 900 amino acids, about 480 amino acids to about 850 amino acids, about 480 amino acids to about 800 amino acids, about 480 amino acids to about 750 amino acids, about 480 amino acids to about 700 amino acids, about 480 amino acids to about 650 amino acids, about 480 amino acids to about 600 amino acids, about 480 amino acids to about 550 amino acids, about 480 amino acids to about 500 amino acids, about 500 amino acids to about 3000 amino acids, about 500 amino acids to about 2500 amino acids, about 500 amino acids to about 2000 amino acids, about 500 amino acids to about 1500 amino acids, about 500 amino acids to about 1000 amino acids, about 500 amino acids to about 950 amino acids, about 500 amino acids to about 900 amino acids, about 500 amino acids to about 850 amino acids, about 500 amino acids to about 800 amino acids, about 500 amino acids to about 750 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 650 amino acids, about 500 amino acids to about 600 amino acids, about 500 amino acids to about 550 amino acids, about 550 amino acids to about 3000 amino acids, about 550 amino acids to about 2500 amino acids, about 550 amino acids to about 2000 amino acids, about 550 amino acids to about 1500 amino acids, about 550 amino acids to about 1000 amino acids, about 550 amino acids to about 950 amino acids, about 550 amino acids to about 900 amino acids, about 550 amino acids to about 850 amino acids, about 550 amino acids to about 800 amino acids, about 550 amino acids to about 750 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 3000 amino acids, about 600 amino acids to about 2500 amino acids, about 600 amino acids to about 2000 amino acids, about 600 amino acids to about 1500 amino acids, about 600 amino acids to about 1000 amino acids, about 600 amino acids to about 950 amino acids, about 600 amino acids to about 900 amino acids, about 600 amino acids to about 850 amino acids, about 600 amino acids to about 800 amino acids, about 600 amino acids to about 750 amino acids, about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 3000 amino acids, about 650 amino acids to about 2500 amino acids, about 650 amino acids to about 2000 amino acids, about 650 amino acids to about 1500 amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to about 950 amino acids, about 650 amino acids to about 900 amino acids, about 650 amino acids to about 850 amino acids, about 650 amino acids to about 800 amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to about 700 amino acids, about 700 amino acids to about 3000 amino acids, about 700 amino acids to about 2500 amino acids, about 700 amino acids to about 2000 amino acids, about 700 amino acids to about 1500 amino acids, about 700 amino acids to about 1000 amino acids, about 700 amino acids to about 950 amino acids, about 700 amino acids to about 900 amino acids, about 700 amino acids to about 850 amino acids, about 700 amino acids to about 800 amino acids, about 700 amino acids to about 750 amino acids, about 750 amino acids to about 3000 amino acids, about 750 amino acids to about 2500 amino acids, about 750 amino acids to about 2000 amino acids, about 750 amino acids to about 1500 amino acids, about 750 amino acids to about 1000 amino acids, about 750 amino acids to about 950 amino acids, about 750 amino acids to about 900 amino acids, about 750 amino acids to about 850 amino acids, about 750 amino acids to about 800 amino acids, about 800 amino acids to about 3000 amino acids, about 800 amino acids to about 2500 amino acids, about 800 amino acids to about 2000 amino acids, about 800 amino acids to about 1500 amino acids, about 800 amino acids to about 1000 amino acids, about 800 amino acids to about 950 amino acids, about 800 amino acids to about 900 amino acids, about 800 amino acids to about 850 amino acids, about 850 amino acids to about 3000 amino acids, about 850 amino acids to about 2500 amino acids, about 850 amino acids to about 2000 amino acids, about 850 amino acids to about 1500 amino acids, about 850 amino acids to about 1000 amino acids, about 850 amino acids to about 950 amino acids, about 850 amino acids to about 900 amino acids, about 900 amino acids to about 3000 amino acids, about 900 amino acids to about 2500 amino acids, about 900 amino acids to about 2000 amino acids, about 900 amino acids to about 1500 amino acids, about 900 amino acids to about 1000 amino acids, about 900 amino acids to about 950 amino acids, about

950 amino acids to about 3000 amino acids, about 950 amino acids to about 2500 amino acids, about 950 amino acids to about 2000 amino acids, about 950 amino acids to about 1500 amino acids, about 950 amino acids to about 1000 amino acids, about 1000 amino acids to about 3000 amino acids, about 1000 amino acids to about 2500 amino acids, about 1000 amino acids to about 2000 amino acids, about 1000 amino acids to about 1500 amino acids, about 1500 amino acids to about 3000 amino acids, about 1500 amino acids to about 2500 amino acids, about 1500 amino acids to about 2000 amino acids, about 2000 amino acids to about 3000 amino acids, about 2000 amino acids to about 2500 amino acids, or about 2500 amino acids to about 3000 amino acids. Diagrams of exemplary multi-chain chimeric polypeptides provided herein are depicted in Figures 1 and 2.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain (e.g., any of the first target-binding domains described herein) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) directly abut each other in the first chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first target-binding domain (e.g., any of the exemplary first target-binding domains described herein) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) in the first chimeric polypeptide.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) directly abut each other in the first chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second domain of the pair of affinity domains (e.g., any of the exemplary second domains of any of the exemplary pairs of affinity domains described herein) and the second target-binding domain (e.g., any of the exemplary second target-binding domains described herein) directly abut each other in the second chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the second domain of the pair of affinity domains (e.g., any of the exemplary second domains of any of the exemplary pairs of affinity domains described herein) and the second targetbinding domain (e.g., any of the exemplary second target-binding domains described herein) in the second chimeric polypeptide.

Non-limiting aspects of these chimeric polypeptides, nucleic acids, vectors, cells, and methods are described below, and can be used in any combination without limitation. Additional aspects of these chimeric polypeptides, nucleic acids, vectors, cells, and methods are known in the art.

Tissue Factor

Human tissue factor is a 263 amino-acid transmembrane protein containing three domains: (1) a219-amino acid N-terminal extracellular domain (residues 1-219); (2) a 22-amino acid transmembrane domain (residues 220-242); and (3) a 21-amino acid cytoplasmic C-terminal tail (residues 242-263) ((UniProtKB Identifier Number: P13726). The cytoplasmic tail contains two phosphorylation sites at Ser253 and Ser258, and one Spalmitoylation site at Cys245. Deletion or mutation of the cytoplasmic domain was not found to affect tissue factor coagulation activity. Tissue factor has one S-palmitoylation site in the intracellular domain of the protein at Cys245. The Cys245 is located at the amino acid terminus of the intracellular domain and close to the membrane surface. The tissue factor transmembrane domain is composed of a single-spanning a-helix.

The extracellular domain of tissue factor, composed of two fibronectin type III domains, is connected to the transmembrane domain through a six-amino acid linker. This linker provides conformational flexibility to decouple the tissue factor extracellular domain from its transmembrane and cytoplasmic domains. Each tissue factor fibronectin type III module is composed of two overlapping β sheets with the top sheet domain containing three antiparallel β-strands and the bottom sheet containing four β-strands. The β-strands are connected by β-loops between strand βΑ and βΒ, βΕ and βϋ, and βΕ and βΕ, all of which are conserved in conformation in the two modules. There are three short a-helix segments connecting the β-strands. A unique feature of tissue factor is a 17amino acid β-hairpin between strand β 10 and strand β 11, which is not a common element of the fibronectin superfamily. The N-terminal domain also contains a 12 amino acid loop between β6Ε and β7Θ that is not present in the C-terminal domain and is unique to tissue factor. Such a fibronectin type III domain structure is a feature of the immunoglobulin-like family of protein folds and is conserved among a wide variety of extracellular proteins.

The zymogen FVII is rapidly converted to FVIIa by limited proteolysis once it binds to tissue to form the active tissue factor-FVIIa complex. The FVIIa, which circulates as an enzyme at a concentration of approximately 0.1 nM (1% of plasma FVII), can also bind directly to tissue factor. The allosteric interaction between tissue factor and FVIIa on the tissue factor-FVIIa complex greatly increases the enzymatic activity of FVIIa: an approximate 20- to 100-fold increase in the rate of hydrolysis of small, chromogenic peptidyl substrates, and nearly a million-fold increase in the rate of activation of the natural macromolecular substrates FIX and FX. In concert with allosteric activation of the active site of FVIIa upon binding to tissue factor, the formation of tissue factor-FVIIa complex on phospholipid bilayer (i.e., upon exposure of phosphatidyl-L-serine on membrane surfaces) increases the rate of FIX or FX activation, in a Ca2+-dependent manner, an additional 1,000-fold. The roughly million-fold overall increase in FX activation by tissue factor-FVIIa-phospholipid complex relative to free FVIIa is a critical regulatory point for the coagulation cascade.

FVII is a ~50 kDa, single-chain polypeptide consisting of 406 amino acid residues, with an N-terminal γ-carboxyglutamate-rich (GLA) domain, two epidermal growth factor-hke domains (EGF1 and EFG2), and a C-terminal serine protease domain. FVII is activated to FVIIa by a specific proteolytic cleavage of the Ile Arg152 bond in the short linker region between the EGF2 and the protease domain. This cleavage results in the light and heavy chains being held together by a single disulfide bond of Cys135 and Cys262. FVIIa binds phospholipid membrane in a Ca2+-dependent manner through its Nterminal GLA-domain. Immediately C-terminal to the GLA domain is an aromatic stack and two EGF domains. The aromatic stack connects the GLA to EGF1 domain which binds a single Ca2+ ion. Occupancy of this Ca2+-binding site increases FVIIa amidolytic activity and tissue factor association. The catalytic triad consist of His193, Asp242, and Ser344, and binding of a single Ca2+ ion within the FVIIa protease domain is critical for its catalytic activity. Proteolytic activation of FVII to FVIIa frees the newly formed amino terminus at He153 to fold back and be inserted into the activation pocket forming a salt bridge with the carboxylate of Asp343 to generate the oxyanion hole. Formation of this salt bridge is critical for FVIIa activity. However, oxyanion hole formation does not occur in free FVIIa upon proteolytic activation. As a result, FVIIa circulates in a zymogen-like state that is poorly recognized by plasma protease inhibitors, allowing it to circulate with a half-life of approximately 90 minutes.

Tissue factor-mediated positioning of the FVIIa active site above the membrane surface is important for FVIIa towards cognate substrates. Free FVIIa adopts a stable, extended structure when bound to the membrane with its active site positioned ~80A above the membrane surface. Upon FVIIa binding to tissue factor, the FVa active site is repositioned ~6A closer to the membrane. This modulation may aid in a proper alignment of the FVIIa catalytic triad with the target substrate cleavage site. Using GLAdomainless FVIIa, it has been shown that the active site was still positioned a similar distance above the membrane, demonstrating that tissue factor is able to fully support FVIIa active site positioning even in the absence of FVIIa-membrane interaction. Additional data showed that tissue factor supported full FVIIa proteolytic activity as long as the tissue factor extracellular domain was tethered in some way to the membrane surface. However, raising the active site of FVIIa greater than 80A above the membrane surface greatly reduced the ability of the tissue factor-FVIIa complex to activate FX but did not diminish tissue factor-FVIIa amidolytic activity.

Alanine scanning mutagenesis has been used to assess the role of specific amino acid side chains in the tissue factor extracellular domain for interaction with FVIIa (Gibbs et al., Biochemistry 33(47): 14003-14010, 1994; Schullek et al., J Biol Chem 269(30): 19399-19403, 1994). Alanine substitution identified a limited number of residue positions at which alanine replacements cause 5- to 10-fold lower affinity for FVIIa binding. Most of these residue side chains were found to be well-exposed to solvent in the crystal structure, concordant with macromolecular ligand interaction. The FVIIa ligand-binding site is located over an extensive region at the boundary between the two modules. In the C-module, residues Arg135 and Phe140 located on the protruding B-C loop provide an independent contact with FVIIa. Leu133 is located at the base of the fingerlike structure and packed into the cleft between the two modules. This provides continuity to a major cluster of important binding residues consisting of Lys20, Thr60, Asp58, and He22. Thr60 is only partially solvent-exposed and may play a local structural role rather than making a significant contact with ligand. The binding site extends onto the concave side of the intermodule angle involving Glu24 and Gin110, and potentially the more distant residue Vai207. The binding region extends from Asp58 onto a convex surface area formed by Lys48, Lys46, Gin37, Asp44, and Trp45. Trp45 and Asp44 do not interact independently with FVIIa, indicating that the mutational effect at the Trp45 position may reflect a structural importance of this side chain for the local packing of the adjacent Asp44 and Gin37 side chain. The interactive area further includes two surfaceexposed aromatic residues, Phe76 and Tyr78, which form part of the hydrophobic cluster in the N-module.

The known physiologic substrates of tissue factor-FVIIa are FVII, FIX, and FX and certain proteinase-activated receptors. Mutational analysis has identified a number of residues that, when mutated, support full FVIIa amidolytic activity towards small peptidyl substrates but are deficient in their ability to support macromolecular substrate (i.e., FVII, FIX, and FX) activation (Ruf et al., J Biol Chem 267(31): 22206-22210, 1992; Ruf et al., J Biol Chem 267(9): 6375-6381, 1992; Huang et al., J Biol Chem 271(36): 21752-21757, 1996; Kirchhofer et al., Biochemistry 39(25): 7380-7387, 2000). The tissue factor loop region at residues 159-165, and residues in or adjacent to this flexible loop have been shown to be critical for the proteolytic activity of the tissue factor-FVIIa complex. This defines the proposed substrate-binding exosite region of tissue factor that is quite distant from the FVIIa active site. A substitution of the glycine residue by a marginally bulkier residue alanine, significantly impairs tissue factor-FVIIa proteolytic activity. This suggests that the flexibility afforded by glycine is critical for the loop of residues 159-165 for tissue factor macromolecular substrate recognition.

The residues Lys165 and Lys166 have also been demonstrated to be important for substrate recognition and binding. Mutation of either of these residues to alanine results in a significant decrease in the tissue factor co-factor function. Lys165 and Lys166 face away from each other, with Lys165 pointing towards FVIIa in most tissue factor-FVIIa structures, and Lys166 pointing into the substrate binding exosite region in the crystal structure. Putative salt bridge formation between Lys165 of and Gia35 of FVIIa would support the notion that tissue factor interaction with the GLA domain of FVIIa modulates substrate recognition. These results suggest that the C-terminal portion of the tissue factor ectodomain directly interacts with the GLA-domain, the possible adjacent EGF1 domains, of FIX and FX, and that the presence of the FVIIa GLA-domain may modulate these interactions either directly or indirectly.

Soluble Tissue Factor Domain

In some embodiments of any of the polypeptides, compositions, or methods described herein, the soluble tissue factor domain can be a wildtype tissue factor polypeptide lacking the signal sequence, the transmembrane domain, and the intracellular domain. In some examples, the soluble tissue factor domain can be a tissue factor mutant, wherein a wildtype tissue factor polypeptide lacking the signal sequence, the transmembrane domain, and the intracellular domain, and has been further modified at selected amino acids. In some examples, the soluble tissue factor domain can be a soluble human tissue factor domain. In some examples, the soluble tissue factor domain can be a soluble mouse tissue factor domain. In some examples, the soluble tissue factor domain can be a soluble rat tissue factor domain. Non-limiting examples of soluble human tissue factor domains, a mouse soluble tissue factor domain, a rat soluble tissue factor domain, and mutant soluble tissue factor domains are shown below.

Exemplary Soluble Human Tissue Factor Domain (SEQ ID NO: 1)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTD TECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQ PTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGK KTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

Exemplary Nucleic Acid Encoding Soluble Human Tissue Factor Domain (SEQ ID NO: 2)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCA ACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACC GTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCAC CGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACC TACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTC CGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGA CCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGT GAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTT CTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGG AAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTT TAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATC CCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGG GCCAAGAAAAGGGCGAGTTCCGGGAG

Exemplary Mutant Soluble Human Tissue Factor Domain (SEQ ID NO: 3)

SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDWKSKCFYTT DTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNL GQPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSS GKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEF RE

Exemplary Mutant Soluble Human Tissue Factor Domain (SEQ ID NO: 4)

SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDAKSKCFYTTD TECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLAENSPEFTPYLETNLG

QPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSG KKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFR E

Exemplary Soluble Mouse Tissue Factor Domain (SEQ ID NO: 5) agipekafnltwistdfktilewqpkptnytytvqisdrsrnwknkcfstt dtecdltdeivkdvtwayeakvlsvprrnsvhgdgdqlvihgeeppftnap kfIpyrdtnlgqpviqqfeqdgrklnvvvkdsltlvrkngtfItlrqvfgk dlgyiityrkgsstgkktnitntnefsidveegvsycffvqamifsrktnq nspgsstveteqwksfIge

Exemplary Soluble Rat Tissue Factor Domain (SEQ ID NO: 6)

Agtppgkafnltwistdfktilewqpkptnytytvqisdrsrnwkykctgt tdtecdltdeivkdvnwtyearvlsvpwrnsthgketlfgthgeeppftna rkfIpyrdtkigqpviqkyeqggtklkvtvkdsftlvrkngtfItlrqvfg ndlgyiltyrkdsstgrktntthtneflidvekgvsycffaqavifsrktn hkspesitkcteqwksvlge

In some embodiments, a soluble tissue factor domain can include a sequence that is at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 1, 3, 4, 5, or 6. In some embodiments, a soluble tissue factor domain can include a sequence of SEQ ID NO: 1, 3, 4, 5, or 6, with one to twenty amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids removed from its N-terminus and/or one to twenty amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids removed from its C-terminus.

As can be appreciated in the art, one skilled in the art would understand that mutation of amino acids that are conserved between different mammalian species is more likely to decrease the activity and/or structural stability of the protein, while mutation of amino acids that are not conserved between different mammalian species is less likely to decrease the activity and/or structural stability of the protein.

In some examples of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain is not capable of binding to Factor Vila. In some examples of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain does not convert inactive Factor X into Factor Xa. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the multichain chimeric polypeptide does not stimulate blood coagulation in a mammal.

In some examples, the soluble tissue factor domain can be a soluble human tissue factor domain. In some embodiments, the soluble tissue factor domain can be a soluble mouse tissue factor domain. In some embodiments, the soluble tissue factor domain can be a soluble rat tissue factor domain.

In some examples, the soluble tissue factor domain does not include one or more (e.g., two, three, four, five, six, or seven) of: a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein; an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein; a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein; an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein; a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein; an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein. In some embodiments, the mutant soluble tissue factor possesses the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

In some examples, the soluble tissue factor domain can be encoded by a nucleic acid including a sequence that is at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 2.

In some embodiments, the soluble tissue factor domain can have a total length of about 20 amino acids to about 220 amino acids, about 20 amino acids to about 215 amino acids, about 20 amino acids to about 210 amino acids, about 20 amino acids to about 205 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 195 amino acids, about 20 amino acids to about 190 amino acids, about 20 amino acids to about 185 amino acids, about 20 amino acids to about 180 amino acids, about 20 amino acids to about 175 amino acids, about 20 amino acids to about 170 amino acids, about 20 amino acids to about 165 amino acids, about 20 amino acids to about 160 amino acids, about 20 amino acids to about 155 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 145 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 135 amino acids, about 20 amino acids to about 130 amino acids, about 20 amino acids to about 125 amino acids, about 20 amino acids to about 120 amino acids, about 20 amino acids to about 115 amino acids, about 20 amino acids to about 110 amino acids, about 20 amino acids to about 105 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 95 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 85 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 75 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 30 amino acids, about 30 amino acids to about 220 amino acids, about 30 amino acids to about 215 amino acids, about 30 amino acids to about 210 amino acids, about 30 amino acids to about 205 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino acids to about 195 amino acids, about 30 amino acids to about 190 amino acids, about 30 amino acids to about 185 amino acids, about 30 amino acids to about 180 amino acids, about 30 amino acids to about 175 amino acids, about 30 amino acids to about 170 amino acids, about 30 amino acids to about 165 amino acids, about 30 amino acids to about 160 amino acids, about 30 amino acids to about 155 amino acids, about 30 amino acids to about 150 amino acids, about 30 amino acids to about 145 amino acids, about 30 amino acids to about 140 amino acids, about 30 amino acids to about 135 amino acids, about 30 amino acids to about 130 amino acids, about 30 amino acids to about 125 amino acids, about 30 amino acids to about 120 amino acids, about 30 amino acids to about 115 amino acids, about 30 amino acids to about 110 amino acids, about 30 amino acids to about 105 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 95 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 85 amino acids, about 30 amino acids to about 80 amino acids, about 30 amino acids to about 75 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about 40 amino acids, about 40 amino acids to about 220 amino acids, about 40 amino acids to about 215 amino acids, about 40 amino acids to about 210 amino acids, about 40 amino acids to about 205 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 195 amino acids, about 40 amino acids to about 190 amino acids, about 40 amino acids to about 185 amino acids, about 40 amino acids to about 180 amino acids, about 40 amino acids to about 175 amino acids, about 40 amino acids to about 170 amino acids, about 40 amino acids to about 165 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 155 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 145 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 135 amino acids, about 40 amino acids to about 130 amino acids, about 40 amino acids to about 125 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 115 amino acids, about 40 amino acids to about 110 amino acids, about 40 amino acids to about 105 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 95 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 85 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 75 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 50 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 215 amino acids, about 50 amino acids to about 210 amino acids, about 50 amino acids to about 205 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 195 amino acids, about 50 amino acids to about 190 amino acids, about 50 amino acids to about 185 amino acids, about 50 amino acids to about 180 amino acids, about 50 amino acids to about 175 amino acids, about 50 amino acids to about 170 amino acids, about 50 amino acids to about 165 amino acids, about 50 amino acids to about 160 amino acids, about 50 amino acids to about 155 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 145 amino acids, about 50 amino acids to about 140 amino acids, about 50 amino acids to about 135 amino acids, about 50 amino acids to about 130 amino acids, about 50 amino acids to about 125 amino acids, about 50 amino acids to about 120 amino acids, about 50 amino acids to about 115 amino acids, about 50 amino acids to about 110 amino acids, about 50 amino acids to about 105 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 95 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 85 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 75 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 220 amino acids, about 60 amino acids to about 215 amino acids, about 60 amino acids to about 210 amino acids, about 60 amino acids to about 205 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 195 amino acids, about 60 amino acids to about 190 amino acids, about 60 amino acids to about 185 amino acids, about 60 amino acids to about 180 amino acids, about 60 amino acids to about 175 amino acids, about 60 amino acids to about 170 amino acids, about 60 amino acids to about 165 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 155 amino acids, about 60 amino acids to about 150 amino acids, about 60 amino acids to about 145 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 135 amino acids, about 60 amino acids to about 130 amino acids, about 60 amino acids to about 125 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 115 amino acids, about 60 amino acids to about 110 amino acids, about 60 amino acids to about 105 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 95 amino acids, about 60 amino acids to about 90 amino acids, about 60 amino acids to about 85 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 75 amino acids, about 60 amino acids to about 70 amino acids, about 70 amino acids to about 220 amino acids, about 70 amino acids to about 215 amino acids, about 70 amino acids to about 210 amino acids, about 70 amino acids to about 205 amino acids, about 70 amino acids to about 200 amino acids, about 70 amino acids to about 195 amino acids, about 70 amino acids to about 190 amino acids, about 70 amino acids to about 185 amino acids, about 70 amino acids to about 180 amino acids, about 70 amino acids to about 175 amino acids, about 70 amino acids to about 170 amino acids, about 70 amino acids to about 165 amino acids, about 70 amino acids to about 160 amino acids, about 70 amino acids to about 155 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 145 amino acids, about 70 amino acids to about 140 amino acids, about 70 amino acids to about 135 amino acids, about 70 amino acids to about 130 amino acids, about 70 amino acids to about 125 amino acids, about 70 amino acids to about 120 amino acids, about 70 amino acids to about 115 amino acids, about 70 amino acids to about 110 amino acids, about 70 amino acids to about 105 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 95 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 85 amino acids, about 70 amino acids to about 80 amino acids, about 80 amino acids to about 220 amino acids, about 80 amino acids to about 215 amino acids, about 80 amino acids to about 210 amino acids, about 80 amino acids to about 205 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino acids to about 195 amino acids, about 80 amino acids to about 190 amino acids, about 80 amino acids to about 185 amino acids, about 80 amino acids to about 180 amino acids, about 80 amino acids to about 175 amino acids, about 80 amino acids to about 170 amino acids, about 80 amino acids to about 165 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 155 amino acids, about 80 amino acids to about 150 amino acids, about 80 amino acids to about 145 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 135 amino acids, about 80 amino acids to about 130 amino acids, about 80 amino acids to about 125 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 115 amino acids, about 80 amino acids to about 110 amino acids, about 80 amino acids to about 105 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 95 amino acids, about 80 amino acids to about 90 amino acids, about 90 amino acids to about 220 amino acids, about 90 amino acids to about 215 amino acids, about 90 amino acids to about 210 amino acids, about 90 amino acids to about 205 amino acids, about 90 amino acids to about 200 amino acids, about 90 amino acids to about 195 amino acids, about 90 amino acids to about 190 amino acids, about 90 amino acids to about 185 amino acids, about 90 amino acids to about 180 amino acids, about 90 amino acids to about 175 amino acids, about 90 amino acids to about 170 amino acids, about 90 amino acids to about 165 amino acids, about 90 amino acids to about 160 amino acids, about 90 amino acids to about 155 amino acids, about 90 amino acids to about 150 amino acids, about 90 amino acids to about 145 amino acids, about 90 amino acids to about 140 amino acids, about 90 amino acids to about 135 amino acids, about 90 amino acids to about 130 amino acids, about 90 amino acids to about 125 amino acids, about 90 amino acids to about 120 amino acids, about 90 amino acids to about 115 amino acids, about 90 amino acids to about 110 amino acids, about 90 amino acids to about 105 amino acids, about 90 amino acids to about 100 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 215 amino acids, about 100 amino acids to about 210 amino acids, about 100 amino acids to about 205 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 195 amino acids, about 100 amino acids to about 190 amino acids, about 100 amino acids to about 185 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 175 amino acids, about 100 amino acids to about 170 amino acids, about 100 amino acids to about 165 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 155 amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to about 145 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 135 amino acids, about 100 amino acids to about 130 amino acids, about 100 amino acids to about 125 amino acids, about 100 amino acids to about 120 amino acids, about 100 amino acids to about 115 amino acids, about 100 amino acids to about 110 amino acids, about 110 amino acids to about 220 amino acids, about 110 amino acids to about 215 amino acids, about 110 amino acids to about 210 amino acids, about 110 amino acids to about 205 amino acids, about 110 amino acids to about 200 amino acids, about 110 amino acids to about 195 amino acids, about 110 amino acids to about 190 amino acids, about 110 amino acids to about 185 amino acids, about 110 amino acids to about 180 amino acids, about 110 amino acids to about 175 amino acids, about 110 amino acids to about 170 amino acids, about 110 amino acids to about 165 amino acids, about 110 amino acids to about 160 amino acids, about 110 amino acids to about 155 amino acids, about 110 amino acids to about 150 amino acids, about 110 amino acids to about 145 amino acids, about 110 amino acids to about 140 amino acids, about 110 amino acids to about 135 amino acids, about 110 amino acids to about 130 amino acids, about 110 amino acids to about 125 amino acids, about 110 amino acids to about 120 amino acids, about 110 amino acids to about 115 amino acids, about 115 amino acids to about 220 amino acids, about 115 amino acids to about 215 amino acids, about 115 amino acids to about 210 amino acids, about 115 amino acids to about 205 amino acids, about 115 amino acids to about 200 amino acids, about 115 amino acids to about 195 amino acids, about 115 amino acids to about 190 amino acids, about 115 amino acids to about 185 amino acids, about 115 amino acids to about 180 amino acids, about 115 amino acids to about 175 amino acids, about 115 amino acids to about 170 amino acids, about 115 amino acids to about 165 amino acids, about 115 amino acids to about 160 amino acids, about 115 amino acids to about 155 amino acids, about 115 amino acids to about 150 amino acids, about 115 amino acids to about 145 amino acids, about 115 amino acids to about 140 amino acids, about 115 amino acids to about 135 amino acids, about 115 amino acids to about 130 amino acids, about 115 amino acids to about 125 amino acids, about 115 amino acids to about 120 amino acids, about 120 amino acids to about 220 amino acids, about 120 amino acids to about 215 amino acids, about 120 amino acids to about 210 amino acids, about 120 amino acids to about 205 amino acids, about 120 amino acids to about 200 amino acids, about 120 amino acids to about 195 amino acids, about 120 amino acids to about 190 amino acids, about 120 amino acids to about 185 amino acids, about 120 amino acids to about 180 amino acids, about 120 amino acids to about 175 amino acids, about 120 amino acids to about 170 amino acids, about 120 amino acids to about 165 amino acids, about 120 amino acids to about 160 amino acids, about 120 amino acids to about 155 amino acids, about 120 amino acids to about 150 amino acids, about 120 amino acids to about 145 amino acids, about 120 amino acids to about 140 amino acids, about 120 amino acids to about 135 amino acids, about 120 amino acids to about 130 amino acids, about 120 amino acids to about 125 amino acids, about 125 amino acids to about 220 amino acids, about 125 amino acids to about 215 amino acids, about 125 amino acids to about 210 amino acids, about 125 amino acids to about 205 amino acids, about 125 amino acids to about 200 amino acids, about 125 amino acids to about 195 amino acids, about 125 amino acids to about 190 amino acids, about 125 amino acids to about 185 amino acids, about 125 amino acids to about 180 amino acids, about 125 amino acids to about 175 amino acids, about 125 amino acids to about 170 amino acids, about 125 amino acids to about 165 amino acids, about 125 amino acids to about 160 amino acids, about 125 amino acids to about 155 amino acids, about 125 amino acids to about 150 amino acids, about 125 amino acids to about 145 amino acids, about 125 amino acids to about 140 amino acids, about 125 amino acids to about 135 amino acids, about 125 amino acids to about 130 amino acids, about 130 amino acids to about 220 amino acids, about 130 amino acids to about 215 amino acids, about 130 amino acids to about 210 amino acids, about 130 amino acids to about 205 amino acids, about 130 amino acids to about 200 amino acids, about 130 amino acids to about 195 amino acids, about 130 amino acids to about 190 amino acids, about 130 amino acids to about 185 amino acids, about 130 amino acids to about 180 amino acids, about 130 amino acids to about 175 amino acids, about 130 amino acids to about 170 amino acids, about 130 amino acids to about 165 amino acids, about 130 amino acids to about 160 amino acids, about 130 amino acids to about 155 amino acids, about 130 amino acids to about 150 amino acids, about 130 amino acids to about 145 amino acids, about 130 amino acids to about 140 amino acids, about 130 amino acids to about 135 amino acids, about 135 amino acids to about 220 amino acids, about 135 amino acids to about 215 amino acids, about 135 amino acids to about 210 amino acids, about 135 amino acids to about 205 amino acids, about 135 amino acids to about 200 amino acids, about 135 amino acids to about 195 amino acids, about 135 amino acids to about 190 amino acids, about 135 amino acids to about 185 amino acids, about 135 amino acids to about 180 amino acids, about 135 amino acids to about 175 amino acids, about 135 amino acids to about 170 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 160 amino acids, about 135 amino acids to about 155 amino acids, about 135 amino acids to about 150 amino acids, about 135 amino acids to about 145 amino acids, about 135 amino acids to about 140 amino acids, about 140 amino acids to about 220 amino acids, about 140 amino acids to about 215 amino acids, about 140 amino acids to about 210 amino acids, about 140 amino acids to about 205 amino acids, about 140 amino acids to about 200 amino acids, about 140 amino acids to about 195 amino acids, about 140 amino acids to about 190 amino acids, about 140 amino acids to about 185 amino acids, about 140 amino acids to about 180 amino acids, about 140 amino acids to about 175 amino acids, about 140 amino acids to about 170 amino acids, about 140 amino acids to about 165 amino acids, about 140 amino acids to about 160 amino acids, about 140 amino acids to about 155 amino acids, about 140 amino acids to about 150 amino acids, about 140 amino acids to about 145 amino acids, about 145 amino acids to about 220 amino acids, about 145 amino acids to about 215 amino acids, about 145 amino acids to about 210 amino acids, about 145 amino acids to about 205 amino acids, about 145 amino acids to about 200 amino acids, about 145 amino acids to about 195 amino acids, about 145 amino acids to about 190 amino acids, about 145 amino acids to about 185 amino acids, about 145 amino acids to about 180 amino acids, about 145 amino acids to about 175 amino acids, about 145 amino acids to about 170 amino acids, about 145 amino acids to about 165 amino acids, about 145 amino acids to about 160 amino acids, about 145 amino acids to about 155 amino acids, about 145 amino acids to about 150 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 215 amino acids, about 150 amino acids to about 210 amino acids, about 150 amino acids to about 205 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 195 amino acids, about 150 amino acids to about 190 amino acids, about 150 amino acids to about 185 amino acids, about 150 amino acids to about 180 amino acids, about 150 amino acids to about 175 amino acids, about 150 amino acids to about 170 amino acids, about 150 amino acids to about 165 amino acids, about 150 amino acids to about 160 amino acids, about 150 amino acids to about 155 amino acids, about 155 amino acids to about 220 amino acids, about 155 amino acids to about 215 amino acids, about 155 amino acids to about 210 amino acids, about 155 amino acids to about 205 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 195 amino acids, about 155 amino acids to about 190 amino acids, about 155 amino acids to about 185 amino acids, about 155 amino acids to about 180 amino acids, about 155 amino acids to about 175 amino acids, about 155 amino acids to about 170 amino acids, about 155 amino acids to about 165 amino acids, about 155 amino acids to about 160 amino acids, about 160 amino acids to about 220 amino acids, about 160 amino acids to about 215 amino acids, about 160 amino acids to about 210 amino acids, about 160 amino acids to about 205 amino acids, about 160 amino acids to about 200 amino acids, about 160 amino acids to about 195 amino acids, about 160 amino acids to about 190 amino acids, about 160 amino acids to about 185 amino acids, about 160 amino acids to about 180 amino acids, about 160 amino acids to about 175 amino acids, about 160 amino acids to about 170 amino acids, about 160 amino acids to about 165 amino acids, about 165 amino acids to about 220 amino acids, about 165 amino acids to about 215 amino acids, about 165 amino acids to about 210 amino acids, about 165 amino acids to about 205 amino acids, about 165 amino acids to about 200 amino acids, about 165 amino acids to about 195 amino acids, about 165 amino acids to about 190 amino acids, about 165 amino acids to about 185 amino acids, about 165 amino acids to about 180 amino acids, about 165 amino acids to about 175 amino acids, about 165 amino acids to about 170 amino acids, about 170 amino acids to about 220 amino acids, about 170 amino acids to about 215 amino acids, about 170 amino acids to about 210 amino acids, about 170 amino acids to about 205 amino acids, about 170 amino acids to about 200 amino acids, about 170 amino acids to about 195 amino acids, about 170 amino acids to about 190 amino acids, about 170 amino acids to about 185 amino acids, about 170 amino acids to about 180 amino acids, about 170 amino acids to about 175 amino acids, about 175 amino acids to about 220 amino acids, about 175 amino acids to about 215 amino acids, about 175 amino acids to about 210 amino acids, about 175 amino acids to about 205 amino acids, about 175 amino acids to about 200 amino acids, about 175 amino acids to about 195 amino acids, about 175 amino acids to about 190 amino acids, about 175 amino acids to about 185 amino acids, about 175 amino acids to about 180 amino acids, about 180 amino acids to about 220 amino acids, about 180 amino acids to about 215 amino acids, about 180 amino acids to about 210 amino acids, about 180 amino acids to about 205 amino acids, about 180 amino acids to about 200 amino acids, about 180 amino acids to about 195 amino acids, about 180 amino acids to about 190 amino acids, about 180 amino acids to about 185 amino acids, about 185 amino acids to about 220 amino acids, about 185 amino acids to about 215 amino acids, about 185 amino acids to about 210 amino acids, about 185 amino acids to about 205 amino acids, about 185 amino acids to about 200 amino acids, about 185 amino acids to about 195 amino acids, about 185 amino acids to about 190 amino acids, about 190 amino acids to about 220 amino acids, about 190 amino acids to about 215 amino acids, about 190 amino acids to about 210 amino acids, about 190 amino acids to about 205 amino acids, about 190 amino acids to about 200 amino acids, about 190 amino acids to about 195 amino acids, about 195 amino acids to about 220 amino acids, about 195 amino acids to about 215 amino acids, about 195 amino acids to about 210 amino acids, about 195 amino acids to about 205 amino acids, about 195 amino acids to about 200 amino acids, about 200 amino acids to about 220 amino acids, about 200 amino acids to about 215 amino acids, about 200 amino acids to about 210 amino acids, about 200 amino acids to about 205 amino acids, about 205 amino acids to about 220 amino acids, about 205 amino acids to about 215 amino acids, about 205 amino acids to about 210 amino acids, about 210 amino acids to about 220 amino acids, about 210 amino acids to about 215 amino acids, or about 215 amino acids to about 220 amino acids.

Linker Sequences

In some embodiments, the linker sequence can be a flexible linker sequence. Non-limiting examples of linker sequences that can be used are described in Klein et al., Protein Engineering, Design & Selection 27(10):325-330, 2014; Priyanka et al., Protein Sci. 22(2):153-167, 2013. In some examples, the linker sequence is a synthetic linker sequence.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide can include one, two, three, four, five, six, seven, eight, nine, or ten linker sequence(s) (e.g., the same or different linker sequences, e.g., any of the exemplary linker sequences described herein or known in the art). In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide can include one, two, three, four, five, six, seven, eight, nine, or ten linker sequence(s) (e.g., the same or different linker sequences, e.g., any of the exemplary linker sequences described herein or known in the art).

In some embodiments, a linker sequence can have a total length of 1 amino acid to about 100 amino acids, 1 amino acid to about 90 amino acids, 1 amino acid to about 80 amino acids, 1 amino acid to about 70 amino acids, 1 amino acid to about 60 amino acids, 1 amino acid to about 50 amino acids, 1 amino acid to about 45 amino acids, 1 amino acid to about 40 amino acids, 1 amino acid to about 35 amino acids, 1 amino acid to about 30 amino acids, 1 amino acid to about 25 amino acids, 1 amino acid to about 24 amino acids, 1 amino acid to about 22 amino acids, 1 amino acid to about 20 amino acids, 1 amino acid to about 18 amino acids, 1 amino acid to about 16 amino acids, 1 amino acid to about 14 amino acids, 1 amino acid to about 12 amino acids, 1 amino acid to about 10 amino acids, 1 amino acid to about 8 amino acids, 1 amino acid to about 6 amino acids, 1 amino acid to about 4 amino acids, about 2 amino acids to about 100 amino acids, about 2 amino acids to about 90 amino acids, about 2 amino acids to about 80 amino acids, about 2 amino acids to about 70 amino acids, about 2 amino acids to about 60 amino acids, about 2 amino acids to about 50 amino acids, about 2 amino acids to about 45 amino acids, about 2 amino acids to about 40 amino acids, about 2 amino acids to about 35 amino acids, about 2 amino acids to about 30 amino acids, about 2 amino acids to about 25 amino acids, about 2 amino acids to about 24 amino acids, about 2 amino acids to about 22 amino acids, about 2 amino acids to about 20 amino acids, about 2 amino acids to about 18 amino acids, about 2 amino acids to about 16 amino acids, about 2 amino acids to about 14 amino acids, about 2 amino acids to about 12 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 8 amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 4 amino acids, about 4 amino acids to about 100 amino acids, about 4 amino acids to about 90 amino acids, about 4 amino acids to about 80 amino acids, about 4 amino acids to about 70 amino acids, about 4 amino acids to about 60 amino acids, about 4 amino acids to about 50 amino acids, about 4 amino acids to about 45 amino acids, about 4 amino acids to about 40 amino acids, about 4 amino acids to about 35 amino acids, about 4 amino acids to about 30 amino acids, about 4 amino acids to about 25 amino acids, about 4 amino acids to about 24 amino acids, about 4 amino acids to about 22 amino acids, about 4 amino acids to about 20 amino acids, about 4 amino acids to about 18 amino acids, about 4 amino acids to about 16 amino acids, about 4 amino acids to about 14 amino acids, about 4 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about 6 amino acids, about 6 amino acids to about 100 amino acids, about 6 amino acids to about 90 amino acids, about 6 amino acids to about 80 amino acids, about 6 amino acids to about 70 amino acids, about 6 amino acids to about 60 amino acids, about 6 amino acids to about 50 amino acids, about 6 amino acids to about 45 amino acids, about 6 amino acids to about 40 amino acids, about 6 amino acids to about 35 amino acids, about 6 amino acids to about 30 amino acids, about 6 amino acids to about 25 amino acids, about 6 amino acids to about 24 amino acids, about 6 amino acids to about 22 amino acids, about 6 amino acids to about 20 amino acids, about 6 amino acids to about 18 amino acids, about 6 amino acids to about 16 amino acids, about 6 amino acids to about 14 amino acids, about 6 amino acids to about 12 amino acids, about 6 amino acids to about 10 amino acids, about 6 amino acids to about 8 amino acids, about 8 amino acids to about 100 amino acids, about 8 amino acids to about 90 amino acids, about 8 amino acids to about 80 amino acids, about 8 amino acids to about 70 amino acids, about 8 amino acids to about 60 amino acids, about 8 amino acids to about 50 amino acids, about 8 amino acids to about 45 amino acids, about 8 amino acids to about 40 amino acids, about 8 amino acids to about 35 amino acids, about 8 amino acids to about 30 amino acids, about 8 amino acids to about 25 amino acids, about 8 amino acids to about 24 amino acids, about 8 amino acids to about 22 amino acids, about 8 amino acids to about 20 amino acids, about 8 amino acids to about 18 amino acids, about 8 amino acids to about 16 amino acids, about 8 amino acids to about 14 amino acids, about 8 amino acids to about 12 amino acids, about 8 amino acids to about 10 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 90 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 70 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 50 amino acids, about 10 amino acids to about 45 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 35 amino acids, about 10 amino acids to about 30 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 24 amino acids, about 10 amino acids to about 22 amino acids, about 10 amino acids to about 20 amino acids, about 10 amino acids to about 18 amino acids, about 10 amino acids to about 16 amino acids, about 10 amino acids to about 14 amino acids, about 10 amino acids to about 12 amino acids, about 12 amino acids to about 100 amino acids, about 12 amino acids to about 90 amino acids, about 12 amino acids to about 80 amino acids, about 12 amino acids to about 70 amino acids, about 12 amino acids to about 60 amino acids, about 12 amino acids to about 50 amino acids, about 12 amino acids to about 45 amino acids, about 12 amino acids to about 40 amino acids, about 12 amino acids to about 35 amino acids, about 12 amino acids to about 30 amino acids, about 12 amino acids to about 25 amino acids, about 12 amino acids to about 24 amino acids, about 12 amino acids to about 22 amino acids, about 12 amino acids to about 20 amino acids, about 12 amino acids to about 18 amino acids, about 12 amino acids to about 16 amino acids, about 12 amino acids to about 14 amino acids, about 14 amino acids to about 100 amino acids, about 14 amino acids to about 90 amino acids, about 14 amino acids to about 80 amino acids, about 14 amino acids to about 70 amino acids, about 14 amino acids to about 60 amino acids, about 14 amino acids to about 50 amino acids, about 14 amino acids to about 45 amino acids, about 14 amino acids to about 40 amino acids, about 14 amino acids to about 35 amino acids, about 14 amino acids to about 30 amino acids, about 14 amino acids to about 25 amino acids, about 14 amino acids to about 24 amino acids, about 14 amino acids to about 22 amino acids, about 14 amino acids to about 20 amino acids, about 14 amino acids to about 18 amino acids, about 14 amino acids to about 16 amino acids, about 16 amino acids to about 100 amino acids, about 16 amino acids to about 90 amino acids, about 16 amino acids to about 80 amino acids, about 16 amino acids to about 70 amino acids, about 16 amino acids to about 60 amino acids, about 16 amino acids to about 50 amino acids, about 16 amino acids to about 45 amino acids, about 16 amino acids to about 40 amino acids, about 16 amino acids to about 35 amino acids, about 16 amino acids to about 30 amino acids, about 16 amino acids to about 25 amino acids, about 16 amino acids to about 24 amino acids, about 16 amino acids to about 22 amino acids, about 16 amino acids to about 20 amino acids, about 16 amino acids to about 18 amino acids, about 18 amino acids to about 100 amino acids, about 18 amino acids to about 90 amino acids, about 18 amino acids to about 80 amino acids, about 18 amino acids to about 70 amino acids, about 18 amino acids to about 60 amino acids, about 18 amino acids to about 50 amino acids, about 18 amino acids to about 45 amino acids, about 18 amino acids to about 40 amino acids, about 18 amino acids to about 35 amino acids, about 18 amino acids to about 30 amino acids, about 18 amino acids to about 25 amino acids, about 18 amino acids to about 24 amino acids, about 18 amino acids to about 22 amino acids, about 18 amino acids to about 20 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 45 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 35 amino acids, about 20 amino acids to about 30 amino acids, about 20 amino acids to about 25 amino acids, about 20 amino acids to about 24 amino acids, about 20 amino acids to about 22 amino acids, about 22 amino acids to about 100 amino acids, about 22 amino acids to about 90 amino acids, about 22 amino acids to about 80 amino acids, about 22 amino acids to about 70 amino acids, about 22 amino acids to about 60 amino acids, about 22 amino acids to about 50 amino acids, about 22 amino acids to about 45 amino acids, about 22 amino acids to about 40 amino acids, about 22 amino acids to about 35 amino acids, about 22 amino acids to about 30 amino acids, about 22 amino acids to about 25 amino acids, about 22 amino acids to about 24 amino acids, about 25 amino acids to about 100 amino acids, about 25 amino acids to about 90 amino acids, about 25 amino acids to about 80 amino acids, about 25 amino acids to about 70 amino acids, about 25 amino acids to about 60 amino acids, about 25 amino acids to about 50 amino acids, about 25 amino acids to about 45 amino acids, about 25 amino acids to about 40 amino acids, about 25 amino acids to about 35 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 80 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about 45 amino acids, about 30 amino acids to about 40 amino acids, about 30 amino acids to about 35 amino acids, about 35 amino acids to about 100 amino acids, about 35 amino acids to about 90 amino acids, about 35 amino acids to about 80 amino acids, about 35 amino acids to about 70 amino acids, about 35 amino acids to about 60 amino acids, about 35 amino acids to about 50 amino acids, about 35 amino acids to about 45 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 50 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 100 amino acids, about 45 amino acids to about 90 amino acids, about 45 amino acids to about 80 amino acids, about 45 amino acids to about 70 amino acids, about 45 amino acids to about 60 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 90 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 70 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 80 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 90 amino acids, or about 90 amino acids to about 100 amino acids.

In some embodiments, the linker is rich in glycine (Gly or G) residues. In some embodiments, the linker is rich in serine (Ser or S) residues. In some embodiments, the linker is rich in glycine and serine residues. In some embodiments, the linker has one or more glycine-serine residue pairs (GS), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs. In some embodiments, the linker has one or more Gly-Gly-Gly-Ser (GGGS) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS sequences. In some embodiments, the linker has one or more Gly-Gly-Gly-Gly-Ser (GGGGS) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS sequences. In some embodiments, the linker has one or more Gly-Gly-Ser-Gly (GGSG) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG sequences.

In some embodiments, the linker sequence can comprise or consist of GGGGSGGGGSGGGGS (SEQ ID NO: 7). In some embodiments, the linker sequence can be encoded by a nucleic acid comprising or consisting of: GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (SEQ ID

NO: 8). In some embodiments, the linker sequence can comprise or consist of: GGGSGGGS (SEQ ID NO: 9).

Target-Binding Domains

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain, the second target-binding domain, and/or the additional one or more target-binding domains can be an antigen-binding domain (e.g., any of the exemplary antigen-binding domains described herein or known in the art), a soluble interleukin or cytokine protein (e.g., any of the exemplary soluble interleukin proteins or soluble cytokine proteins described herein), and a soluble interleukin or cytokine receptor (e.g., any of the exemplary soluble interleukin receptors or soluble cytokine receptors described herein).

In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or more of the first target-binding domain (e.g., any of the exemplary first target binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary second target binding domains described herein or known in the art), and the one or more additional target binding domains can each, independently, bind specifically to a target selected from the group of: bind specifically to a target selected from the group consisting of: CD16a, CD28, CD3 (e.g., one or more of CD3a, CD3β, CD35, CD38, and CD3y), CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein (e.g., ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6), HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF- β receptor II (TGF- β RII), a ligand of TGF- β RIII, a ligand of DNAM-1, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD28.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain, the second target-binding domain, and/or the one or more additional target-binding domains can each independent have a total number of amino acids of about 5 amino acids to about 1000 amino acids, about 5 amino acids to about 950 amino acids, about 5 amino acids to about 900 amino acids, about 5 amino acids to about 850 amino acids, about 5 amino acids to about 800 amino acids, about 5 amino acids to about 750 amino acids, about 5 amino acids to about 700 amino acids, about 5 amino acids to about 650 amino acids, about 5 amino acids to about 600 amino acids, about 5 amino acids to about 550 amino acids, about 5 amino acids to about 500 amino acids, about 5 amino acids to about 450 amino acids, about 5 amino acids to about 400 amino acids, about 5 amino acids to about 350 amino acids, about 5 amino acids to about 300 amino acids, about 5 amino acids to about 280 amino acids, about 5 amino acids to about 260 amino acids, about 5 amino acids to about 240 amino acids, about 5 amino acids to about 220 amino acids, about 5 amino acids to about 200 amino acids, about 5 amino acids to about 195 amino acids, about 5 amino acids to about 190 amino acids, about 5 amino acids to about 185 amino acids, about 5 amino acids to about 180 amino acids, about 5 amino acids to about 175 amino acids, about 5 amino acids to about 170 amino acids, about 5 amino acids to about 165 amino acids, about 5 amino acids to about 160 amino acids, about 5 amino acids to about 155 amino acids, about 5 amino acids to about 150 amino acids, about 5 amino acids to about 145 amino acids, about 5 amino acids to about 140 amino acids, about 5 amino acids to about 135 amino acids, about 5 amino acids to about 130 amino acids, about 5 amino acids to about 125 amino acids, about 5 amino acids to about 120 amino acids, about 5 amino acids to about 115 amino acids, about 5 amino acids to about 110 amino acids, about 5 amino acids to about 105 amino acids, about 5 amino acids to about 100 amino acids, about 5 amino acids to about 95 amino acids, about 5 amino acids to about 90 amino acids, about 5 amino acids to about 85 amino acids, about 5 amino acids to about 80 amino acids, about 5 amino acids to about 75 amino acids, about 5 amino acids to about 70 amino acids, about 5 amino acids to about 65 amino acids, about 5 amino acids to about 60 amino acids, about 5 amino acids to about 55 amino acids, about 5 amino acids to about 50 amino acids, about 5 amino acids to about 45 amino acids, about 5 amino acids to about 40 amino acids, about 5 amino acids to about 35 amino acids, about 5 amino acids to about 30 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 15 amino acids, about 5 amino acids to about 10 amino acids, about 10 amino acids to about 1000 amino acids, about 10 amino acids to about 950 amino acids, about 10 amino acids to about 900 amino acids, about 10 amino acids to about 850 amino acids, about 10 amino acids to about 800 amino acids, about 10 amino acids to about 750 amino acids, about 10 amino acids to about 700 amino acids, about 10 amino acids to about 650 amino acids, about 10 amino acids to about 600 amino acids, about 10 amino acids to about 550 amino acids, about 10 amino acids to about 500 amino acids, about 10 amino acids to about 450 amino acids, about 10 amino acids to about 400 amino acids, about 10 amino acids to about 350 amino acids, about 10 amino acids to about 300 amino acids, about 10 amino acids to about 280 amino acids, about 10 amino acids to about 260 amino acids, about 10 amino acids to about 240 amino acids, about 10 amino acids to about 220 amino acids, about 10 amino acids to about 200 amino acids, about 10 amino acids to about 195 amino acids, about 10 amino acids to about 190 amino acids, about 10 amino acids to about 185 amino acids, about 10 amino acids to about 180 amino acids, about 10 amino acids to about 175 amino acids, about 10 amino acids to about 170 amino acids, about 10 amino acids to about 165 amino acids, about 10 amino acids to about 160 amino acids, about 10 amino acids to about 155 amino acids, about 10 amino acids to about 150 amino acids, about 10 amino acids to about 145 amino acids, about 10 amino acids to about 140 amino acids, about 10 amino acids to about 135 amino acids, about 10 amino acids to about 130 amino acids, about 10 amino acids to about 125 amino acids, about 10 amino acids to about 120 amino acids, about 10 amino acids to about 115 amino acids, about 10 amino acids to about 110 amino acids, about 10 amino acids to about 105 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 95 amino acids, about 10 amino acids to about 90 amino acids, about 10 amino acids to about 85 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 75 amino acids, about 10 amino acids to about 70 amino acids, about 10 amino acids to about 65 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 55 amino acids, about 10 amino acids to about 50 amino acids, about 10 amino acids to about 45 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 35 amino acids, about 10 amino acids to about 30 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 20 amino acids, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 1000 amino acids, about 15 amino acids to about 950 amino acids, about 15 amino acids to about 900 amino acids, about 15 amino acids to about 850 amino acids, about 15 amino acids to about 800 amino acids, about 15 amino acids to about 750 amino acids, about 15 amino acids to about 700 amino acids, about 15 amino acids to about 650 amino acids, about 15 amino acids to about 600 amino acids, about 15 amino acids to about 550 amino acids, about 15 amino acids to about 500 amino acids, about 15 amino acids to about 450 amino acids, about 15 amino acids to about 400 amino acids, about 15 amino acids to about 350 amino acids, about 15 amino acids to about 300 amino acids, about 15 amino acids to about 280 amino acids, about 15 amino acids to about 260 amino acids, about 15 amino acids to about 240 amino acids, about 15 amino acids to about 220 amino acids, about 15 amino acids to about 200 amino acids, about 15 amino acids to about 195 amino acids, about 15 amino acids to about 190 amino acids, about 15 amino acids to about 185 amino acids, about 15 amino acids to about 180 amino acids, about 15 amino acids to about 175 amino acids, about 15 amino acids to about 170 amino acids, about 15 amino acids to about 165 amino acids, about 15 amino acids to about 160 amino acids, about 15 amino acids to about 155 amino acids, about 15 amino acids to about 150 amino acids, about 15 amino acids to about 145 amino acids, about 15 amino acids to about 140 amino acids, about 15 amino acids to about 135 amino acids, about 15 amino acids to about 130 amino acids, about 15 amino acids to about 125 amino acids, about 15 amino acids to about 120 amino acids, about 15 amino acids to about 115 amino acids, about 15 amino acids to about 110 amino acids, about 15 amino acids to about 105 amino acids, about 15 amino acids to about 100 amino acids, about 15 amino acids to about 95 amino acids, about 15 amino acids to about 90 amino acids, about 15 amino acids to about 85 amino acids, about 15 amino acids to about 80 amino acids, about 15 amino acids to about 75 amino acids, about 15 amino acids to about 70 amino acids, about 15 amino acids to about 65 amino acids, about 15 amino acids to about 60 amino acids, about 15 amino acids to about 55 amino acids, about 15 amino acids to about 50 amino acids, about 15 amino acids to about 45 amino acids, about 15 amino acids to about 40 amino acids, about 15 amino acids to about 35 amino acids, about 15 amino acids to about 30 amino acids, about 15 amino acids to about 25 amino acids, about 15 amino acids to about 20 amino acids, about 20 amino acids to about 1000 amino acids, about 20 amino acids to about 950 amino acids, about 20 amino acids to about 900 amino acids, about 20 amino acids to about 850 amino acids, about 20 amino acids to about 800 amino acids, about 20 amino acids to about 750 amino acids, about 20 amino acids to about 700 amino acids, about 20 amino acids to about 650 amino acids, about 20 amino acids to about 600 amino acids, about 20 amino acids to about 550 amino acids, about 20 amino acids to about 500 amino acids, about 20 amino acids to about 450 amino acids, about 20 amino acids to about 400 amino acids, about 20 amino acids to about 350 amino acids, about 20 amino acids to about 300 amino acids, about 20 amino acids to about 280 amino acids, about 20 amino acids to about 260 amino acids, about 20 amino acids to about 240 amino acids, about 20 amino acids to about 220 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 195 amino acids, about 20 amino acids to about 190 amino acids, about 20 amino acids to about 185 amino acids, about 20 amino acids to about 180 amino acids, about 20 amino acids to about 175 amino acids, about 20 amino acids to about 170 amino acids, about 20 amino acids to about 165 amino acids, about 20 amino acids to about 160 amino acids, about 20 amino acids to about 155 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 145 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 135 amino acids, about 20 amino acids to about 130 amino acids, about 20 amino acids to about 125 amino acids, about 20 amino acids to about 120 amino acids, about 20 amino acids to about 115 amino acids, about 20 amino acids to about 110 amino acids, about 20 amino acids to about 105 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 95 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 85 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 75 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 65 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 55 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 45 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 35 amino acids, about 20 amino acids to about 30 amino acids, about 20 amino acids to about 25 amino acids, about 25 amino acids to about 1000 amino acids, about 25 amino acids to about 950 amino acids, about 25 amino acids to about 900 amino acids, about 25 amino acids to about 850 amino acids, about 25 amino acids to about 800 amino acids, about 25 amino acids to about 750 amino acids, about 25 amino acids to about 700 amino acids, about 25 amino acids to about 650 amino acids, about 25 amino acids to about 600 amino acids, about 25 amino acids to about 550 amino acids, about 25 amino acids to about 500 amino acids, about 25 amino acids to about 450 amino acids, about 25 amino acids to about 400 amino acids, about 25 amino acids to about 350 amino acids, about 25 amino acids to about 300 amino acids, about 25 amino acids to about 280 amino acids, about 25 amino acids to about 260 amino acids, about 25 amino acids to about 240 amino acids, about 25 amino acids to about 220 amino acids, about 25 amino acids to about 200 amino acids, about 25 amino acids to about 195 amino acids, about 25 amino acids to about 190 amino acids, about 25 amino acids to about 185 amino acids, about 25 amino acids to about 180 amino acids, about 25 amino acids to about 175 amino acids, about 25 amino acids to about 170 amino acids, about 25 amino acids to about 165 amino acids, about 25 amino acids to about 160 amino acids, about 25 amino acids to about 155 amino acids, about 25 amino acids to about 150 amino acids, about 25 amino acids to about 145 amino acids, about 25 amino acids to about 140 amino acids, about 25 amino acids to about 135 amino acids, about 25 amino acids to about 130 amino acids, about 25 amino acids to about 125 amino acids, about 25 amino acids to about 120 amino acids, about 25 amino acids to about 115 amino acids, about 25 amino acids to about 110 amino acids, about 25 amino acids to about 105 amino acids, about 25 amino acids to about 100 amino acids, about 25 amino acids to about 95 amino acids, about 25 amino acids to about 90 amino acids, about 25 amino acids to about 85 amino acids, about 25 amino acids to about 80 amino acids, about 25 amino acids to about 75 amino acids, about 25 amino acids to about 70 amino acids, about 25 amino acids to about 65 amino acids, about 25 amino acids to about 60 amino acids, about 25 amino acids to about 55 amino acids, about 25 amino acids to about 50 amino acids, about 25 amino acids to about 45 amino acids, about 25 amino acids to about 40 amino acids, about 25 amino acids to about 35 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 1000 amino acids, about 30 amino acids to about 950 amino acids, about 30 amino acids to about 900 amino acids, about 30 amino acids to about 850 amino acids, about 30 amino acids to about 800 amino acids, about 30 amino acids to about 750 amino acids, about 30 amino acids to about 700 amino acids, about 30 amino acids to about 650 amino acids, about 30 amino acids to about 600 amino acids, about 30 amino acids to about 550 amino acids, about 30 amino acids to about 500 amino acids, about 30 amino acids to about 450 amino acids, about 30 amino acids to about 400 amino acids, about 30 amino acids to about 350 amino acids, about 30 amino acids to about 300 amino acids, about 30 amino acids to about 280 amino acids, about 30 amino acids to about 260 amino acids, about 30 amino acids to about 240 amino acids, about 30 amino acids to about 220 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino acids to about 195 amino acids, about 30 amino acids to about 190 amino acids, about 30 amino acids to about 185 amino acids, about 30 amino acids to about 180 amino acids, about 30 amino acids to about 175 amino acids, about 30 amino acids to about 170 amino acids, about 30 amino acids to about 165 amino acids, about 30 amino acids to about 160 amino acids, about 30 amino acids to about 155 amino acids, about 30 amino acids to about 150 amino acids, about 30 amino acids to about 145 amino acids, about 30 amino acids to about 140 amino acids, about 30 amino acids to about 135 amino acids, about 30 amino acids to about 130 amino acids, about 30 amino acids to about 125 amino acids, about 30 amino acids to about 120 amino acids, about 30 amino acids to about 115 amino acids, about 30 amino acids to about 110 amino acids, about 30 amino acids to about 105 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 95 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 85 amino acids, about 30 amino acids to about 80 amino acids, about 30 amino acids to about 75 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 65 amino acids, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 55 amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about 45 amino acids, about 30 amino acids to about 40 amino acids, about 30 amino acids to about 35 amino acids, about 35 amino acids to about 1000 amino acids, about 35 amino acids to about 950 amino acids, about 35 amino acids to about 900 amino acids, about 35 amino acids to about 850 amino acids, about 35 amino acids to about 800 amino acids, about 35 amino acids to about 750 amino acids, about 35 amino acids to about 700 amino acids, about 35 amino acids to about 650 amino acids, about 35 amino acids to about 600 amino acids, about 35 amino acids to about 550 amino acids, about 35 amino acids to about 500 amino acids, about 35 amino acids to about 450 amino acids, about 35 amino acids to about 400 amino acids, about 35 amino acids to about 350 amino acids, about 35 amino acids to about 300 amino acids, about 35 amino acids to about 280 amino acids, about 35 amino acids to about 260 amino acids, about 35 amino acids to about 240 amino acids, about 35 amino acids to about 220 amino acids, about 35 amino acids to about 200 amino acids, about 35 amino acids to about 195 amino acids, about 35 amino acids to about 190 amino acids, about 35 amino acids to about 185 amino acids, about 35 amino acids to about 180 amino acids, about 35 amino acids to about 175 amino acids, about 35 amino acids to about 170 amino acids, about 35 amino acids to about 165 amino acids, about 35 amino acids to about 160 amino acids, about 35 amino acids to about 155 amino acids, about 35 amino acids to about 150 amino acids, about 35 amino acids to about 145 amino acids, about 35 amino acids to about 140 amino acids, about 35 amino acids to about 135 amino acids, about 35 amino acids to about 130 amino acids, about 35 amino acids to about 125 amino acids, about 35 amino acids to about 120 amino acids, about 35 amino acids to about 115 amino acids, about 35 amino acids to about 110 amino acids, about 35 amino acids to about 105 amino acids, about 35 amino acids to about 100 amino acids, about 35 amino acids to about 95 amino acids, about 35 amino acids to about 90 amino acids, about 35 amino acids to about 85 amino acids, about 35 amino acids to about 80 amino acids, about 35 amino acids to about 75 amino acids, about 35 amino acids to about 70 amino acids, about 35 amino acids to about 65 amino acids, about 35 amino acids to about 60 amino acids, about 35 amino acids to about 55 amino acids, about 35 amino acids to about 50 amino acids, about 35 amino acids to about 45 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 1000 amino acids, about 40 amino acids to about 950 amino acids, about 40 amino acids to about 900 amino acids, about 40 amino acids to about 850 amino acids, about 40 amino acids to about 800 amino acids, about 40 amino acids to about 750 amino acids, about 40 amino acids to about 700 amino acids, about 40 amino acids to about 650 amino acids, about 40 amino acids to about 600 amino acids, about 40 amino acids to about 550 amino acids, about 40 amino acids to about 500 amino acids, about 40 amino acids to about 450 amino acids, about 40 amino acids to about 400 amino acids, about 40 amino acids to about 350 amino acids, about 40 amino acids to about 300 amino acids, about 40 amino acids to about 280 amino acids, about 40 amino acids to about 260 amino acids, about 40 amino acids to about 240 amino acids, about 40 amino acids to about 220 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 195 amino acids, about 40 amino acids to about 190 amino acids, about 40 amino acids to about 185 amino acids, about 40 amino acids to about 180 amino acids, about 40 amino acids to about 175 amino acids, about 40 amino acids to about 170 amino acids, about 40 amino acids to about 165 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 155 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 145 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 135 amino acids, about 40 amino acids to about 130 amino acids, about 40 amino acids to about 125 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 115 amino acids, about 40 amino acids to about 110 amino acids, about 40 amino acids to about 105 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 95 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 85 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 75 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 65 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 55 amino acids, about 40 amino acids to about 50 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 1000 amino acids, about 45 amino acids to about 950 amino acids, about 45 amino acids to about 900 amino acids, about 45 amino acids to about 850 amino acids, about 45 amino acids to about 800 amino acids, about 45 amino acids to about 750 amino acids, about 45 amino acids to about 700 amino acids, about 45 amino acids to about 650 amino acids, about 45 amino acids to about 600 amino acids, about 45 amino acids to about 550 amino acids, about 45 amino acids to about 500 amino acids, about 45 amino acids to about 450 amino acids, about 45 amino acids to about 400 amino acids, about 45 amino acids to about 350 amino acids, about 45 amino acids to about 300 amino acids, about 45 amino acids to about 280 amino acids, about 45 amino acids to about 260 amino acids, about 45 amino acids to about 240 amino acids, about 45 amino acids to about 220 amino acids, about 45 amino acids to about 200 amino acids, about 45 amino acids to about 195 amino acids, about 45 amino acids to about 190 amino acids, about 45 amino acids to about 185 amino acids, about 45 amino acids to about 180 amino acids, about 45 amino acids to about 175 amino acids, about 45 amino acids to about 170 amino acids, about 45 amino acids to about 165 amino acids, about 45 amino acids to about 160 amino acids, about 45 amino acids to about 155 amino acids, about 45 amino acids to about 150 amino acids, about 45 amino acids to about 145 amino acids, about 45 amino acids to about 140 amino acids, about 45 amino acids to about 135 amino acids, about 45 amino acids to about 130 amino acids, about 45 amino acids to about 125 amino acids, about 45 amino acids to about 120 amino acids, about 45 amino acids to about 115 amino acids, about 45 amino acids to about 110 amino acids, about 45 amino acids to about 105 amino acids, about 45 amino acids to about 100 amino acids, about 45 amino acids to about 95 amino acids, about 45 amino acids to about 90 amino acids, about 45 amino acids to about 85 amino acids, about 45 amino acids to about 80 amino acids, about 45 amino acids to about 75 amino acids, about 45 amino acids to about 70 amino acids, about 45 amino acids to about 65 amino acids, about 45 amino acids to about 60 amino acids, about 45 amino acids to about 55 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 1000 amino acids, about 50 amino acids to about 950 amino acids, about 50 amino acids to about 900 amino acids, about 50 amino acids to about 850 amino acids, about 50 amino acids to about 800 amino acids, about 50 amino acids to about 750 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 450 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 350 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 280 amino acids, about 50 amino acids to about 260 amino acids, about 50 amino acids to about 240 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 195 amino acids, about 50 amino acids to about 190 amino acids, about 50 amino acids to about 185 amino acids, about 50 amino acids to about 180 amino acids, about 50 amino acids to about 175 amino acids, about 50 amino acids to about 170 amino acids, about 50 amino acids to about 165 amino acids, about 50 amino acids to about 160 amino acids, about 50 amino acids to about 155 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 145 amino acids, about 50 amino acids to about 140 amino acids, about 50 amino acids to about 135 amino acids, about 50 amino acids to about 130 amino acids, about 50 amino acids to about 125 amino acids, about 50 amino acids to about 120 amino acids, about 50 amino acids to about 115 amino acids, about 50 amino acids to about 110 amino acids, about 50 amino acids to about 105 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 95 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 85 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 75 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 65 amino acids, about 50 amino acids to about 60 amino acids, about 50 amino acids to about 55 amino acids, about 55 amino acids to about 1000 amino acids, about 55 amino acids to about 950 amino acids, about 55 amino acids to about 900 amino acids, about 55 amino acids to about 850 amino acids, about 55 amino acids to about 800 amino acids, about 55 amino acids to about 750 amino acids, about 55 amino acids to about 700 amino acids, about 55 amino acids to about 650 amino acids, about 55 amino acids to about 600 amino acids, about 55 amino acids to about 550 amino acids, about 55 amino acids to about 500 amino acids, about 55 amino acids to about 450 amino acids, about 55 amino acids to about 400 amino acids, about 55 amino acids to about 350 amino acids, about 55 amino acids to about 300 amino acids, about 55 amino acids to about 280 amino acids, about 55 amino acids to about 260 amino acids, about 55 amino acids to about 240 amino acids, about 55 amino acids to about 220 amino acids, about 55 amino acids to about 200 amino acids, about 55 amino acids to about 195 amino acids, about 55 amino acids to about 190 amino acids, about 55 amino acids to about 185 amino acids, about 55 amino acids to about 180 amino acids, about 55 amino acids to about 175 amino acids, about 55 amino acids to about 170 amino acids, about 55 amino acids to about 165 amino acids, about 55 amino acids to about 160 amino acids, about 55 amino acids to about 155 amino acids, about 55 amino acids to about 150 amino acids, about 55 amino acids to about 145 amino acids, about 55 amino acids to about 140 amino acids, about 55 amino acids to about 135 amino acids, about 55 amino acids to about 130 amino acids, about 55 amino acids to about 125 amino acids, about 55 amino acids to about 120 amino acids, about 55 amino acids to about 115 amino acids, about 55 amino acids to about 110 amino acids, about 55 amino acids to about 105 amino acids, about 55 amino acids to about 100 amino acids, about 55 amino acids to about 95 amino acids, about 55 amino acids to about 90 amino acids, about 55 amino acids to about 85 amino acids, about 55 amino acids to about 80 amino acids, about 55 amino acids to about 75 amino acids, about 55 amino acids to about 70 amino acids, about 55 amino acids to about 65 amino acids, about 55 amino acids to about 60 amino acids, about 60 amino acids to about 1000 amino acids, about 60 amino acids to about 950 amino acids, about 60 amino acids to about 900 amino acids, about 60 amino acids to about 850 amino acids, about 60 amino acids to about 800 amino acids, about 60 amino acids to about 750 amino acids, about 60 amino acids to about 700 amino acids, about 60 amino acids to about 650 amino acids, about 60 amino acids to about 600 amino acids, about 60 amino acids to about 550 amino acids, about 60 amino acids to about 500 amino acids, about 60 amino acids to about 450 amino acids, about 60 amino acids to about 400 amino acids, about 60 amino acids to about 350 amino acids, about 60 amino acids to about 300 amino acids, about 60 amino acids to about 280 amino acids, about 60 amino acids to about 260 amino acids, about 60 amino acids to about 240 amino acids, about 60 amino acids to about 220 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 195 amino acids, about 60 amino acids to about 190 amino acids, about 60 amino acids to about 185 amino acids, about 60 amino acids to about 180 amino acids, about 60 amino acids to about 175 amino acids, about 60 amino acids to about 170 amino acids, about 60 amino acids to about 165 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 155 amino acids, about 60 amino acids to about 150 amino acids, about 60 amino acids to about 145 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 135 amino acids, about 60 amino acids to about 130 amino acids, about 60 amino acids to about 125 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 115 amino acids, about 60 amino acids to about 110 amino acids, about 60 amino acids to about 105 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 95 amino acids, about 60 amino acids to about 90 amino acids, about 60 amino acids to about 85 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 75 amino acids, about 60 amino acids to about 70 amino acids, about 60 amino acids to about 65 amino acids, about 65 amino acids to about 1000 amino acids, about 65 amino acids to about 950 amino acids, about 65 amino acids to about 900 amino acids, about 65 amino acids to about 850 amino acids, about 65 amino acids to about 800 amino acids, about 65 amino acids to about 750 amino acids, about 65 amino acids to about 700 amino acids, about 65 amino acids to about 650 amino acids, about 65 amino acids to about 600 amino acids, about 65 amino acids to about 550 amino acids, about 65 amino acids to about 500 amino acids, about 65 amino acids to about 450 amino acids, about 65 amino acids to about 400 amino acids, about 65 amino acids to about 350 amino acids, about 65 amino acids to about 300 amino acids, about 65 amino acids to about 280 amino acids, about 65 amino acids to about 260 amino acids, about 65 amino acids to about 240 amino acids, about 65 amino acids to about 220 amino acids, about 65 amino acids to about 200 amino acids, about 65 amino acids to about 195 amino acids, about 65 amino acids to about 190 amino acids, about 65 amino acids to about 185 amino acids, about 65 amino acids to about 180 amino acids, about 65 amino acids to about 175 amino acids, about 65 amino acids to about 170 amino acids, about 65 amino acids to about 165 amino acids, about 65 amino acids to about 160 amino acids, about 65 amino acids to about 155 amino acids, about 65 amino acids to about 150 amino acids, about 65 amino acids to about 145 amino acids, about 65 amino acids to about 140 amino acids, about 65 amino acids to about 135 amino acids, about 65 amino acids to about 130 amino acids, about 65 amino acids to about 125 amino acids, about 65 amino acids to about 120 amino acids, about 65 amino acids to about 115 amino acids, about 65 amino acids to about 110 amino acids, about 65 amino acids to about 105 amino acids, about 65 amino acids to about 100 amino acids, about 65 amino acids to about 95 amino acids, about 65 amino acids to about 90 amino acids, about 65 amino acids to about 85 amino acids, about 65 amino acids to about 80 amino acids, about 65 amino acids to about 75 amino acids, about 65 amino acids to about 70 amino acids, about 70 amino acids to about 1000 amino acids, about 70 amino acids to about 950 amino acids, about 70 amino acids to about 900 amino acids, about 70 amino acids to about 850 amino acids, about 70 amino acids to about 800 amino acids, about 70 amino acids to about 750 amino acids, about 70 amino acids to about 700 amino acids, about 70 amino acids to about 650 amino acids, about 70 amino acids to about 600 amino acids, about 70 amino acids to about 550 amino acids, about 70 amino acids to about 500 amino acids, about 70 amino acids to about 450 amino acids, about 70 amino acids to about 400 amino acids, about 70 amino acids to about 350 amino acids, about 70 amino acids to about 300 amino acids, about 70 amino acids to about 280 amino acids, about 70 amino acids to about 260 amino acids, about 70 amino acids to about 240 amino acids, about 70 amino acids to about 220 amino acids, about 70 amino acids to about 200 amino acids, about 70 amino acids to about 195 amino acids, about 70 amino acids to about 190 amino acids, about 70 amino acids to about 185 amino acids, about 70 amino acids to about 180 amino acids, about 70 amino acids to about 175 amino acids, about 70 amino acids to about 170 amino acids, about 70 amino acids to about 165 amino acids, about 70 amino acids to about 160 amino acids, about 70 amino acids to about 155 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 145 amino acids, about 70 amino acids to about 140 amino acids, about 70 amino acids to about 135 amino acids, about 70 amino acids to about 130 amino acids, about 70 amino acids to about 125 amino acids, about 70 amino acids to about 120 amino acids, about 70 amino acids to about 115 amino acids, about 70 amino acids to about 110 amino acids, about 70 amino acids to about 105 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 95 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 85 amino acids, about 70 amino acids to about 80 amino acids, about 70 amino acids to about 75 amino acids, about 75 amino acids to about 1000 amino acids, about 75 amino acids to about 950 amino acids, about 75 amino acids to about 900 amino acids, about 75 amino acids to about 850 amino acids, about 75 amino acids to about 800 amino acids, about 75 amino acids to about 750 amino acids, about 75 amino acids to about 700 amino acids, about 75 amino acids to about 650 amino acids, about 75 amino acids to about 600 amino acids, about 75 amino acids to about 550 amino acids, about 75 amino acids to about 500 amino acids, about 75 amino acids to about 450 amino acids, about 75 amino acids to about 400 amino acids, about 75 amino acids to about 350 amino acids, about 75 amino acids to about 300 amino acids, about 75 amino acids to about 280 amino acids, about 75 amino acids to about 260 amino acids, about 75 amino acids to about 240 amino acids, about 75 amino acids to about 220 amino acids, about 75 amino acids to about 200 amino acids, about 75 amino acids to about 195 amino acids, about 75 amino acids to about 190 amino acids, about 75 amino acids to about 185 amino acids, about 75 amino acids to about 180 amino acids, about 75 amino acids to about 175 amino acids, about 75 amino acids to about 170 amino acids, about 75 amino acids to about 165 amino acids, about 75 amino acids to about 160 amino acids, about 75 amino acids to about 155 amino acids, about 75 amino acids to about 150 amino acids, about 75 amino acids to about 145 amino acids, about 75 amino acids to about 140 amino acids, about 75 amino acids to about 135 amino acids, about 75 amino acids to about 130 amino acids, about 75 amino acids to about 125 amino acids, about 75 amino acids to about 120 amino acids, about 75 amino acids to about 115 amino acids, about 75 amino acids to about 110 amino acids, about 75 amino acids to about 105 amino acids, about 75 amino acids to about 100 amino acids, about 75 amino acids to about 95 amino acids, about 75 amino acids to about 90 amino acids, about 75 amino acids to about 85 amino acids, about 75 amino acids to about 80 amino acids, about 80 amino acids to about 1000 amino acids, about 80 amino acids to about 950 amino acids, about 80 amino acids to about 900 amino acids, about 80 amino acids to about 850 amino acids, about 80 amino acids to about 800 amino acids, about 80 amino acids to about 750 amino acids, about 80 amino acids to about 700 amino acids, about 80 amino acids to about 650 amino acids, about 80 amino acids to about 600 amino acids, about 80 amino acids to about 550 amino acids, about 80 amino acids to about 500 amino acids, about 80 amino acids to about 450 amino acids, about 80 amino acids to about 400 amino acids, about 80 amino acids to about 350 amino acids, about 80 amino acids to about 300 amino acids, about 80 amino acids to about 280 amino acids, about 80 amino acids to about 260 amino acids, about 80 amino acids to about 240 amino acids, about 80 amino acids to about 220 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino acids to about 195 amino acids, about 80 amino acids to about 190 amino acids, about 80 amino acids to about 185 amino acids, about 80 amino acids to about 180 amino acids, about 80 amino acids to about 175 amino acids, about 80 amino acids to about 170 amino acids, about 80 amino acids to about 165 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 155 amino acids, about 80 amino acids to about 150 amino acids, about 80 amino acids to about 145 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 135 amino acids, about 80 amino acids to about 130 amino acids, about 80 amino acids to about 125 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 115 amino acids, about 80 amino acids to about 110 amino acids, about 80 amino acids to about 105 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 95 amino acids, about 80 amino acids to about 90 amino acids, about 80 amino acids to about 85 amino acids, about 85 amino acids to about 1000 amino acids, about 85 amino acids to about 950 amino acids, about 85 amino acids to about 900 amino acids, about 85 amino acids to about 850 amino acids, about 85 amino acids to about 800 amino acids, about 85 amino acids to about 750 amino acids, about 85 amino acids to about 700 amino acids, about 85 amino acids to about 650 amino acids, about 85 amino acids to about 600 amino acids, about 85 amino acids to about 550 amino acids, about 85 amino acids to about 500 amino acids, about 85 amino acids to about 450 amino acids, about 85 amino acids to about 400 amino acids, about 85 amino acids to about 350 amino acids, about 85 amino acids to about 300 amino acids, about 85 amino acids to about 280 amino acids, about 85 amino acids to about 260 amino acids, about 85 amino acids to about 240 amino acids, about 85 amino acids to about 220 amino acids, about 85 amino acids to about 200 amino acids, about 85 amino acids to about 195 amino acids, about 85 amino acids to about 190 amino acids, about 85 amino acids to about 185 amino acids, about 85 amino acids to about 180 amino acids, about 85 amino acids to about 175 amino acids, about 85 amino acids to about 170 amino acids, about 85 amino acids to about 165 amino acids, about 85 amino acids to about 160 amino acids, about 85 amino acids to about 155 amino acids, about 85 amino acids to about 150 amino acids, about 85 amino acids to about 145 amino acids, about 85 amino acids to about 140 amino acids, about 85 amino acids to about 135 amino acids, about 85 amino acids to about 130 amino acids, about 85 amino acids to about 125 amino acids, about 85 amino acids to about 120 amino acids, about 85 amino acids to about 115 amino acids, about 85 amino acids to about 110 amino acids, about 85 amino acids to about 105 amino acids, about 85 amino acids to about 100 amino acids, about 85 amino acids to about 95 amino acids, about 85 amino acids to about 90 amino acids, about 90 amino acids to about 1000 amino acids, about 90 amino acids to about 950 amino acids, about 90 amino acids to about 900 amino acids, about 90 amino acids to about 850 amino acids, about 90 amino acids to about 800 amino acids, about 90 amino acids to about 750 amino acids, about 90 amino acids to about 700 amino acids, about 90 amino acids to about 650 amino acids, about 90 amino acids to about 600 amino acids, about 90 amino acids to about 550 amino acids, about 90 amino acids to about 500 amino acids, about 90 amino acids to about 450 amino acids, about 90 amino acids to about 400 amino acids, about 90 amino acids to about 350 amino acids, about 90 amino acids to about 300 amino acids, about 90 amino acids to about 280 amino acids, about 90 amino acids to about 260 amino acids, about 90 amino acids to about 240 amino acids, about 90 amino acids to about 220 amino acids, about 90 amino acids to about 200 amino acids, about 90 amino acids to about 195 amino acids, about 90 amino acids to about 190 amino acids, about 90 amino acids to about 185 amino acids, about 90 amino acids to about 180 amino acids, about 90 amino acids to about 175 amino acids, about 90 amino acids to about 170 amino acids, about 90 amino acids to about 165 amino acids, about 90 amino acids to about 160 amino acids, about 90 amino acids to about 155 amino acids, about 90 amino acids to about 150 amino acids, about 90 amino acids to about 145 amino acids, about 90 amino acids to about 140 amino acids, about 90 amino acids to about 135 amino acids, about 90 amino acids to about 130 amino acids, about 90 amino acids to about 125 amino acids, about 90 amino acids to about 120 amino acids, about 90 amino acids to about 115 amino acids, about 90 amino acids to about 110 amino acids, about 90 amino acids to about 105 amino acids, about 90 amino acids to about 100 amino acids, about 90 amino acids to about 95 amino acids, about 95 amino acids to about 1000 amino acids, about 95 amino acids to about 950 amino acids, about 95 amino acids to about 900 amino acids, about 95 amino acids to about 850 amino acids, about 95 amino acids to about 800 amino acids, about 95 amino acids to about 750 amino acids, about 95 amino acids to about 700 amino acids, about 95 amino acids to about 650 amino acids, about 95 amino acids to about 600 amino acids, about 95 amino acids to about 550 amino acids, about 95 amino acids to about 500 amino acids, about 95 amino acids to about 450 amino acids, about 95 amino acids to about 400 amino acids, about 95 amino acids to about 350 amino acids, about 95 amino acids to about 300 amino acids, about 95 amino acids to about 280 amino acids, about 95 amino acids to about 260 amino acids, about 95 amino acids to about 240 amino acids, about 95 amino acids to about 220 amino acids, about 95 amino acids to about 200 amino acids, about 95 amino acids to about 195 amino acids, about 95 amino acids to about 190 amino acids, about 95 amino acids to about 185 amino acids, about 95 amino acids to about 180 amino acids, about 95 amino acids to about 175 amino acids, about 95 amino acids to about 170 amino acids, about 95 amino acids to about 165 amino acids, about 95 amino acids to about 160 amino acids, about 95 amino acids to about 155 amino acids, about 95 amino acids to about 150 amino acids, about 95 amino acids to about 145 amino acids, about 95 amino acids to about 140 amino acids, about 95 amino acids to about 135 amino acids, about 95 amino acids to about 130 amino acids, about 95 amino acids to about 125 amino acids, about 95 amino acids to about 120 amino acids, about 95 amino acids to about 115 amino acids, about 95 amino acids to about 110 amino acids, about 95 amino acids to about 105 amino acids, about 95 amino acids to about 100 amino acids, about 100 amino acids to about 1000 amino acids, about 100 amino acids to about 950 amino acids, about 100 amino acids to about 900 amino acids, about 100 amino acids to about 850 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 450 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 350 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 280 amino acids, about 100 amino acids to about 260 amino acids, about 100 amino acids to about 240 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 195 amino acids, about 100 amino acids to about 190 amino acids, about 100 amino acids to about 185 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 175 amino acids, about 100 amino acids to about 170 amino acids, about 100 amino acids to about 165 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 155 amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to about 145 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 135 amino acids, about 100 amino acids to about 130 amino acids, about 100 amino acids to about 125 amino acids, about 100 amino acids to about 120 amino acids, about 100 amino acids to about 115 amino acids, about 100 amino acids to about 110 amino acids, about 100 amino acids to about 105 amino acids, about 105 amino acids to about 1000 amino acids, about 105 amino acids to about 950 amino acids, about 105 amino acids to about 900 amino acids, about 105 amino acids to about 850 amino acids, about 105 amino acids to about 800 amino acids, about 105 amino acids to about 750 amino acids, about 105 amino acids to about 700 amino acids, about 105 amino acids to about 650 amino acids, about 105 amino acids to about 600 amino acids, about 105 amino acids to about 550 amino acids, about 105 amino acids to about 500 amino acids, about 105 amino acids to about 450 amino acids, about 105 amino acids to about 400 amino acids, about 105 amino acids to about 350 amino acids, about 105 amino acids to about 300 amino acids, about 105 amino acids to about 280 amino acids, about 105 amino acids to about 260 amino acids, about 105 amino acids to about 240 amino acids, about 105 amino acids to about 220 amino acids, about 105 amino acids to about 200 amino acids, about 105 amino acids to about 195 amino acids, about 105 amino acids to about 190 amino acids, about 105 amino acids to about 185 amino acids, about 105 amino acids to about 180 amino acids, about 105 amino acids to about 175 amino acids, about 105 amino acids to about 170 amino acids, about 105 amino acids to about 165 amino acids, about 105 amino acids to about 160 amino acids, about 105 amino acids to about 155 amino acids, about 105 amino acids to about 150 amino acids, about 105 amino acids to about 145 amino acids, about 105 amino acids to about 140 amino acids, about 105 amino acids to about 135 amino acids, about 105 amino acids to about 130 amino acids, about 105 amino acids to about 125 amino acids, about 105 amino acids to about 120 amino acids, about 105 amino acids to about 115 amino acids, about 105 amino acids to about 110 amino acids, about 110 amino acids to about 1000 amino acids, about 110 amino acids to about 950 amino acids, about 110 amino acids to about 900 amino acids, about 110 amino acids to about 850 amino acids, about 110 amino acids to about 800 amino acids, about 110 amino acids to about 750 amino acids, about 110 amino acids to about 700 amino acids, about 110 amino acids to about 650 amino acids, about 110 amino acids to about 600 amino acids, about 110 amino acids to about 550 amino acids, about 110 amino acids to about 500 amino acids, about 110 amino acids to about 450 amino acids, about 110 amino acids to about 400 amino acids, about 110 amino acids to about 350 amino acids, about 110 amino acids to about 300 amino acids, about 110 amino acids to about 280 amino acids, about 110 amino acids to about 260 amino acids, about 110 amino acids to about 240 amino acids, about 110 amino acids to about 220 amino acids, about 110 amino acids to about 200 amino acids, about 110 amino acids to about 195 amino acids, about 110 amino acids to about 190 amino acids, about 110 amino acids to about 185 amino acids, about 110 amino acids to about 180 amino acids, about 110 amino acids to about 175 amino acids, about 110 amino acids to about 170 amino acids, about 110 amino acids to about 165 amino acids, about 110 amino acids to about 160 amino acids, about 110 amino acids to about 155 amino acids, about 110 amino acids to about 150 amino acids, about 110 amino acids to about 145 amino acids, about 110 amino acids to about 140 amino acids, about 110 amino acids to about 135 amino acids, about 110 amino acids to about 130 amino acids, about 110 amino acids to about 125 amino acids, about 110 amino acids to about 120 amino acids, about 110 amino acids to about 115 amino acids, about 115 amino acids to about 1000 amino acids, about 115 amino acids to about 950 amino acids, about 115 amino acids to about 900 amino acids, about 115 amino acids to about 850 amino acids, about 115 amino acids to about 800 amino acids, about 115 amino acids to about 750 amino acids, about 115 amino acids to about 700 amino acids, about 115 amino acids to about 650 amino acids, about 115 amino acids to about 600 amino acids, about 115 amino acids to about 550 amino acids, about 115 amino acids to about 500 amino acids, about 115 amino acids to about 450 amino acids, about 115 amino acids to about 400 amino acids, about 115 amino acids to about 350 amino acids, about 115 amino acids to about 300 amino acids, about 115 amino acids to about 280 amino acids, about 115 amino acids to about 260 amino acids, about 115 amino acids to about 240 amino acids, about 115 amino acids to about 220 amino acids, about 115 amino acids to about 200 amino acids, about 115 amino acids to about 195 amino acids, about 115 amino acids to about 190 amino acids, about 115 amino acids to about 185 amino acids, about 115 amino acids to about 180 amino acids, about 115 amino acids to about 175 amino acids, about 115 amino acids to about 170 amino acids, about 115 amino acids to about 165 amino acids, about 115 amino acids to about 160 amino acids, about 115 amino acids to about 155 amino acids, about 115 amino acids to about 150 amino acids, about 115 amino acids to about 145 amino acids, about 115 amino acids to about 140 amino acids, about 115 amino acids to about 135 amino acids, about 115 amino acids to about 130 amino acids, about 115 amino acids to about 125 amino acids, about 115 amino acids to about 120 amino acids, about 120 amino acids to about 1000 amino acids, about 120 amino acids to about 950 amino acids, about 120 amino acids to about 900 amino acids, about 120 amino acids to about 850 amino acids, about 120 amino acids to about 800 amino acids, about 120 amino acids to about 750 amino acids, about 120 amino acids to about 700 amino acids, about 120 amino acids to about 650 amino acids, about 120 amino acids to about 600 amino acids, about 120 amino acids to about 550 amino acids, about 120 amino acids to about 500 amino acids, about 120 amino acids to about 450 amino acids, about 120 amino acids to about 400 amino acids, about 120 amino acids to about 350 amino acids, about 120 amino acids to about 300 amino acids, about 120 amino acids to about 280 amino acids, about 120 amino acids to about 260 amino acids, about 120 amino acids to about 240 amino acids, about 120 amino acids to about 220 amino acids, about 120 amino acids to about 200 amino acids, about 120 amino acids to about 195 amino acids, about 120 amino acids to about 190 amino acids, about 120 amino acids to about 185 amino acids, about 120 amino acids to about 180 amino acids, about 120 amino acids to about 175 amino acids, about 120 amino acids to about 170 amino acids, about 120 amino acids to about 165 amino acids, about 120 amino acids to about 160 amino acids, about 120 amino acids to about 155 amino acids, about 120 amino acids to about 150 amino acids, about 120 amino acids to about 145 amino acids, about 120 amino acids to about 140 amino acids, about 120 amino acids to about 135 amino acids, about 120 amino acids to about 130 amino acids, about 120 amino acids to about 125 amino acids, about 125 amino acids to about 1000 amino acids, about 125 amino acids to about 950 amino acids, about 125 amino acids to about 900 amino acids, about 125 amino acids to about 850 amino acids, about 125 amino acids to about 800 amino acids, about 125 amino acids to about 750 amino acids, about 125 amino acids to about 700 amino acids, about 125 amino acids to about 650 amino acids, about 125 amino acids to about 600 amino acids, about 125 amino acids to about 550 amino acids, about 125 amino acids to about 500 amino acids, about 125 amino acids to about 450 amino acids, about 125 amino acids to about 400 amino acids, about 125 amino acids to about 350 amino acids, about 125 amino acids to about 300 amino acids, about 125 amino acids to about 280 amino acids, about 125 amino acids to about 260 amino acids, about 125 amino acids to about 240 amino acids, about 125 amino acids to about 220 amino acids, about 125 amino acids to about 200 amino acids, about 125 amino acids to about 195 amino acids, about 125 amino acids to about 190 amino acids, about 125 amino acids to about 185 amino acids, about 125 amino acids to about 180 amino acids, about 125 amino acids to about 175 amino acids, about 125 amino acids to about 170 amino acids, about 125 amino acids to about 165 amino acids, about 125 amino acids to about 160 amino acids, about 125 amino acids to about 155 amino acids, about 125 amino acids to about 150 amino acids, about 125 amino acids to about 145 amino acids, about 125 amino acids to about 140 amino acids, about 125 amino acids to about 135 amino acids, about 125 amino acids to about 130 amino acids, about 130 amino acids to about 1000 amino acids, about 130 amino acids to about 950 amino acids, about 130 amino acids to about 900 amino acids, about 130 amino acids to about 850 amino acids, about 130 amino acids to about 800 amino acids, about 130 amino acids to about 750 amino acids, about 130 amino acids to about 700 amino acids, about 130 amino acids to about 650 amino acids, about 130 amino acids to about 600 amino acids, about 130 amino acids to about 550 amino acids, about 130 amino acids to about 500 amino acids, about 130 amino acids to about 450 amino acids, about 130 amino acids to about 400 amino acids, about 130 amino acids to about 350 amino acids, about 130 amino acids to about 300 amino acids, about 130 amino acids to about 280 amino acids, about 130 amino acids to about 260 amino acids, about 130 amino acids to about 240 amino acids, about 130 amino acids to about 220 amino acids, about 130 amino acids to about 200 amino acids, about 130 amino acids to about 195 amino acids, about 130 amino acids to about 190 amino acids, about 130 amino acids to about 185 amino acids, about 130 amino acids to about 180 amino acids, about 130 amino acids to about 175 amino acids, about 130 amino acids to about 170 amino acids, about 130 amino acids to about 165 amino acids, about 130 amino acids to about 160 amino acids, about 130 amino acids to about 155 amino acids, about 130 amino acids to about 150 amino acids, about 130 amino acids to about 145 amino acids, about 130 amino acids to about 140 amino acids, about 130 amino acids to about 135 amino acids, about 135 amino acids to about 1000 amino acids, about 135 amino acids to about 950 amino acids, about 135 amino acids to about 900 amino acids, about 135 amino acids to about 850 amino acids, about 135 amino acids to about 800 amino acids, about 135 amino acids to about 750 amino acids, about 135 amino acids to about 700 amino acids, about 135 amino acids to about 650 amino acids, about 135 amino acids to about 600 amino acids, about 135 amino acids to about 550 amino acids, about 135 amino acids to about 500 amino acids, about 135 amino acids to about 450 amino acids, about 135 amino acids to about 400 amino acids, about 135 amino acids to about 350 amino acids, about 135 amino acids to about 300 amino acids, about 135 amino acids to about 280 amino acids, about 135 amino acids to about 260 amino acids, about 135 amino acids to about 240 amino acids, about 135 amino acids to about 220 amino acids, about 135 amino acids to about 200 amino acids, about 135 amino acids to about 195 amino acids, about 135 amino acids to about 190 amino acids, about 135 amino acids to about 185 amino acids, about 135 amino acids to about 180 amino acids, about 135 amino acids to about 175 amino acids, about 135 amino acids to about 170 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 160 amino acids, about 135 amino acids to about 155 amino acids, about 135 amino acids to about 150 amino acids, about 135 amino acids to about 145 amino acids, about 135 amino acids to about 140 amino acids, about 140 amino acids to about 1000 amino acids, about 140 amino acids to about 950 amino acids, about 140 amino acids to about 900 amino acids, about 140 amino acids to about 850 amino acids, about 140 amino acids to about 800 amino acids, about 140 amino acids to about 750 amino acids, about 140 amino acids to about 700 amino acids, about 140 amino acids to about 650 amino acids, about 140 amino acids to about 600 amino acids, about 140 amino acids to about 550 amino acids, about 140 amino acids to about 500 amino acids, about 140 amino acids to about 450 amino acids, about 140 amino acids to about 400 amino acids, about 140 amino acids to about 350 amino acids, about 140 amino acids to about 300 amino acids, about 140 amino acids to about 280 amino acids, about 140 amino acids to about 260 amino acids, about 140 amino acids to about 240 amino acids, about 140 amino acids to about 220 amino acids, about 140 amino acids to about 200 amino acids, about 140 amino acids to about 195 amino acids, about 140 amino acids to about 190 amino acids, about 140 amino acids to about 185 amino acids, about 140 amino acids to about 180 amino acids, about 140 amino acids to about 175 amino acids, about 140 amino acids to about 170 amino acids, about 140 amino acids to about 165 amino acids, about 140 amino acids to about 160 amino acids, about 140 amino acids to about 155 amino acids, about 140 amino acids to about 150 amino acids, about 140 amino acids to about 145 amino acids, about 145 amino acids to about 1000 amino acids, about 145 amino acids to about 950 amino acids, about 145 amino acids to about 900 amino acids, about 145 amino acids to about 850 amino acids, about 145 amino acids to about 800 amino acids, about 145 amino acids to about 750 amino acids, about 145 amino acids to about 700 amino acids, about 145 amino acids to about 650 amino acids, about 145 amino acids to about 600 amino acids, about 145 amino acids to about 550 amino acids, about 145 amino acids to about 500 amino acids, about 145 amino acids to about 450 amino acids, about 145 amino acids to about 400 amino acids, about 145 amino acids to about 350 amino acids, about 145 amino acids to about 300 amino acids, about 145 amino acids to about 280 amino acids, about 145 amino acids to about 260 amino acids, about 145 amino acids to about 240 amino acids, about 145 amino acids to about 220 amino acids, about 145 amino acids to about 200 amino acids, about 145 amino acids to about 195 amino acids, about 145 amino acids to about 190 amino acids, about 145 amino acids to about 185 amino acids, about 145 amino acids to about 180 amino acids, about 145 amino acids to about 175 amino acids, about 145 amino acids to about 170 amino acids, about 145 amino acids to about 165 amino acids, about 145 amino acids to about 160 amino acids, about 145 amino acids to about 155 amino acids, about 145 amino acids to about 150 amino acids, about 150 amino acids to about 1000 amino acids, about 150 amino acids to about 950 amino acids, about 150 amino acids to about 900 amino acids, about 150 amino acids to about 850 amino acids, about 150 amino acids to about 800 amino acids, about 150 amino acids to about 750 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 450 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 350 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 280 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 240 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 195 amino acids, about 150 amino acids to about 190 amino acids, about 150 amino acids to about 185 amino acids, about 150 amino acids to about 180 amino acids, about 150 amino acids to about 175 amino acids, about 150 amino acids to about 170 amino acids, about 150 amino acids to about 165 amino acids, about 150 amino acids to about 160 amino acids, about 150 amino acids to about 155 amino acids, about 155 amino acids to about 1000 amino acids, about 155 amino acids to about 950 amino acids, about 155 amino acids to about 900 amino acids, about 155 amino acids to about 850 amino acids, about 155 amino acids to about 800 amino acids, about 155 amino acids to about 750 amino acids, about 155 amino acids to about 700 amino acids, about 155 amino acids to about 650 amino acids, about 155 amino acids to about 600 amino acids, about 155 amino acids to about 550 amino acids, about 155 amino acids to about 500 amino acids, about 155 amino acids to about 450 amino acids, about 155 amino acids to about 400 amino acids, about 155 amino acids to about 350 amino acids, about 155 amino acids to about 300 amino acids, about 155 amino acids to about 280 amino acids, about 155 amino acids to about 260 amino acids, about 155 amino acids to about 240 amino acids, about 155 amino acids to about 220 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 195 amino acids, about 155 amino acids to about 190 amino acids, about 155 amino acids to about 185 amino acids, about 155 amino acids to about 180 amino acids, about 155 amino acids to about 175 amino acids, about 155 amino acids to about 170 amino acids, about 155 amino acids to about 165 amino acids, about 155 amino acids to about 160 amino acids, about 160 amino acids to about 1000 amino acids, about 160 amino acids to about 950 amino acids, about 160 amino acids to about 900 amino acids, about 160 amino acids to about 850 amino acids, about 160 amino acids to about 800 amino acids, about 160 amino acids to about 750 amino acids, about 160 amino acids to about 700 amino acids, about 160 amino acids to about 650 amino acids, about 160 amino acids to about 600 amino acids, about 160 amino acids to about 550 amino acids, about 160 amino acids to about 500 amino acids, about 160 amino acids to about 450 amino acids, about 160 amino acids to about 400 amino acids, about 160 amino acids to about 350 amino acids, about 160 amino acids to about 300 amino acids, about 160 amino acids to about 280 amino acids, about 160 amino acids to about 260 amino acids, about 160 amino acids to about 240 amino acids, about 160 amino acids to about 220 amino acids, about 160 amino acids to about 200 amino acids, about 160 amino acids to about 195 amino acids, about 160 amino acids to about 190 amino acids, about 160 amino acids to about 185 amino acids, about 160 amino acids to about 180 amino acids, about 160 amino acids to about 175 amino acids, about 160 amino acids to about 170 amino acids, about 160 amino acids to about 165 amino acids, about 165 amino acids to about 1000 amino acids, about 165 amino acids to about 950 amino acids, about 165 amino acids to about 900 amino acids, about 165 amino acids to about 850 amino acids, about 165 amino acids to about 800 amino acids, about 165 amino acids to about 750 amino acids, about 165 amino acids to about 700 amino acids, about 165 amino acids to about 650 amino acids, about 165 amino acids to about 600 amino acids, about 165 amino acids to about 550 amino acids, about 165 amino acids to about 500 amino acids, about 165 amino acids to about 450 amino acids, about 165 amino acids to about 400 amino acids, about 165 amino acids to about 350 amino acids, about 165 amino acids to about 300 amino acids, about 165 amino acids to about 280 amino acids, about 165 amino acids to about 260 amino acids, about 165 amino acids to about 240 amino acids, about 165 amino acids to about 220 amino acids, about 165 amino acids to about 200 amino acids, about 165 amino acids to about 195 amino acids, about 165 amino acids to about 190 amino acids, about 165 amino acids to about 185 amino acids, about 165 amino acids to about 180 amino acids, about 165 amino acids to about 175 amino acids, about 165 amino acids to about 170 amino acids, about 170 amino acids to about 1000 amino acids, about 170 amino acids to about 950 amino acids, about 170 amino acids to about 900 amino acids, about 170 amino acids to about 850 amino acids, about 170 amino acids to about 800 amino acids, about 170 amino acids to about 750 amino acids, about 170 amino acids to about 700 amino acids, about 170 amino acids to about 650 amino acids, about 170 amino acids to about 600 amino acids, about 170 amino acids to about 550 amino acids, about 170 amino acids to about 500 amino acids, about 170 amino acids to about 450 amino acids, about 170 amino acids to about 400 amino acids, about 170 amino acids to about 350 amino acids, about 170 amino acids to about 300 amino acids, about 170 amino acids to about 280 amino acids, about 170 amino acids to about 260 amino acids, about 170 amino acids to about 240 amino acids, about 170 amino acids to about 220 amino acids, about 170 amino acids to about 200 amino acids, about 170 amino acids to about 195 amino acids, about 170 amino acids to about 190 amino acids, about 170 amino acids to about 185 amino acids, about 170 amino acids to about 180 amino acids, about 170 amino acids to about 175 amino acids, about 175 amino acids to about 1000 amino acids, about 175 amino acids to about 950 amino acids, about 175 amino acids to about 900 amino acids, about 175 amino acids to about 850 amino acids, about 175 amino acids to about 800 amino acids, about 175 amino acids to about 750 amino acids, about 175 amino acids to about 700 amino acids, about 175 amino acids to about 650 amino acids, about 175 amino acids to about 600 amino acids, about 175 amino acids to about 550 amino acids, about 175 amino acids to about 500 amino acids, about 175 amino acids to about 450 amino acids, about 175 amino acids to about 400 amino acids, about 175 amino acids to about 350 amino acids, about 175 amino acids to about 300 amino acids, about 175 amino acids to about 280 amino acids, about 175 amino acids to about 260 amino acids, about 175 amino acids to about 240 amino acids, about 175 amino acids to about 220 amino acids, about 175 amino acids to about 200 amino acids, about 175 amino acids to about 195 amino acids, about 175 amino acids to about 190 amino acids, about 175 amino acids to about 185 amino acids, about 175 amino acids to about 180 amino acids, about 180 amino acids to about 1000 amino acids, about 180 amino acids to about 950 amino acids, about 180 amino acids to about 900 amino acids, about 180 amino acids to about 850 amino acids, about 180 amino acids to about 800 amino acids, about 180 amino acids to about 750 amino acids, about 180 amino acids to about 700 amino acids, about 180 amino acids to about 650 amino acids, about 180 amino acids to about 600 amino acids, about 180 amino acids to about 550 amino acids, about 180 amino acids to about 500 amino acids, about 180 amino acids to about 450 amino acids, about 180 amino acids to about 400 amino acids, about 180 amino acids to about 350 amino acids, about 180 amino acids to about 300 amino acids, about 180 amino acids to about 280 amino acids, about 180 amino acids to about 260 amino acids, about 180 amino acids to about 240 amino acids, about 180 amino acids to about 220 amino acids, about 180 amino acids to about 200 amino acids, about 180 amino acids to about 195 amino acids, about 180 amino acids to about 190 amino acids, about 180 amino acids to about 185 amino acids, about 185 amino acids to about 1000 amino acids, about 185 amino acids to about 950 amino acids, about 185 amino acids to about 900 amino acids, about 185 amino acids to about 850 amino acids, about 185 amino acids to about 800 amino acids, about 185 amino acids to about 750 amino acids, about 185 amino acids to about 700 amino acids, about 185 amino acids to about 650 amino acids, about 185 amino acids to about 600 amino acids, about 185 amino acids to about 550 amino acids, about 185 amino acids to about 500 amino acids, about 185 amino acids to about 450 amino acids, about 185 amino acids to about 400 amino acids, about 185 amino acids to about 350 amino acids, about 185 amino acids to about 300 amino acids, about 185 amino acids to about 280 amino acids, about 185 amino acids to about 260 amino acids, about 185 amino acids to about 240 amino acids, about 185 amino acids to about 220 amino acids, about 185 amino acids to about 200 amino acids, about 185 amino acids to about 195 amino acids, about 185 amino acids to about 190 amino acids, about 190 amino acids to about 1000 amino acids, about 190 amino acids to about 950 amino acids, about 190 amino acids to about 900 amino acids, about 190 amino acids to about 850 amino acids, about 190 amino acids to about 800 amino acids, about 190 amino acids to about 750 amino acids, about 190 amino acids to about 700 amino acids, about 190 amino acids to about 650 amino acids, about 190 amino acids to about 600 amino acids, about 190 amino acids to about 550 amino acids, about 190 amino acids to about 500 amino acids, about 190 amino acids to about 450 amino acids, about 190 amino acids to about 400 amino acids, about 190 amino acids to about 350 amino acids, about 190 amino acids to about 300 amino acids, about 190 amino acids to about 280 amino acids, about 190 amino acids to about 260 amino acids, about 190 amino acids to about 240 amino acids, about 190 amino acids to about 220 amino acids, about 190 amino acids to about 200 amino acids, about 190 amino acids to about 195 amino acids, about 195 amino acids to about 1000 amino acids, about 195 amino acids to about 950 amino acids, about 195 amino acids to about 900 amino acids, about 195 amino acids to about 850 amino acids, about 195 amino acids to about 800 amino acids, about 195 amino acids to about 750 amino acids, about 195 amino acids to about 700 amino acids, about 195 amino acids to about 650 amino acids, about 195 amino acids to about 600 amino acids, about 195 amino acids to about 550 amino acids, about 195 amino acids to about 500 amino acids, about 195 amino acids to about 450 amino acids, about 195 amino acids to about 400 amino acids, about 195 amino acids to about 350 amino acids, about 195 amino acids to about 300 amino acids, about 195 amino acids to about 280 amino acids, about 195 amino acids to about 260 amino acids, about 195 amino acids to about 240 amino acids, about 195 amino acids to about 220 amino acids, about 195 amino acids to about 200 amino acids, about 200 amino acids to about 1000 amino acids, about 200 amino acids to about 950 amino acids, about 200 amino acids to about 900 amino acids, about 200 amino acids to about 850 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 450 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 350 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 280 amino acids, about 200 amino acids to about 260 amino acids, about 200 amino acids to about 240 amino acids, about 200 amino acids to about 220 amino acids, about 220 amino acids to about 1000 amino acids, about 220 amino acids to about 950 amino acids, about 220 amino acids to about 900 amino acids, about 220 amino acids to about 850 amino acids, about 220 amino acids to about 800 amino acids, about 220 amino acids to about 750 amino acids, about 220 amino acids to about 700 amino acids, about 220 amino acids to about 650 amino acids, about 220 amino acids to about 600 amino acids, about 220 amino acids to about 550 amino acids, about 220 amino acids to about 500 amino acids, about 220 amino acids to about 450 amino acids, about 220 amino acids to about 400 amino acids, about 220 amino acids to about 350 amino acids, about 220 amino acids to about 300 amino acids, about 220 amino acids to about 280 amino acids, about 220 amino acids to about 260 amino acids, about 220 amino acids to about 240 amino acids, about 240 amino acids to about 1000 amino acids, about 240 amino acids to about 950 amino acids, about 240 amino acids to about 900 amino acids, about 240 amino acids to about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 650 amino acids, about 240 amino acids to about 600 amino acids, about 240 amino acids to about 550 amino acids, about 240 amino acids to about 500 amino acids, about 240 amino acids to about 450 amino acids, about 240 amino acids to about 400 amino acids, about 240 amino acids to about 350 amino acids, about 240 amino acids to about 300 amino acids, about 240 amino acids to about 280 amino acids, about 240 amino acids to about 260 amino acids, about 260 amino acids to about 1000 amino acids, about 260 amino acids to about 950 amino acids, about 260 amino acids to about 900 amino acids, about 260 amino acids to about 850 amino acids, about 260 amino acids to about 800 amino acids, about 260 amino acids to about 750 amino acids, about 260 amino acids to about 700 amino acids, about 260 amino acids to about 650 amino acids, about 260 amino acids to about 600 amino acids, about 260 amino acids to about 550 amino acids, about 260 amino acids to about 500 amino acids, about 260 amino acids to about 450 amino acids, about 260 amino acids to about 400 amino acids, about 260 amino acids to about 350 amino acids, about 260 amino acids to about 300 amino acids, about 260 amino acids to about 280 amino acids, about 280 amino acids to about 1000 amino acids, about 280 amino acids to about 950 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids to about 850 amino acids, about 280 amino acids to about 800 amino acids, about 280 amino acids to about 750 amino acids, about 280 amino acids to about 700 amino acids, about 280 amino acids to about 650 amino acids, about 280 amino acids to about 600 amino acids, about 280 amino acids to about 550 amino acids, about 280 amino acids to about 500 amino acids, about 280 amino acids to about 450 amino acids, about 280 amino acids to about 400 amino acids, about 280 amino acids to about 350 amino acids, about 280 amino acids to about 300 amino acids, about 300 amino acids to about 1000 amino acids, about 300 amino acids to about 950 amino acids, about 300 amino acids to about 900 amino acids, about 300 amino acids to about 850 amino acids, about 300 amino acids to about 800 amino acids, about 300 amino acids to about 750 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 450 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 350 amino acids, about 350 amino acids to about 1000 amino acids, about 350 amino acids to about 950 amino acids, about 350 amino acids to about 900 amino acids, about 350 amino acids to about 850 amino acids, about 350 amino acids to about 800 amino acids, about 350 amino acids to about 750 amino acids, about 350 amino acids to about 700 amino acids, about 350 amino acids to about 650 amino acids, about 350 amino acids to about 600 amino acids, about 350 amino acids to about 550 amino acids, about 350 amino acids to about 500 amino acids, about 350 amino acids to about 450 amino acids, about 350 amino acids to about 400 amino acids, about 400 amino acids to about 1000 amino acids, about 400 amino acids to about 950 amino acids, about 400 amino acids to about 900 amino acids, about 400 amino acids to about 850 amino acids, about 400 amino acids to about 800 amino acids, about 400 amino acids to about 750 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 450 amino acids, about 450 amino acids to about 1000 amino acids, about 450 amino acids to about 950 amino acids, about 450 amino acids to about 900 amino acids, about 450 amino acids to about 850 amino acids, about 450 amino acids to about 800 amino acids, about 450 amino acids to about 750 amino acids, about 450 amino acids to about 700 amino acids, about 450 amino acids to about 650 amino acids, about 450 amino acids to about 600 amino acids, about 450 amino acids to about 550 amino acids, about 450 amino acids to about 500 amino acids, about 500 amino acids to about 1000 amino acids, about 500 amino acids to about 950 amino acids, about 500 amino acids to about 900 amino acids, about 500 amino acids to about 850 amino acids, about 500 amino acids to about 800 amino acids, about 500 amino acids to about 750 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 650 amino acids, about 500 amino acids to about 600 amino acids, about 500 amino acids to about 550 amino acids, about 550 amino acids to about 1000 amino acids, about 550 amino acids to about 950 amino acids, about 550 amino acids to about 900 amino acids, about 550 amino acids to about 850 amino acids, about 550 amino acids to about 800 amino acids, about 550 amino acids to about 750 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 1000 amino acids, about 600 amino acids to about 950 amino acids, about 600 amino acids to about 900 amino acids, about 600 amino acids to about 850 amino acids, about 600 amino acids to about 800 amino acids, about 600 amino acids to about 750 amino acids, about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to about 950 amino acids, about 650 amino acids to about 900 amino acids, about 650 amino acids to about 850 amino acids, about 650 amino acids to about 800 amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to about 700 amino acids, about 700 amino acids to about 1000 amino acids, about 700 amino acids to about 950 amino acids, about 700 amino acids to about 900 amino acids, about 700 amino acids to about 850 amino acids, about 700 amino acids to about 800 amino acids, about 700 amino acids to about 750 amino acids, about 750 amino acids to about 1000 amino acids, about 750 amino acids to about 950 amino acids, about 750 amino acids to about 900 amino acids, about 750 amino acids to about 850 amino acids, about 750 amino acids to about 800 amino acids, about 800 amino acids to about 1000 amino acids, about 800 amino acids to about 950 amino acids, about 800 amino acids to about 900 amino acids, about 800 amino acids to about 850 amino acids, about 850 amino acids to about 1000 amino acids, about 850 amino acids to about 950 amino acids, about 850 amino acids to about 900 amino acids, about 900 amino acids to about 1000 amino acids, about 900 amino acids to about 950 amino acids, or about 950 amino acids to about 1000 amino acids.

Any of the target-binding domains described herein can bind to its target with a dissociation equilibrium constant (Kd) of less than 1 x 10'7M, less than 1 x 10'8M, less than 1 x 10'9M, less than 1 x 10'10M, less than 1 x 10'11 M, less than 1 x 10'12M, or less than 1 x 10'13 M. In some embodiments, the antigen-binding protein construct provided herein can bind to an identifying antigen with a Kd of about 1 x 10’3 M to about 1 x 10’5 M, about 1 x IO'4 M to about 1 x 10'6M, about 1 x 10'5M to about 1 x IO'7 M, about 1 x IO'6 M to about 1 x IO'8 M, about 1 x IO'7 M to about 1 x 10'9M, about 1 x 10'8M to about 1 x 10'10 M, or about 1 x IO'9 M to about 1 x 10M (inclusive).

Any of the target-binding domains described herein can bind to its target with a Kd of between about 1 pM to about 30 nM (e.g., about 1 pM to about 25 nM, about 1 pM to about 20 nM, about 1 pM to about 15 nM, about 1 pM to about 10 nM, about 1 pM to about 5 nM, about 1 pM to about 2 nM, about 1 pM to about 1 nM, about 1 pM to about 950 pM, about 1 pM to about 900 pM, about 1 pM to about 850 pM, about 1 pM to about

800 pM, about 1 pM to about 750 pM, about 1 pM to about 700 pM, about 1 pM to about

650 pM, about 1 pM to about 600 pM, about 1 pM to about 550 pM, about 1 pM to about

500 pM, about 1 pM to about 450 pM, about 1 pM to about 400 pM, about 1 pM to about

350 pM, about 1 pM to about 300 pM, about 1 pM to about 250 pM, about 1 pM to about 200 pM, about 1 pM to about 150 pM, about 1 pM to about 100 pM, about 1 pM to about 90 pM, about 1 pM to about 80 pM, about 1 pM to about 70 pM, about 1 pM to about 60 pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM, about 1 pM to about 30 pM, about 1 pM to about 20 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 1 pM to about 4 pM, about 1 pM to about 3 pM, about 1 pM to about 2 pM, about 2 pM to about 30 nM, about 2 pM to about 25 nM, about 2 pM to about 20 nM, about 2 pM to about 15 nM, about 2 pM to about 10 nM, about 2 pM to about 5 nM, about 2 pM to about 2 nM, about 2 pM to about 1 nM, about 2 pM to about 950 pM, about 2 pM to about 900 pM, about 2 pM to about 850 pM, about 2 pM to about 800 pM, about 2 pM to about 750 pM, about 2 pM to about 700 pM, about 2 pM to about 650 pM, about 2 pM to about 600 pM, about 2 pM to about 550 pM, about 2 pM to about 500 pM, about 2 pM to about 450 pM, about 2 pM to about 400 pM, about 2 pM to about 350 pM, about 2 pM to about 300 pM, about 2 pM to about 250 pM, about 2 pM to about 200 pM, about 2 pM to about 150 pM, about 2 pM to about 100 pM, about 2 pM to about 90 pM, about 2 pM to about 80 pM, about 2 pM to about 70 pM, about 2 pM to about 60 pM, about 2 pM to about 50 pM, about 2 pM to about 40 pM, about 2 pM to about 30 pM, about 2 pM to about 20 pM, about 2 pM to about 10 pM, about 2 pM to about 5 pM, about 2 pM to about 4 pM, about 2 pM to about 3 pM, about 5 pM to about 30 nM, about 5 pM to about 25 nM, about 5 pM to about 20 nM, about 5 pM to about 15 nM, about 5 pM to about 10 nM, about 5 pM to about 5 nM, about 5 pM to about 2 nM, about 5 pM to about 1 nM, about 5 pM to about 950 pM, about 5 pM to about 900 pM, about 5 pM to about 850 pM, about 5 pM to about 800 pM, about 5 pM to about 750 pM, about 5 pM to about 700 pM, about 5 pM to about 650 pM, about 5 pM to about 600 pM, about 5 pM to about 550 pM, about 5 pM to about 500 pM, about 5 pM to about 450 pM, about 5 pM to about 400 pM, about 5 pM to about 350 pM, about 5 pM to about 300 pM, about 5 pM to about 250 pM, about 5 pM to about 200 pM, about 5 pM to about 150 pM, about 5 pM to about 100 pM, about 5 pM to about 90 pM, about 5 pM to about 80 pM, about 5 pM to about 70 pM, about 5 pM to about 60 pM, about 5 pM to about 50 pM, about 5 pM to about 40 pM, about 5 pM to about 30 pM, about 5 pM to about 20 pM, about 5 pM to about 10 pM, about 10 pM to about 30 nM, about 10 pM to about 25 nM, about 10 pM to about 20 nM, about 10 pM to about 15 nM, about 10 pM to about 10 nM, about 10 pM to about 5 nM, about 10 pM to about 2 nM, about 10 pM to about 1 nM, about 10 pM to about 950 pM, about 10 pM to about 900 pM, about 10 pM to about 850 pM, about 10 pM to about 800 pM, about 10 pM to about 750 pM, about 10 pM to about 700 pM, about 10 pM to about 650 pM, about 10 pM to about 600 pM, about 10 pM to about 550 pM, about 10 pM to about 500 pM, about 10 pM to about 450 pM, about 10 pM to about 400 pM, about 10 pM to about 350 pM, about 10 pM to about 300 pM, about 10 pM to about 250 pM, about 10 pM to about 200 pM, about 10 pM to about 150 pM, about 10 pM to about 100 pM, about 10 pM to about 90 pM, about 10 pM to about 80 pM, about 10 pM to about 70 pM, about 10 pM to about 60 pM, about 10 pM to about 50 pM, about 10 pM to about 40 pM, about 10 pM to about 30 pM, about 10 pM to about 20 pM, about 15 pM to about 30 nM, about 15 pM to about 25 nM, about 15 pM to about 20 nM, about 15 pM to about 15 nM, about 15 pM to about 10 nM, about 15 pM to about 5 nM, about 15 pM to about 2 nM, about 15 pM to about 1 nM, about 15 pM to about 950 pM, about 15 pM to about 900 pM, about 15 pM to about 850 pM, about 15 pM to about 800 pM, about 15 pM to about

750 pM, about 15 pM to about 700 pM, about 15 pM to about 650 pM, about 15 pM to about 600 pM, about 15 pM to about 550 pM, about 15 pM to about 500 pM, about 15 pM to about 450 pM, about 15 pM to about 400 pM, about 15 pM to about 350 pM, about 15 pM to about 300 pM, about 15 pM to about 250 pM, about 15 pM to about 200 pM, about 15 pM to about 150 pM, about 15 pM to about 100 pM, about 15 pM to about 90 pM, about 15 pM to about 80 pM, about 15 pM to about 70 pM, about 15 pM to about 60 pM, about 15 pM to about 50 pM, about 15 pM to about 40 pM, about 15 pM to about 30 pM, about 15 pM to about 20 pM, about 20 pM to about 30 nM, about 20 pM to about 25 nM, about 20 pM to about 20 nM, about 20 pM to about 15 nM, about 20 pM to about 10 nM, about 20 pM to about 5 nM, about 20 pM to about 2 nM, about 20 pM to about 1 nM, about 20 pM to about 950 pM, about 20 pM to about 900 pM, about 20 pM to about 850 pM, about 20 pM to about 800 pM, about 20 pM to about 750 pM, about 20 pM to about 700 pM, about 20 pM to about 650 pM, about 20 pM to about 600 pM, about 20 pM to about 550 pM, about 20 pM to about 500 pM, about 20 pM to about 450 pM, about 20 pM to about 400 pM, about 20 pM to about 350 pM, about 20 pM to about 300 pM, about 20 pM to about 250 pM, about 20 pM to about 20 pM, about 200 pM to about 150 pM, about 20 pM to about 100 pM, about 20 pM to about 90 pM, about 20 pM to about 80 pM, about 20 pM to about 70 pM, about 20 pM to about 60 pM, about 20 pM to about 50 pM, about 20 pM to about 40 pM, about 20 pM to about 30 pM, about 30 pM to about 30 nM, about 30 pM to about 25 nM, about 30 pM to about 30 nM, about 30 pM to about 15 nM, about 30 pM to about 10 nM, about 30 pM to about 5 nM, about 30 pM to about 2 nM, about 30 pM to about 1 nM, about 30 pM to about 950 pM, about 30 pM to about 900 pM, about 30 pM to about 850 pM, about 30 pM to about 800 pM, about 30 pM to about 750 pM, about 30 pM to about 700 pM, about 30 pM to about 650 pM, about 30 pM to about 600 pM, about 30 pM to about 550 pM, about 30 pM to about 500 pM, about 30 pM to about 450 pM, about 30 pM to about 400 pM, about 30 pM to about 350 pM, about 30 pM to about 300 pM, about 30 pM to about 250 pM, about 30 pM to about 200 pM, about 30 pM to about 150 pM, about 30 pM to about 100 pM, about 30 pM to about 90 pM, about 30 pM to about 80 pM, about 30 pM to about 70 pM, about 30 pM to about 60 pM, about 30 pM to about 50 pM, about 30 pM to about 40 pM, about 40 pM to about 30 nM, about 40 pM to about 25 nM, about 40 pM to about 30 nM, about 40 pM to about nM, about 40 pM to about 10 nM, about 40 pM to about 5 nM, about 40 pM to about 2 nM, about 40 pM to about 1 nM, about 40 pM to about 950 pM, about 40 pM to about 900 pM, about 40 pM to about 850 pM, about 40 pM to about 800 pM, about 40 pM to about 750 pM, about 40 pM to about 700 pM, about 40 pM to about 650 pM, about 40 pM to about 600 pM, about 40 pM to about 550 pM, about 40 pM to about 500 pM, about 40 pM to about 450 pM, about 40 pM to about 400 pM, about 40 pM to about 350 pM, about 40 pM to about 300 pM, about 40 pM to about 250 pM, about 40 pM to about 200 pM, about 40 pM to about 150 pM, about 40 pM to about 100 pM, about 40 pM to about 90 pM, about 40 pM to about 80 pM, about 40 pM to about 70 pM, about 40 pM to about 60 pM, about 40 pM to about 50 pM, about 50 pM to about 30 nM, about 50 pM to about 25 nM, about 50 pM to about 30 nM, about 50 pM to about 15 nM, about 50 pM to about 10 nM, about 50 pM to about 5 nM, about 50 pM to about 2 nM, about 50 pM to about 1 nM, about 50 pM to about 950 pM, about 50 pM to about 900 pM, about 50 pM to about 850 pM, about 50 pM to about 800 pM, about 50 pM to about 750 pM, about 50 pM to about 700 pM, about 50 pM to about 650 pM, about 50 pM to about 600 pM, about 50 pM to about 550 pM, about 50 pM to about 500 pM, about 50 pM to about 450 pM, about 50 pM to about 400 pM, about 50 pM to about 350 pM, about 50 pM to about 300 pM, about 50 pM to about 250 pM, about 50 pM to about 200 pM, about 50 pM to about 150 pM, about 50 pM to about 100 pM, about 50 pM to about 90 pM, about 50 pM to about 80 pM, about 50 pM to about 70 pM, about 50 pM to about 60 pM, about 60 pM to about 30 nM, about 60 pM to about 25 nM, about 60 pM to about 30 nM, about 60 pM to about 15 nM, about 60 pM to about 10 nM, about 60 pM to about 5 nM, about 60 pM to about 2 nM, about 60 pM to about 1 nM, about 60 pM to about 950 pM, about 60 pM to about 900 pM, about 60 pM to about 850 pM, about 60 pM to about 800 pM, about 60 pM to about 750 pM, about 60 pM to about 700 pM, about 60 pM to about 650 pM, about 60 pM to about 600 pM, about 60 pM to about 550 pM, about 60 pM to about 500 pM, about 60 pM to about 450 pM, about 60 pM to about 400 pM, about 60 pM to about 350 pM, about 60 pM to about 300 pM, about 60 pM to about 250 pM, about 60 pM to about 200 pM, about 60 pM to about 150 pM, about 60 pM to about 100 pM, about 60 pM to about 90 pM, about 60 pM to about 80 pM, about 60 pM to about 70 pM, about 70 pM to about 30 nM, about 70 pM to about 25 nM, about 70 pM to about 30 nM, about 70 pM to about nM, about 70 pM to about 10 nM, about 70 pM to about 5 nM, about 70 pM to about 2 nM, about 70 pM to about 1 nM, about 70 pM to about 950 pM, about 70 pM to about 900 pM, about 70 pM to about 850 pM, about 70 pM to about 800 pM, about 70 pM to about 750 pM, about 70 pM to about 700 pM, about 70 pM to about 650 pM, about 70 pM to about 600 pM, about 70 pM to about 550 pM, about 70 pM to about 500 pM, about 70 pM to about 450 pM, about 70 pM to about 400 pM, about 70 pM to about 350 pM, about 70 pM to about 300 pM, about 70 pM to about 250 pM, about 70 pM to about 200 pM, about 70 pM to about 150 pM, about 70 pM to about 100 pM, about 70 pM to about 90 pM, about 70 pM to about 80 pM, about 80 pM to about 30 nM, about 80 pM to about 25 nM, about 80 pM to about 30 nM, about 80 pM to about 15 nM, about 80 pM to about 10 nM, about 80 pM to about 5 nM, about 80 pM to about 2 nM, about 80 pM to about 1 nM, about 80 pM to about 950 pM, about 80 pM to about 900 pM, about 80 pM to about 850 pM, about 80 pM to about 800 pM, about 80 pM to about 750 pM, about 80 pM to about 700 pM, about 80 pM to about 650 pM, about 80 pM to about 600 pM, about 80 pM to about 550 pM, about 80 pM to about 500 pM, about 80 pM to about 450 pM, about 80 pM to about 400 pM, about 80 pM to about 350 pM, about 80 pM to about 300 pM, about 80 pM to about 250 pM, about 80 pM to about 200 pM, about 80 pM to about 150 pM, about 80 pM to about 100 pM, about 80 pM to about 90 pM, about 90 pM to about 30 nM, about 90 pM to about 25 nM, about 90 pM to about 30 nM, about 90 pM to about 15 nM, about 90 pM to about 10 nM, about 90 pM to about 5 nM, about 90 pM to about 2 nM, about 90 pM to about 1 nM, about 90 pM to about 950 pM, about 90 pM to about 900 pM, about 90 pM to about 850 pM, about 90 pM to about 800 pM, about 90 pM to about 750 pM, about 90 pM to about 700 pM, about 90 pM to about 650 pM, about 90 pM to about 600 pM, about 90 pM to about 550 pM, about 90 pM to about 500 pM, about 90 pM to about 450 pM, about 90 pM to about 400 pM, about 90 pM to about 350 pM, about 90 pM to about 300 pM, about 90 pM to about 250 pM, about 90 pM to about 200 pM, about 90 pM to about 150 pM, about 90 pM to about 100 pM, about 100 pM to about 30 nM, about 100 pM to about 25 nM, about 100 pM to about 30 nM, about 100 pM to about 15 nM, about 100 pM to about 10 nM, about 100 pM to about 5 nM, about 100 pM to about 2 nM, about 100 pM to about 1 nM, about 100 pM to about 950 pM, about 100 pM to about 900 pM, about 100 pM to about 850 pM, about 100 pM to about

800 pM, about 100 pM to about 750 pM, about 100 pM to about 700 pM, about 100 pM to about 650 pM, about 100 pM to about 600 pM, about 100 pM to about 550 pM, about 100 pM to about 500 pM, about 100 pM to about 450 pM, about 100 pM to about 400 pM, about 100 pM to about 350 pM, about 100 pM to about 300 pM, about 100 pM to about 250 pM, about 100 pM to about 200 pM, about 100 pM to about 150 pM, about 150 pM to about 30 nM, about 150 pM to about 25 nM, about 150 pM to about 30 nM, about 150 pM to about 15 nM, about 150 pM to about 10 nM, about 150 pM to about 5 nM, about 150 pM to about 2 nM, about 150 pM to about 1 nM, about 150 pM to about 950 pM, about 150 pM to about 900 pM, about 150 pM to about 850 pM, about 150 pM to about 800 pM, about 150 pM to about 750 pM, about 150 pM to about 700 pM, about 150 pM to about 650 pM, about 150 pM to about 600 pM, about 150 pM to about 550 pM, about 150 pM to about 500 pM, about 150 pM to about 450 pM, about 150 pM to about 400 pM, about 150 pM to about 350 pM, about 150 pM to about 300 pM, about 150 pM to about 250 pM, about 150 pM to about 200 pM, about 200 pM to about 30 nM, about 200 pM to about 25 nM, about 200 pM to about 30 nM, about 200 pM to about 15 nM, about 200 pM to about 10 nM, about 200 pM to about 5 nM, about 200 pM to about 2 nM, about 200 pM to about 1 nM, about 200 pM to about 950 pM, about 200 pM to about 900 pM, about 200 pM to about 850 pM, about 200 pM to about 800 pM, about 200 pM to about 750 pM, about 200 pM to about 700 pM, about 200 pM to about 650 pM, about 200 pM to about 600 pM, about 200 pM to about 550 pM, about 200 pM to about 500 pM, about 200 pM to about 450 pM, about 200 pM to about 400 pM, about 200 pM to about 350 pM, about 200 pM to about 300 pM, about 200 pM to about 250 pM, about 300 pM to about 30 nM, about 300 pM to about 25 nM, about 300 pM to about 30 nM, about 300 pM to about 15 nM, about 300 pM to about 10 nM, about 300 pM to about 5 nM, about 300 pM to about 2 nM, about 300 pM to about 1 nM, about 300 pM to about 950 pM, about 300 pM to about 900 pM, about 300 pM to about 850 pM, about 300 pM to about 800 pM, about 300 pM to about 750 pM, about 300 pM to about 700 pM, about 300 pM to about 650 pM, about 300 pM to about 600 pM, about 300 pM to about 550 pM, about 300 pM to about 500 pM, about 300 pM to about 450 pM, about 300 pM to about 400 pM, about 300 pM to about 350 pM, about 400 pM to about 30 nM, about 400 pM to about 25 nM, about 400 pM to about 30 nM, about 400 pM to about nM, about 400 pM to about 10 nM, about 400 pM to about 5 nM, about 400 pM to about 2 nM, about 400 pM to about 1 nM, about 400 pM to about 950 pM, about 400 pM to about 900 pM, about 400 pM to about 850 pM, about 400 pM to about 800 pM, about 400 pM to about 750 pM, about 400 pM to about 700 pM, about 400 pM to about 650 pM, about 400 pM to about 600 pM, about 400 pM to about 550 pM, about 400 pM to about 500 pM, about 500 pM to about 30 nM, about 500 pM to about 25 nM, about 500 pM to about 30 nM, about 500 pM to about 15 nM, about 500 pM to about 10 nM, about 500 pM to about 5 nM, about 500 pM to about 2 nM, about 500 pM to about 1 nM, about 500 pM to about 950 pM, about 500 pM to about 900 pM, about 500 pM to about 850 pM, about 500 pM to about 800 pM, about 500 pM to about 750 pM, about 500 pM to about 700 pM, about 500 pM to about 650 pM, about 500 pM to about 600 pM, about 500 pM to about 550 pM, about 600 pM to about 30 nM, about 600 pM to about 25 nM, about 600 pM to about 30 nM, about 600 pM to about 15 nM, about 600 pM to about 10 nM, about 600 pM to about 5 nM, about 600 pM to about 2 nM, about 600 pM to about 1 nM, about 600 pM to about 950 pM, about 600 pM to about 900 pM, about 600 pM to about 850 pM, about 600 pM to about 800 pM, about 600 pM to about 750 pM, about 600 pM to about 700 pM, about 600 pM to about 650 pM, about 700 pM to about 30 nM, about 700 pM to about 25 nM, about 700 pM to about 30 nM, about 700 pM to about 15 nM, about 700 pM to about 10 nM, about 700 pM to about 5 nM, about 700 pM to about 2 nM, about 700 pM to about 1 nM, about 700 pM to about 950 pM, about 700 pM to about 900 pM, about 700 pM to about 850 pM, about 700 pM to about 800 pM, about 700 pM to about 750 pM, about 800 pM to about 30 nM, about 800 pM to about 25 nM, about 800 pM to about 30 nM, about 800 pM to about 15 nM, about 800 pM to about 10 nM, about 800 pM to about 5 nM, about 800 pM to about 2 nM, about 800 pM to about 1 nM, about 800 pM to about 950 pM, about 800 pM to about 900 pM, about 800 pM to about 850 pM, about 900 pM to about 30 nM, about 900 pM to about 25 nM, about 900 pM to about 30 nM, about 900 pM to about 15 nM, about 900 pM to about 10 nM, about 900 pM to about 5 nM, about 900 pM to about 2 nM, about 900 pM to about 1 nM, about 900 pM to about 950 pM, about 1 nM to about 30 nM, about 1 nM to about 25 nM, about 1 nM to about 20 nM, about 1 nM to about 15 nM, about 1 nM to about 10 nM, about 1 nM to about 5 nM, about 2 nM to about 30 nM, about 2 nM to about 25 nM, about 2 nM to about 20 nM, about 2 nM to about 15 nM, about 2 nM to about 10 nM, about 2 nM to about 5 nM, about 4 nM to about 30 nM, about 4 nM to about 25 nM, about 4 nM to about 20 nM, about 4 nM to about 15 nM, about 4 nM to about 10 nM, about 4 nM to about 5 nM, about 5 nM to about 30 nM, about 5 nM to about 25 nM, about 5 nM to about 20 nM, about 5 nM to about 15 nM, about 5 nM to about 10 nM, about 10 nM to about 30 nM, about 10 nM to about 25 nM, about 10 nM to about 20 nM, about 10 nM to about 15 nM, about 15 nM to about 30 nM, about 15 nM to about 25 nM, about 15 nM to about 20 nM, about 20 nM to about 30 nM, and about 20 nM to about 25 nM).

Any of the target-binding domains described herein can bind to its target with a Kd of between about 1 nM to about 10 nM (e.g., about 1 nM to about 9 nM, about 1 nM to about 8 nM, about 1 nM to about 7 nM, about 1 nM to about 6 nM, about 1 nM to about 5 nM, about 1 nM to about 4 nM, about 1 nM to about 3 nM, about 1 nM to about 2 nM, about 2 nM to about 10 nM, about 2 nM to about 9 nM, about 2 nM to about 8 nM, about 2 nM to about 7 nM, about 2 nM to about 6 nM, about 2 nM to about 5 nM, about 2 nM to about 4 nM, about 2 nM to about 3 nM, about 3 nM to about 10 nM, about 3 nM to about 9 nM, about 3 nM to about 8 nM, about 3 nM to about 7 nM, about 3 nM to about 6 nM, about 3 nM to about 5 nM, about 3 nM to about 4 nM, about 4 nM to about 10 nM, about 4 nM to about 9 nM, about 4 nM to about 8 nM, about 4 nM to about 7 nM, about 4 nM to about 6 nM, about 4 nM to about 5 nM, about 5 nM to about 10 nM, about 5 nM to about 9 nM, about 5 nM to about 8 nM, about 5 nM to about 7 nM, about 5 nM to about 6 nM, about 6 nM to about 10 nM, about 6 nM to about 9 nM, about 6 nM to about 8 nM, about 6 nM to about 7 nM, about 7 nM to about 10 nM, about 7 nM to about 9 nM, about 7 nM to about 8 nM, about 8 nM to about 10 nM, about 8 nM to about 9 nM, and about 9 nM to about 10 nM).

A variety of different methods known in the art can be used to determine the Kd values of any of the antigen-binding protein constructs described herein (e.g., an electrophoretic mobility shift assay, a filter binding assay, surface plasmon resonance, and a biomolecular binding kinetics assay, etc.).

Antigen-Binding Domains

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second target-binding domain bind specifically to the same antigen. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain include the same amino acid sequence.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second target-binding domain bind specifically to different antigens.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target-binding domain and the second target-binding domain is an antigen-binding domain. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second target-binding domain are each antigen-binding domains.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the antigen-binding domain includes or is a scFv or a single domain antibody (e.g., a VHH or a VNAR domain).

In some examples, an antigen-binding domain (e.g., any of the antigen-binding domains described herein) can bind specifically to any one of CD 16a (see, e.g., those described in U.S. Patent No. 9,035,026), CD28 (see, e.g., those described in U.S. Patent No. 7,723,482), CD3 (see, e.g., those described in U.S. Patent No. 9,226,962), CD33 (see, e.g., those described in U.S. Patent No. 8,759,494), CD20 (see, e.g., those described in WO 2014/026054), CD19 (see, e.g., those described in U.S. Patent No. 9,701,758), CD22 (see, e.g., those described in WO 2003/104425), CD123 (see, e.g., those described in WO 2014/130635), IL-1R (see, e.g., those described in U.S. Patent No. 8,741,604), IL1 (see, e.g., those described in WO 2014/095808), VEGF (see, e.g., those described in U.S. Patent No. 9,090,684), IL-6R (see, e.g., those described in U.S. Patent No. 7,482,436), IL-4 (see, e.g., those described in U.S. Patent Application Publication No. 2012/0171197), IL-10 (see, e.g., those described in U.S. Patent Application Publication

No. 2016/0340413), PDL-1 (see, e.g., those described in Drees et al., Protein Express. Purif. 94:60-66, 2014), TIGIT (see, e.g., those described in U.S. Patent Application Publication No. 2017/0198042), PD-1 (see, e.g., those described in U.S. Patent No. 7,488,802), TIM3 (see, e.g., those described in U.S. Patent No. 8,552,156), CTLA4 (see, e.g., those described in WO 2012/120125), MICA (see, e.g., those described in WO 2016/154585), MICB (see, e.g., those described in U.S. Patent No. 8,753,640), IL-6 (see, e.g., those described in Gejima et al., Human Antibodies 11(4):121-129, 2002), IL-8 (see, e.g., those described in U.S. Patent No. 6,117,980), TNFa (see, e.g., those described in Geng et al., Immunol. Res. 62(3):377-385, 2015), CD26a (see, e.g., those described in WO 2017/189526), CD36 (see, e.g., those described in U.S. Patent Application Publication No. 2015/0259429), ULBP2 (see, e.g., those described in U.S. Patent No. 9,273,136), CD30 (see, e.g., those described in Homach et al., Scand. J. Immunol. 48(5):497-501, 1998), CD200 (see, e.g., those described in U.S. Patent No. 9,085,623), IGF-1R (see, e.g., those described in U.S. Patent Application Publication No. 2017/0051063), MUC4AC (see, e.g., those described in WO 2012/170470), MUC5AC (see, e.g., those described in U.S. Patent No. 9,238,084), Trop-2 (see, e.g., those described in WO 2013/068946), CMET (see, e.g., those described in Edwardraja et al., Biotechnol. Bioeng. 106(3):367-375, 2010), EGFR (see, e.g., those described in Akbari et al., ProteinExpr. Purif. 127:8-15, 2016), HER1 (see, e.g., those described in U.S. Patent Application Publication No. 2013/0274446), HER2 (see, e.g., those described in Cao et al., Biotechnol. Lett. 37(7):1347-1354, 2015), HER3 (see, e.g., those described in U.S. Patent No. 9,505,843), PSMA (see, e.g., those described in Parker et al., Protein Expr.

Purif. 89(2):136-145, 2013), CEA (see, e.g., those described in WO 1995/015341), B7H3 (see, e.g., those described in U.S. Patent No. 9,371,395), EPCAM (see, e.g., those described in WO 2014/159531), BCMA (see, e.g., those described in Smith et al., Mol. Ther. 26(6):1447-1456, 2018), P-cadherin (see, e.g., those described in U.S. Patent No. 7,452,537), CEACAM5 (see, e.g., those described in U.S. Patent No. 9,617,345), a UL16-binding protein (see, e.g., those described in WO 2017/083612), HLA-DR (see, e.g., Pistillo et al., Exp. Clin. Immunogenet. 14(2):123-130, 1997), DLL4 (see, e.g., those described in WO 2014/007513), TYRO3 (see, e.g., those described in WO 2016/166348), AXL (see, e.g., those described in WO 2012/175692), MER (see, e.g., those described in

WO 2016/106221), CD122 (see, e.g., those described in U.S. Patent Application Publication No. 2016/0367664), CD155 (see, e.g., those described in WO 2017/149538), or PDGF-DD (see, e.g., those described in U.S. Patent No. 9,441,034).

The antigen-binding domains present in any of the multi-chain chimeric polypeptides described herein are each independently selected from the group consisting of: a VHH domain, a VNAR domain, and a scFv. In some embodiments, any of the antigen-binding domains described herein is a BiTe, a (scFv)2, a nanobody, a nanobodyHSA, a DART, a TandAb, a scDiabody, a scDiabody-CH3, scFv-CH-CL-scFv, a HSAbody, scDiabody-HAS, or a tandem-scFv. Additional examples of antigen-binding domains that can be used in any of the multi-chain chimeric polypeptide are known in the art.

A VHH domain is a single monomeric variable antibody domain that can be found in camelids. A VNAR domain is a single monomeric variable antibody domain that can be found in cartilaginous fish. Non-limiting aspects of VHH domains and Vnar domains are described in, e.g., Cromie et al., Curr Top. Med. Chem. 15:2543-2557, 2016; De Genst et al., Dev. Comp. Immunol. 30:187-198, 2006; De Meyer et al., Trends Biotechnol. 32:263-270, 2014; Kijanka et al., Nanomedicine 10:161-174, 2015; Kovaleva et al., Expert. Opin. Biol. Ther. 14:1527-1539, 2014; Krah et al., ImmunopharmacoL ImmunotoxicoL 38:21-28, 2016; Mujic-Delic etal., Trends Pharmacol. Sci. 35:247-255, 2014; Muyldermans, J. Biotechnol. 74:277-302, 2001; Muyldermans et al., Trends Biochem. Sci. 26:230-235, 2001; Muyldermans, Ann. Rev. Biochem. 82:775-797, 2013; Rahbarizadeh et al., Immunol. Invest. 40:299-338, 2011; Van Audenhove et al., EBioMedicine 8:40-48, 2016; Van Bockstaele et al., Curr. Opin. Investig. Drugs 10:12121224, 2009; Vincke et al., Methods Mol. Biol. 911:15-26, 2012; and Wesolowski et al., Med. Microbiol. Immunol. 198:157-174, 2009.

In some embodiments, each of the antigen-binding domains in the multi-chain chimeric polypeptides described herein are both VHH domains, or at least one antigenbinding domain is a VHH domain. In some embodiments, each of the antigen-binding domains in the multi-chain chimeric polypeptides described herein are both VNAR domains, or at least one antigen-binding domain is a VNAR domain. In some embodiments, each of the antigen-binding domains in the multi-chain chimeric polypeptides described herein are both scFv domains, or at least one antigen-binding domain is a scFv domain.

In some embodiments, two or more of polypeptides present in the multi-chain chimeric polypeptide can assemble (e.g., non-covalently assemble) to form any of the antigen-binding domains described herein, e.g., an antigen-binding fragment of an antibody (e.g., any of the antigen-binding fragments of an antibody described herein), a VHH-scAb, a VHH-Fab, a Dual scFab, a F(ab’)2, a diabody, a crossMab, a DAF (two-inone), a DAF (four-in-one), a DutaMab, a DT-IgG, a knobs-in-holes common light chain, a knobs-in-holes assembly, a charge pair, a Fab-arm exchange, a SEEDbody, a LUZ-Y, a Fcab, a κλ-body, an orthogonal Fab, a DVD-IgG, a IgG(H)-scFv, a scFv-(H)IgG, IgG(L)scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgGscFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, a triple body, a miniantibody, a minibody, a TriBi minibody, scFv-CH3 KIH, Fab-scFv, a F(ab’)2scFv2, a scFv-KIH, a Fab-scFv-Fc, a tetravalent HCAb, a scDiabody-Fc, aDiabody-Fc, a tandem scFv-Fc, an Intrabody, a dock and lock, a ImmTAC, an IgG-IgG conjugate, a Cov-X-Body, and a scFvl-PEG-scFv2. See, e.g., Spiess et al., Mol. Immunol. 67:95-106, 2015, incorporated in its entirety herewith, for a description of these elements. Nonlimiting examples of an antigen-binding fragment of an antibody include an Fv fragment, a Fab fragment, a F(ab')2 fragment, and a Fab' fragment. Additional examples of an antigen-binding fragment of an antibody is an antigen-binding fragment of an IgG (e.g., an antigen-binding fragment of IgGl, IgG2, IgG3, or IgG4) (e.g., an antigen-binding fragment of a human or humanized IgG, e.g., human or humanized IgGl, IgG2, IgG3, or IgG4); an antigen-binding fragment of an IgA (e.g., an antigen-binding fragment of IgAl or IgA2) (e.g., an antigen-binding fragment of a human or humanized IgA, e.g., a human or humanized IgAl or IgA2); an antigen-binding fragment of an IgD (e.g., an antigenbinding fragment of a human or humanized IgD); an antigen-binding fragment of an IgE (e.g., an antigen-binding fragment of a human or humanized IgE); or an antigen-binding fragment of an IgM (e.g., an antigen-binding fragment of a human or humanized IgM).

An “Fv” fragment includes a non-covalently-linked dimer of one heavy chain variable domain and one light chain variable domain.

A “Fab” fragment includes, the constant domain of the light chain and the first constant domain (Chi) of the heavy chain, in addition to the heavy and light chain variable domains of the Fv fragment.

A “F(ab')2” fragment includes two Fab fragments joined, near the hinge region, by disulfide bonds.

A “dual variable domain immunoglobulin” or “DVD-Ig” refers to multivalent and multispecific binding proteins as described, e.g., in DiGiammarino et al., Methods Mol. Biol. 899:145-156, 2012; Jakob etal.,M4fe 5:358-363, 2013; and U.S. Patent Nos. 7,612,181; 8,258,268; 8,586,714; 8,716,450; 8,722,855; 8,735,546; and 8,822,645, each of which is incorporated by reference in its entirety.

DARTs are described in, e.g., Garber, Nature Reviews Drug Discovery 13:799801, 2014.

In some embodiments of any of the antigen-binding domains described herein can bind to an antigen selected from the group consisting of: a protein, a carbohydrate, a lipid, and a combination thereof.

Additional examples and aspects of antigen-binding domains are known in the art.

Soluble Interleukin or Cytokine Protein

In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target-binding domain and the second target-binding domain can be a soluble interleukin protein or soluble cytokine protein. In some embodiments, the soluble interleukin or soluble cytokine protein is selected from the group of: IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L. Non-limiting examples of soluble IL-2, IL-3, IL-7, IL-8, IL-10, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3Lare provided below.

Human Soluble IL-2 (SEQ ID NO: 9) aptssstkkt qlqlehllld Iqmilnginn yknpkltrml tfkfympkka telkhlqcle eelkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr witfcqsiis tit

Human Soluble IL-3 (SEQ ID NO: 10) apmtqttplkt swvncsnmid eiithlkqpp Iplldfnnln gedqdilmen nlrrpnleaf nravkslqna saiesilknl Ipclplataa ptrhpihikd gdwnefrrkl tfylktlena qaqqttlsla if

Human Soluble IL-7 (SEQ ID NO: 11) dcdiegkdgkqyesv Imvsidqlld smkeigsncl nnefnffkrh icdankegmf Ifraarklrq flkmnstgdf dlhllkvseg ttillnctgq vkgrkpaalg eaqptkslee nkslkeqkkl ndlcflkrll qeiktcwnki Imgtkeh

Human Soluble IL-8 (SEQ ID NO: 12) egavlprsak elrcqcikty skpfhpkfik elrviesgph canteiivkl sdgrelcldp kenwvqrvve kflkraens

Human Soluble IL-10 (SEQ ID NO: 13) spgqgtqsensc thfpgnlpnm Irdlrdafsr vktffqmkdq Idnlllkesl ledfkgylgc qalsemiqfy leevmpqaen qdpdikahvn slgenlktlr Irlrrchrfl pcenkskave qvknafnklq ekgiykamse fdifinyiea ymtmkirn

Human Soluble IL-15 (SEQ ID NO: 14)

Nwvnvisdlkki edliqsmhid atlytesdvh psckvtamkc fllelqvisl esgdasihdt venliilann slssngnvte sgckeceele eknikeflqs fvhivqmfin ts

Human Soluble IL-17 (SEQ ID NO: 15) gitiprn pgcpnsedkn fprtvmvnln ihnrntntnp krssdyynrs tspwnlhrne dperypsviw eakcrhlgci nadgnvdyhm nsvpiqqeil vlrrepphcp nsfrlekilv svgctcvtpi vhhva

Human Soluble IL-18 (SEQ ID NO: 16) yfgklesklsvirn Indqvlfidq gnrplfedmt dsdcrdnapr tifiismykd sqprgmavti svkcekistl scenkiisfk emnppdnikd tksdiiffqr svpghdnkmq fesssyegyf lacekerdlf klilkkedel gdrsimftvq ned

Human Soluble PDGF-DD (SEQ ID NO: 17) rdtsatpqsasi kalrnanlrr desnhltdly rrdetiqvkg ngyvqsprfp nsyprnlllt wrlhsqentr iqlvfdnqfg leeaendicr ydfvevedis etstiirgrw cghkevppri ksrtnqikit fksddyfvak pgfkiyysll edfqpaaase tnwesvtssi sgvsynspsv tdptliadal dkkiaefdtv edllkyfnpe swqedlenmy Idtpryrgrs yhdrkskvdl drlnddakry sctprnysvn ireelklanv vffprcllvq rcggncgcgt vnwrsctcns gktvkkyhev Iqfepghikr rgraktmalv diqldhherc dcicssrppr

Human Soluble SCF (SEQ ID NO: 18) egicrnrvtnnvkdv tklvanlpkd ymitlkyvpg mdvlpshcwi semvvqlsds ltdildkfsn iseglsnysi idklvnivdd Ivecvkenss kdlkksfksp eprlftpeef frifnrsida fkdfvvaset sdcvvsstls pekdsrvsvt kpfmlppvaa sslrndssss nrkaknppgd sslhwaamal palfsliigf afgalywkkr qpsltraven iqineednei smlqekeref qev

Human Soluble FLT3L (SEQ ID NO: 19) tqdcsfqhspissd favkirelsd yllqdypvtv asnlqdeelc gglwrlvlaq rwmerlktva gskmqgller vnteihfvtk cafqpppscl rfvqtnisrl Iqetseqlva Ikpwitrqnf srclelqcqp dsstlpppws prpleatapt apqpplllll llpvglllla aawclhwqrt rrrtprpgeq vppvpspqdl llveh

Non-limiting examples of soluble MICA, MICB, ULBP1, ULBP2, ULBP3,

ULBP4, ULBP5, and ULBP6 are provided below.

Human Soluble MICA (SEQ ID NO: 20) ephslry nltvlswdgs vqsgfltevh Idgqpflrcd rqkcrakpqg qwaedvlgnk ihednstrss edamktkthy itvtcrasgf ricqgeeqrf fviiifyvrc msdlgstgst twdretrdlt qhfyydgelf hamhadclqe ypwnitlswr tcymehsgnh ckkktsaaeg ega gngkdlrmtl Isqnletkew Irrylksgvv qdgvslshdt sthpvpsgkv pelvslqvld ahikdqkegl tmpqssraqt Irrtvppmvn qqwgdvlpdg Ivlqshwqtf qhpvgtsdhr hslqeirvce lamnvrnfIk vtrseasegn ngtyqtwvat hvsavaaaai datqlgfqpl

Human Soluble MICB (SEQ ID NO: 21) aephslry nlmvlsqdes vqsgflaegh Idgqpflryd rqkrrakpqg qwaedvlgak ihedsstrgs edamktkthy itvtcrassf rirqgeeqrf viiiilcvpc daaqlgfqpl twdtetedlt rhfyydgelf ramqadclqk yprnitltwr tcymehsgnh ckkktsaaeg msatgstgst engqdlrrtl Isqnletqes Iqrylksgva qdgvslshnt gthpvpsgkv pelvslqvld ega thikdqkggl tvpqssraqt irrtvppmvn qqwgdvlpdg Ivlqsqrtdf qhpvgtgdhr hslqeirvce lamnvtnfwk vtcsevsegn ngtyqtwvat pyvsaampcf

Human Soluble ULBP1 (SEQ ID NO: 22) wvdthclcydfiit pksrpepqwc evqglvderp flhydcvnhk akafaslgkk vnvtktweeq tetlrdvvdf Ikgqlldiqv enlipieplt Iqarmscehe ahghgrgswq flfngqkfll fdsnnrkwta Ihpgakkmte kweknrdvtm ffqkislgdc kmwleeflmy weqmldptkp pslapg

Human Soluble ULBP2 (SEQ ID NO: 23) gradphslcyditvi pkfrpgprwc avqgqvdekt flhydcgnkt vtpvsplgkk Invttawkaq npvlrevvdi Iteqlrdiql enytpkeplt Iqarmsceqk aeghssgswq fsfdgqifll fdsekrmwtt vhpgarkmke kwendkvvam sfhyfsmgdc igwledflmg mdstlepsag aplams

Human Soluble ULBP3 (SEQ ID NO: 24) dahslwynfti ihlprhgqqw cevqsqvdqk nflsydcgsd kvlsmghlee qlyatdawgk qlemlrevgq rlrleladte ledftpsgpl tlqvrmscec eadgyirgsw qfsfdgrkfl Ifdsnnrkwt vvhagarrmk ekwekdsglt tffkmvsmrd ckswlrdflm hrkkrlepta pptmapg

Human Soluble ULBP4 (SEQ ID NO: 25) hslcfnftik slsrpgqpwc eaqvflnknl flqynsdnnm vkplgllgkk vyatstwgel tqtlgevgrd Irmllcdikp qiktsdpstl qvemfcqrea erctgaswqf atngeksllf damnmtwtvi nheaskiket wkkdrgleky frklskgdcd hwlreflghw eampeptvsp vnasdihwss sslpdrwiil gafillvlmg ivlicvwwqn gewqaglwpl rts

Human Soluble ULBP5 (SEQ ID NO: 26) gladp hslcyditvi pkfrpgprwc avqgqvdekt flhydcgskt vtpvsplgkk Invttawkaq npvlrevvdi Iteqlldiql enyipkeplt Iqarmsceqk aeghgsgswq Isfdgqifll fdsenrmwtt vhpgarkmke kwendkdmtm sfhyismgdc tgwledflmg mdstlepsag apptmssg

Human Soluble ULBP6 (SEQ ID NO: 27) rrddp hslcyditvi pkfrpgprwc avqgqvdekt flhydcgnkt vtpvsplgkk Invtmawkaq npvlrevvdi Iteqlldiql enytpkeplt Iqarmsceqk aeghssgswq fsidgqtfll fdsekrmwtt vhpgarkmke kwendkdvam sfhyismgdc igwledflmg mdstlepsag aplamssg

Additional examples of soluble interleukin proteins and soluble cytokine proteins are known in the art.

Soluble Receptor

In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin receptor, a soluble cytokine receptor or a ligand receptor. In some embodiments, the soluble receptor is a soluble TGF-β receptor II (TGF-β RII) (see, e.g., those described in Yung et al., Am. J. Resp. Crit. CareMed. 194(9):1140-1151, 2016), a soluble ΤΟΡ-βΚΙΙΙ (see, e.g., those described in Heng et al., Placenta 57:320, 2017), a soluble NKG2D (see, e.g., Cosman et al., Immunity 14(2):123-133, 2001; Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018; doi: 10.3389/f1mmu.2018.01150), a soluble NKp30 (see, e.g., Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018; doi: 10.3389/f1mmu.2018.01150), a soluble NKp44 (see, e.g., those described in Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018; doi: 10.3389/f1mmu.2018.01150), a soluble NKp46 (see, e.g., Mandelboim etal., Nature 409:1055-1060, 2001; Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018; doi: 10.3389/f1mmu.2018.01150), a soluble DNAM-1 (see, e.g., those described in Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018; doi: 10.3389/f1mmu.2018.01150), a scMHCI (see, e.g., those described in Washburn et al., PLoS One 6(3):el8439, 2011), a scMHCII (see, e.g., those described in Bishwajit et al., Cellular Immunol. 170(1):25-33, 1996), a scTCR (see, e.g., those described in Weber et al., Nature 356(6372):793-796, 1992), a soluble CD155 (see, e.g., those described in Tahara-Hanaoka et al., Int. Immunol. 16(4):533-538, 2004), or a soluble CD28 (see, e.g., Hebbar et al., Clin. Exp. Immunol. 136:388-392, 2004).

Additional examples of soluble interleukin receptors and soluble cytokine receptors are known in the art.

Additional Antigen-Binding Domains

In some embodiments of any of the multi-chain chimeric polypeptides, the first chimeric polypeptide further includes one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding domain(s) (e.g., any of the exemplary targetbinding domains described herein or known in the art), where at least one of the one or more additional antigen-binding domain(s) is positioned between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein). In some embodiments, the first chimeric polypeptide can further include a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the at least one of the one or more additional targetbinding domain(s) (e.g., any of the exemplary target-binding domains described herein or known in the art), and/or a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the at least one of the one or more additional target-binding domain(s) (e.g., any of the exemplary target-binding domains described herein or known in the art) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein).

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further includes one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding domains at the N-terminal and/or C-terminal end of the first chimeric polypeptide. In some embodiments, at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) directly abuts the first domain of the pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein). In some embodiments, the at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) directly abuts the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) and the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art).

In some embodiments of any of the multi-chain chimeric polypeptides described herein, at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) is disposed at the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) is positioned between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the at least one additional target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) of the one or more additional target-binding domains disposed at the N-terminus directly abuts the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) or the first domain of the pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the linker sequences described herein or known in the art) disposed between the at least one additional target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) and the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) or the first domain of the pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the at least one additional target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) of the one or more additional target-binding domains disposed at the C-terminus directly abuts the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) or the first domain of the pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) disposed between the at least one additional target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) and the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) or the first domain of the pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) positioned between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the first domain of the pair of affinity domains (e.g., any of the first domains described herein or any of the exemplary pairs of affinity domains described herein), directly abuts the soluble tissue factor domain and/or the first domain of the pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) disposed (i) between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) positioned between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein), and/or (ii) between the first domain of the pair of affinity domains and the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) at the Nterminal end and/or the C-terminal end of the second chimeric polypeptide. In some embodiments, at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) directly abuts the second domain of the pair of affinity domains (e.g., any of the exemplary second domains of any of the exemplary pairs of affinity domains described herein) in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional targetbinding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) and the second domain of the pair of affinity domains (e.g., any of the second domains described herein of any of the exemplary pairs of affinity domains described herein) in the second chimeric polypeptide. In some embodiments, at least one of the one or more additional target-binding domains (e.g., any of the exemplary targetbinding domains described herein or known in the art) directly abuts the second targetbinding domain (e.g., any of the target-binding domains described herein or known in the art) in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target-binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) and the second target-binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) in the second chimeric polypeptide.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. In some embodiments, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope. In some embodiments, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains include the same amino acid sequence. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same antigen. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same epitope. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each include the same amino acid sequence.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to different antigens. In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains is an antigenbinding domain. In some embodiments, the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains are each an antigen-binding domain (e.g., a scFv or a single-domain antibody).

Pairs of Affinity Domains

In some embodiments, a multi-chain chimeric polypeptide includes: 1) a first chimeric polypeptide that includes a first domain of a pair of affinity domains, and 2) a second chimeric polypeptide that includes a second domain of a pair of affinity domains such that the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains. In some embodiments, the pair of affinity domains is a sushi domain from an alpha chain of human IL-15 receptor (IL15Ra) and a soluble IL-15. A sushi domain, also known as a short consensus repeat or type 1 glycoprotein motif, is a common motif in protein-protein interaction. Sushi domains have been identified on a number of proteinbinding molecules, including complement components Clr, Cis, factor H, and C2m, as well as the nonimmunologic molecules factor XIII and 32-gly coprotein. Atypical Sushi domain has approximately 60 amino acid residues and contains four cysteines (Ranganathan, Pac. Symp Biocomput. 2000:155-67). The first cysteine can form a disulfide bond with the third cysteine, and the second cysteine can form a disulfide bridge with the fourth cysteine. In some embodiments in which one member of the pair of affinity domains is a soluble IL-15, the soluble IL 15 has a D8N or D8A amino acid substitution. In some embodiments in which one member of the pair of affinity domains is an alpha chain of human IL-15 receptor (IL15Ra), the human IL15Ra is a mature fulllength IL15Ra. In some embodiments, the pair of affinity domains is barnase and barnstar. In some embodiments, the pair of affinity domains is a PKA and an AKAP. In some embodiments, the pair of affinity domains is an adapter/docking tag module based on mutated RNase I fragments (Rossi, Proc Natl Acad Set USA. 103:6841-6846, 2006; Sharkey et al., Cancer Res. 68:5282-5290, 2008; Rossi et al., Trends Pharmacol Sci. 33:474-481, 2012) or SNARE modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25 (Deyev et al., Nat Biotechnol. 1486-1492, 2003).

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide includes a first domain of a pair of affinity domains and a second chimeric polypeptide of the multi-chain chimeric polypeptide includes a second domain of a pair of affinity domains, wherein the first domain of the pair of affinity domains and the second domain of the pair of affinity domains bind to each other with a dissociation equilibrium constant (Kd) of less than 1 x IO'7 M, less than 1 x IO'8 M, less than 1 x IO'9 M, less than 1 x 10'10M, less than 1 x 10'11 M, less than 1 x IO'12 M, or less than 1 x 10'13 M. In some embodiments, the first domain of the pair of affinity domains and the second domain of the pair of affinity domains bind to each other with a Kd of about 1 x IO’4 M to about 1 x IO'6 M, about 1 x 10'5 M to about 1 x IO'7 M, about 1 x IO'6 M to about 1 x IO'8

M, about 1 x IO'7 M to about 1 x IO'9 M, about 1 x IO'8 M to about 1 x 10'10 M, about 1 x IO'9 M to about 1 x 10'11 M, about 1 x 10'10 M to about 1 x IO'12 M, about 1 x 10'11 M to about 1 x 10'13 M, about 1 x IO'4 M to about 1 x 10'5 M, about 1 x 10'5 M to about 1x10' 6 M, about 1 x IO'6 M to about 1 x IO'7 M, about 1 x IO'7 M to about 1 x IO'8 M, about 1 x IO'8 M to about 1 x IO'9 M, about 1 x IO'9 M to about 1 x 10'10 M, about 1 x 10'10 M to about 1 x 10'11 M, about 1 x 10'11 M to about 1 x IO'12 M, or about 1 x IO'12 M to about 1 x 10'13 M (inclusive). Any of a variety of different methods known in the art can be used to determine the Kd value of the binding of the first domain of the pair of affinity domains and the second domain of the pair of affinity domains (e.g., an electrophoretic mobility shift assay, a filter binding assay, surface plasmon resonance, and a biomolecular binding kinetics assay, etc.).

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide includes a first domain of a pair of affinity domains and a second chimeric polypeptide of the multi-chain chimeric polypeptide includes a second domain of a pair of affinity domains, wherein the first domain of the pair of affinity domains, the second domain of the pair of affinity domains, or both is about 10 to 100 amino acids in length. For example, a first domain of a pair of affinity domains, a second domain of a pair of affinity domains, or both can be about 10 to 100 amino acids in length, about 15 to 100 amino acids in length, about 20 to 100 amino acids in length, about 25 to 100 amino acids in length, about 30 to 100 amino acids in length, about 35 to 100 amino acids in length, about 40 to 100 amino acids in length, about 45 to 100 amino acids in length, about 50 to 100 amino acids in length, about 55 to 100 amino acids in length, about 60 to 100 amino acids in length, about 65 to 100 amino acids in length, about 70 to 100 amino acids in length, about 75 to 100 amino acids in length, about 80 to 100 amino acids in length, about 85 to 100 amino acids in length, about 90 to 100 amino acids in length, about 95 to 100 amino acids in length, about 10 to 95 amino acids in length, about 10 to 90 amino acids in length, about 10 to 85 amino acids in length, about 10 to 80 amino acids in length, about 10 to 75 amino acids in length, about 10 to 70 amino acids in length, about 10 to 65 amino acids in length, about 10 to 60 amino acids in length, about 10 to 55 amino acids in length, about 10 to 50 amino acids in length, about 10 to 45 amino acids in length, about 10 to 40 amino acids in length, about 10 to 35 amino acids in length, about to 30 amino acids in length, about 10 to 25 amino acids in length, about 10 to 20 amino acids in length, about 10 to 15 amino acids in length, about 20 to 30 amino acids in length, about 30 to 40 amino acids in length, about 40 to 50 amino acids in length, about 50 to 60 amino acids in length, about 60 to 70 amino acids in length, about 70 to 80 amino acids in length, about 80 to 90 amino acids in length, about 90 to 100 amino acids in length, about 20 to 90 amino acids in length, about 30 to 80 amino acids in length, about 40 to 70 amino acids in length, about 50 to 60 amino acids in length, or any range in between. In some embodiments, a first domain of a pair of affinity domains, a second domain of a pair of affinity domains, or both is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length.

In some embodiments, any of the first and/or second domains of a pair of affinity domains disclosed herein can include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at its N-terminus and/or C-terminus, so long as the function of the first and/or second domains of a pair of affinity domains remains intact. For example, a sushi domain from an alpha chain of human IL-15 receptor (IL15Ra) can include one or more additional amino acids at the N-terminus and/or the C-terminus, while still retaining the ability to bind to a soluble IL-15. Additionally or alternatively, a soluble IL-15 can include one or more additional amino acids at the N-terminus and/or the C-terminus, while still retaining the ability to bind to a sushi domain from an alpha chain of human IL-15 receptor (IL15Ra).

Anon-limiting example of a sushi domain from an alpha chain of IL-15 receptor alpha (IL15Ra) can include a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAH WTTPSLKCIR (SEQ ID NO: 28). In some embodiments, a sushi domain from an alpha chain of IL15Ra can be encoded by a nucleic acid including ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAG CTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGA AGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGT GGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 29).

In some embodiments, a soluble IL-15 can include a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGD ASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINT S (SEQ ID NO: 14). In some embodiments, a soluble IL-15 can be encoded by a nucleic acid including the sequence of AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTC CATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAA GGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAG CGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATA ACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGA AGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTG TCCAGATGTTCATCAATACCTCC (SEQ ID NO: 30).

Signal Sequence

In some embodiments, a multi-chain chimeric polypeptide includes a first chimeric polypeptide that includes a signal sequence at its N-terminal end. In some embodiments, a multi-chain chimeric polypeptide includes a second chimeric polypeptide that includes a signal sequence at its N-terminal end. In some embodiments, both the first chimeric polypeptide of a multi-chain chimeric polypeptide and a second chimeric polypeptide of the multi-chain chimeric polypeptide include a signal sequence. As will be understood by those of ordinary skill in the art, a signal sequence is an amino acid sequence that is present at the N-terminus of a number of endogenously produced proteins that directs the protein to the secretory pathway (e.g., the protein is directed to reside in certain intracellular organelles, to reside in the cell membrane, or to be secreted from the cell). Signal sequences are heterogeneous and differ greatly in their primary amino acid sequences. However, signal sequences are typically 16 to 30 amino acids in length and include a hydrophilic, usually positively charged N-terminal region, a central hydrophobic domain, and a C-terminal region that contains the cleavage site for signal peptidase.

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both includes a signal sequence having an amino acid sequence MKWVTFISLLFLFSSAYS (SEQ ID NO: 31). In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both includes a signal sequence encoded by the nucleic acid sequence ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (SEQ ID NO: 32), ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC (SEQ ID NO: 33), or ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (SEQ ID NO: 34).

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both includes a signal sequence having an amino acid sequence MKCLLYLAFLFLGVNC (SEQ ID NO: 35). In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both includes a signal sequence having an amino acid sequence MGQIVTMFEALPHIIDEVINIVIIVLIIITSIKAVYNFATCGILALVSFLFLAGRSCG (SEQ ID NO: 36). In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both includes a signal sequence having an amino acid sequence MPNHQSGSPTGSSDLLLSGI<I<QRPHLALRRI<RRREMRI<INRI<VRRMNLAPII<EI< TAWQHLQALISEAEEVLKTSQTPQNSLTLFLALLSVLGPPVTG (SEQ ID NO: 37). In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both includes a signal sequence having an amino acid sequence MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS (SEQ ID NO: 38). Those of ordinary skill in the art will be aware of other appropriate signal sequences for use in a first chimeric polypeptide and/or a second chimeric polypeptide of multi-chain chimeric polypeptides described herein.

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both includes a signal sequence that is about 10 to 100 amino acids in length. For example, a signal sequence can be about 10 to 100 amino acids in length, about 15 to 100 amino acids in length, about 20 to 100 amino acids in length, about 25 to 100 amino acids in length, about 30 to 100 amino acids in length, about 35 to 100 amino acids in length, about 40 to 100 amino acids in length, about 45 to 100 amino acids in length, about 50 to 100 amino acids in length, about 55 to 100 amino acids in length, about 60 to 100 amino acids in length, about 65 to 100 amino acids in length, about 70 to 100 amino acids in length, about 75 to 100 amino acids in length, about 80 to 100 amino acids in length, about 85 to 100 amino acids in length, about 90 to 100 amino acids in length, about 95 to 100 amino acids in length, about 10 to 95 amino acids in length, about 10 to 90 amino acids in length, about 10 to 85 amino acids in length, about 10 to 80 amino acids in length, about 10 to 75 amino acids in length, about 10 to 70 amino acids in length, about 10 to 65 amino acids in length, about 10 to 60 amino acids in length, about 10 to 55 amino acids in length, about 10 to 50 amino acids in length, about 10 to 45 amino acids in length, about 10 to 40 amino acids in length, about 10 to 35 amino acids in length, about 10 to 30 amino acids in length, about 10 to 25 amino acids in length, about 10 to 20 amino acids in length, about 10 to 15 amino acids in length, about 20 to 30 amino acids in length, about 30 to 40 amino acids in length, about 40 to 50 amino acids in length, about 50 to 60 amino acids in length, about 60 to 70 amino acids in length, about 70 to 80 amino acids in length, about 80 to 90 amino acids in length, about 90 to 100 amino acids in length, about 20 to 90 amino acids in length, about 30 to 80 amino acids in length, about 40 to 70 amino acids in length, about 50 to 60 amino acids in length, or any range in between. In some embodiments, a signal sequence is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length.

In some embodiments, any of the signal sequences disclosed herein can include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at its N-terminus and/or C-terminus, so long as the function of the signal sequence remains intact. For example, a signal sequence having the amino acid sequence MKCLLYLAFLFLGVNC (SEQ ID NO: 35) can include one or more additional amino acids at the N-terminus or C-terminus, while still retaining the ability to direct a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both to the secretory pathway.

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both includes a signal sequence that directs the multi-chain chimeric polypeptide into the extracellular space. Such embodiments are useful in producing multi-chain chimeric polypeptides that are relatively easy to be isolated and/or purified.

Peptide Tags

In some embodiments, a multi-chain chimeric polypeptide includes a first chimeric polypeptide that includes a peptide tag (e.g., at the N-terminal end or the Cterminal end of the first chimeric polypeptide). In some embodiments, a multi-chain chimeric polypeptide includes a second chimeric polypeptide that includes a peptide tag (e.g., at the N-terminal end or the C-terminal end of the second chimeric polypeptide). In some embodiments, both the first chimeric polypeptide of a multi-chain chimeric polypeptide and a second chimeric polypeptide of the multi-chain chimeric polypeptide include a peptide tag. In some embodiments, a first chimeric polypeptide of a multichain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both include two or more peptide tags.

Exemplary peptide tags that can be included in a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both include, without limitation, AviTag (GLNDIFEAQKIEWHE; SEQ ID NO: 39), a calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 40), a polyglutamate tag (EEEEEE; SEQ ID NO: 41), an E-tag (GAPVPYPDPLEPR; SEQ ID NO: 42), a FLAGtag (DYKDDDDK; SEQ ID NO: 43), an HA-tag, a peptide from hemagglutinin (YPYDVPDYA; SEQ ID NO: 44), a his-tag (HHHHH (SEQ ID NO: 45); HHHHHH (SEQ ID NO: 46); HHHHHHH (SEQ ID NO: 47); HHHHHHHH (SEQ ID NO: 48);

HHHHHHHHH (SEQ ID NO: 49); or HHHHHHHHHH (SEQ ID NO: 50)), a myc-tag (EQKLISEEDL; SEQ ID NO: 51), NE־tag (TKENPRSNQEESYDDNES; SEQ ID NO: 52), S-tag, (KETAAAKFERQHMDS; SEQ ID NO: 53), SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP; SEQ ID NO: 54), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 55), Softag 3 (TQDPSRVG; SEQ ID NO: 56), Spot-tag (PDRVRAVSHWSS; SEQ ID NO: 57), Strep-tag (WSHPQFEK; SEQ ID NO: 58), TC tag (CCPGCC; SEQ ID NO: 59), Ty tag (EVHTNQDPLD; SEQ ID NO: 60), V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 61), VSV-tag (YTDIEMNRLGK; SEQ ID NO: 62), and Xpress tag (DLYDDDDK; SEQ ID NO: 63). In some embodiments, tissue factor protein is a peptide tag.

Peptide tags that can be included in a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both can be used in any of a variety of applications related to the multichain chimeric polypeptide. For example, a peptide tag can be used in the purification of a multi-chain chimeric polypeptide. As one non-limiting example, a first chimeric polypeptide of a multi-chain chimeric polypeptide (e.g., a recombinantly expressed first chimeric polypeptide), a second chimeric polypeptide of the multi-chain chimeric polypeptide (e.g., a recombinantly expressed second chimeric polypeptide), or both can include a myc tag; the multi-chain chimeric polypeptide that includes the myc-tagged first chimeric polypeptide, the myc-tagged second chimeric polypeptide, or both can be purified using an antibody that recognizes the myc tag(s). One non-limiting example of an antibody that recognizes a myc tag is 9E10, available from the non-commercial Developmental Studies Hybridoma Bank. As another non-limiting example, a first chimeric polypeptide of a multi-chain chimeric polypeptide (e.g., a recombinantly expressed first chimeric polypeptide), a second chimeric polypeptide of the multi-chain chimeric polypeptide (e.g., a recombinantly expressed second chimeric polypeptide), or both can include a histidine tag; the multi-chain chimeric polypeptide that includes the histidine-tagged first chimeric polypeptide, the histidine-tagged second chimeric polypeptide, or both can be purified using a nickel or cobalt chelate. Those of ordinary skill in the art will be aware of other suitable tags and agent that bind those tags for use in purifying multi-chain chimeric polypeptide. In some embodiments, a peptide tag is removed from the first chimeric polypeptide and/or the second chimeric polypeptide of the multi-chain chimeric polypeptide after purification. In some embodiments, a peptide tag is not removed from the first chimeric polypeptide and/or the second chimeric polypeptide of the multi-chain chimeric polypeptide after purification.

Peptide tags that can be included in a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both can be used, for example, in immunoprecipitation of the multi-chain chimeric polypeptide, imaging of the multi-chain chimeric polypeptide (e.g., via Western blotting, ELISA, flow cytometry, and/or immunocytochemistry), and/or solubilization of the multi-chain chimeric polypeptide.

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both includes a peptide tag that is about 10 to 100 amino acids in length. For example, a peptide tag can be about 10 to 100 amino acids in length, about 15 to 100 amino acids in length, about 20 to 100 amino acids in length, about 25 to 100 amino acids in length, about 30 to 100 amino acids in length, about 35 to 100 amino acids in length, about 40 to 100 amino acids in length, about 45 to 100 amino acids in length, about 50 to 100 amino acids in length, about 55 to 100 amino acids in length, about 60 to 100 amino acids in length, about 65 to 100 amino acids in length, about 70 to 100 amino acids in length, about 75 to 100 amino acids in length, about 80 to 100 amino acids in length, about 85 to 100 amino acids in length, about 90 to 100 amino acids in length, about 95 to 100 amino acids in length, about 10 to 95 amino acids in length, about 10 to 90 amino acids in length, about 10 to 85 amino acids in length, about 10 to 80 amino acids in length, about 10 to 75 amino acids in length, about 10 to 70 amino acids in length, about 10 to 65 amino acids in length, about 10 to 60 amino acids in length, about 10 to 55 amino acids in length, about 10 to 50 amino acids in length, about 10 to 45 amino acids in length, about 10 to 40 amino acids in length, about 10 to 35 amino acids in length, about 10 to 30 amino acids in length, about 10 to 25 amino acids in length, about 10 to 20 amino acids in length, about 10 to 15 amino acids in length, about 20 to 30 amino acids in length, about 30 to 40 amino acids in length, about 40 to 50 amino acids in length, about 50 to 60 amino acids in length, about 60 to 70 amino acids in length, about 70 to 80 amino acids in length, about 80 to 90 amino acids in length, about 90 to 100 amino acids in length, about 20 to 90 amino acids in length, about 30 to 80 amino acids in length, about 40 to 70 amino acids in length, about 50 to 60 amino acids in length, or any range in between. In some embodiments, a peptide tag is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length.

Peptide tags included in a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both can be of any suitable length. For example, peptide tags can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids in length. In embodiments in which a multi-chain chimeric polypeptide includes two or more peptide tags, the two or more peptide tags can be of the same or different lengths. In some embodiments, any of the peptide tags disclosed herein may include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at the N-terminus and/or C-terminus, so long as the function of the peptide tag remains intact. For example, a myc tag having the amino acid sequence EQKLISEEDL (SEQ ID NO: 64) can include one or more additional amino acids (e.g., at the N-terminus and/or the C- terminus of the peptide tag), while still retaining the ability to be bound by an antibody (e.g., 9E10).

Exemplary Multi-Chain Chimeric Polypeptides- Type A

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each independently bind specifically to a receptor of IL-18 or a receptor of IL-12. In some examples of these multi-chain chimeric polypeptides, the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target-binding domain and the second target-binding domain is an agonistic antigen-binding domain. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain are each agonistic antigen-binding domains. In some embodiments of these multi-chain chimeric polypeptides, the antigen-binding domain includes a scFv or single-domain antibody.

In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target-binding domain and the second target-binding domain is a soluble IL-15 or a soluble IL-18. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain are each independently a soluble IL-15 or a soluble IL-18. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain both bind specifically to a receptor of IL-18 or a receptor of IL-12. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain include the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain binds specifically to a receptor for IL-12, and the second target-binding domain binds specifically to a receptor for IL-18. In some embodiments of these multichain chimeric polypeptides, the first target-binding domain binds specifically to a receptor for IL-18, and the second target-binding domain bind specifically to a receptor for IL-12.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain includes a soluble IL-18 (e.g., a soluble human IL-18).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-18 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQ PRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKM QFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 16).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-18 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACC AAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGAC TCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAA GGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAA AATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACC CCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCC GTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCT ACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAA GGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT (SEQ ID NO: 65).

In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain includes a soluble IL-12 (e.g., a soluble human IL-12). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-15 includes a sequence of soluble human ΙΕ-12β (p40) and a sequence of soluble human IL12a (p35). In some embodiments of these multi-chain chimeric polypeptides, the soluble IL-15 human IL-15 further includes a linker sequence (e.g., any of the exemplary linker sequences described herein) between the sequence of soluble ΙΕ-12β (p40) and the sequence of soluble human IL-12a (p35). In some examples of these multi-chain chimeric polypeptides, the linker sequence comprises GGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, the sequence of soluble human ΙΕ-12β (p40) comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLT IQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLR CEAKNYSGRFTCWWLTTISTDLTF S VKS SRGS SDPQGVTCGAATLS AERVRGDN KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKN LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKT SATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 66).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human ΙΕ-12β (p40) is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCG ATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGG CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACC CTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACA AGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGA CGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAG ACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTG GCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGA AGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGA GGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAG ATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGAC GCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGA CATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATA GCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCA CAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGG AGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCG GAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGC TGGTCCGAGTGGGCCAGCGTGCCTTGTTCC (SEQ ID NO: 67).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-12a (p35) includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKD KTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKM YQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPD FYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 68).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-12a (p35) is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCA CAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAG ACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCA CCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAA GAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTT GTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCT ACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCT GCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTG ATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGA AGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCAT TTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCT ATTTAAACGCCAGC (SEQ ID NO: 69).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQ PRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKM QFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDSGTTNTVAAYN LTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKD VKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGT

KVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEF LIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLK KIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLI ILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 70).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACC AAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGAC TCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAA GGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAA AATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACC CCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCC GTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCT ACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAA GGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCA ACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACAC CGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACC ACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGA CCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGG TTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCG AGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAA GGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACAC CTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATT ACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGA GTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCT GTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGT GCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCA GCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCAC TTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAAT GTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATC CACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAA CGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAA GAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCA ATACCTCC (SEQ ID NO: 71).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD CRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKD TKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIM FTVQNEDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKS KCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP YLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYY WKS S S SGKKTAKTNTNEFLID VDKGENYCF S VQ AVIP SRTVNRK STD SP VECMG QEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLEL QVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV HIVQMFINTS (SEQ ID NO: 72).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACC AAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGAC TCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAA GGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAA AATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACC CCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCC GTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCT ACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAA GGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCA ACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACAC CGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACC ACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGA CCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGG TTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCG AGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAA GGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACAC CTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATT

ACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGA GTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCT GTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGT GCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCA GCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCAC TTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAAT GTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATC CACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAA CGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAA GAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCA ATACCTCC (SEQ ID NO: 73).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLT IQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLR CEAKNYSGRFTCWWLTTISTDLTF S VKS SRGS SDPQGVTCGAATLS AERVRGDN KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKN LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKT SATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPV ATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTV EACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEF KTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTK IKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHADIWVKSYSLYSRERYICN SGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 74).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCG ATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGG CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACC CTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACA AGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGA CGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAG

ACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTG GCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGA AGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGA GGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAG ATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGAC GCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGA CATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATA GCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCA CAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGG AGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCG GAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGC TGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAG GTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATCC CGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGC AACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCG AGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGG CTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAA ACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTAT GATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGG AGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTT TTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACT TCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTAC AAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCG TGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGCATTACATGCCCCCCT CCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACA GCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCA GCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC AACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 75).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKW VTFISLLFLF S S AYSIWELKKD VYVVELDW YPD APGEMVVLTCDTPEEDGI TWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIW STDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTF S VKS SRGS SDPQG VTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYE NYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQV QGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGG GSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYP CTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMM

ALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNS ETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHA DIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 76).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCG ATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGG CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACC CTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACA AGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGA CGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAG ACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTG GCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGA AGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGA GGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAG ATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGAC GCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGA CATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATA GCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCA CAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGG AGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCG GAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGC TGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAG GTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATCC CGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGC AACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCG AGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGG CTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAA ACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTAT GATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGG AGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTT TTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACT TCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTAC AAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCG TGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGCATTACATGCCCCCCT CCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACA

GCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCA GCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC AACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 77).

Exemplary Multi-Chain Chimeric Polypeptides- Type B

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each independently bind specifically to a receptor of IL-21 or to TGF-β. In some examples of these multi-chain chimeric polypeptides, the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target-binding domain and the second target-binding domain is a soluble IL-21 (e.g., a soluble human IL-21 polypeptide) or a soluble TGF-β receptor (e.g., a soluble ΤΟΡΚβΚΙΙ receptor). In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain are each independently a soluble IL-21 or a soluble TGF-β receptor (e.g., a soluble TGFR^RII receptor). In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain both bind specifically to a receptor of IL-21 or to TGF-β. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain include the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain binds specifically to a receptor for IL-21, and the second target-binding domain binds specifically to TGF-β. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain binds specifically to TGF-β, and the second target-binding domain bind specifically to a receptor for IL-21.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain includes a soluble IL-21 (e.g., a soluble human IL-21). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 78).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 79).

In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain includes a soluble TGF-β receptor (e.g., a soluble ΤΟΡΚβΚΙΙ receptor (e.g., a soluble human ΤΟΡΚβΚΙΙ receptor)). In some embodiments of these multi-chain chimeric polypeptides, the soluble human ΤΟΡΚβΚΙΙ includes a first sequence of soluble human ΤΟΡΚβΚΙΙ and a second sequence of soluble human TGFRJ3RII. In some embodiments of these multi-chain chimeric polypeptides, the soluble human ΤΟΡΚβΚΙΙ includes a linker disposed between the first sequence of soluble human ΤΟΡΚβΚΙΙ and the second sequence of soluble human ΤΟΡΚβΚΙΙ. In some examples of these multi-chain chimeric polypeptides, the linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human ΤΟΡΚβΚΙΙ receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 80).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human ΤΟΡΚβΚΙΙ receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 81).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGFRpRII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 82).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGFRpRII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCA TCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGA GAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGAT TTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGA AGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGA CAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 83).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRpRII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC CAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 84).

In some embodiments of these multi-chain chimeric polypeptides, the human

TGFpRII receptor includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPD (SEQ ID NO: 85).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER

FKSLLQKMIHQHLSSRTHGSEDSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQ VYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVEST GSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLS LRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRT VNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHP SCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECE ELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 86).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCTCCGGCACCACCAAT ACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTC TGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCAC CAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGT GATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGG TCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCT CTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCA GCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTC GAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGG ATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCC TCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGG ACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTAC CGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAA GGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATC GAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGA CGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGC AAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAA TTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGT CCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTC TGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 87).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED VETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLT CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSSGTTNTVAAYNLTW KSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQ TYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVN VTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDV DKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIED LIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAN NSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 88).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC CCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTC GACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC CCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAA GGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGT GAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAG GCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCC CCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATC AGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCTCCGGCACCACCAA TACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATT CTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCA CCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGT GTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCG GGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGC CTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGC CAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCG TCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCG GGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCT CCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGT GGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGT

ACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAA AGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGAT CGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCG ACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTG CAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGA ATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAG TCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTT CTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 89).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLT ECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 90).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC CAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTC

AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGACATCACGTGTCCTCCTCCTATGTCC GTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGC GGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGAC GGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGT CTCAAATGTATTAGA (SEQ ID NO: 91).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLF S S AYSIPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCDVRF ST CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRER YICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 92).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC CATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGAC AACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT CAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCC ATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGAC GAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACG ACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAA GAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAAC GACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTG GCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGT GCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTG AAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAA CCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCT

CAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGG AAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGA CGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGAC CTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTA GCGAGGAATACAATACCAGCAACCCCGACATCACGTGTCCTCCTCCTATGTC CGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAG CGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGAC GGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGT CTCAAATGTATTAGA (SEQ ID NO: 93).

Exemplary Multi-Chain Chimeric Polypeptides- Type C

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each independently bind specifically to a receptor of IL-7 or a receptor of IL-21. In some examples of these multi-chain chimeric polypeptides, the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target-binding domain and the second target-binding domain is a soluble IL-21 (e.g., a soluble human IL-21 polypeptide) or a soluble IL-7 (e.g., a soluble human IL-7 polypeptide). In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain are each independently a soluble IL-21 or a soluble IL-7. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain both bind specifically to a receptor of IL-21 or a receptor of IL-7. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain include the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain binds specifically to a receptor for IL-21, and the second target-binding domain binds specifically to a receptor for IL-7. In some embodiments of these multichain chimeric polypeptides, the first target-binding domain binds specifically to a receptor for IL-7, and the second target-binding domain binds specifically to a receptor for IL-21.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain includes a soluble IL-21 (e.g., a soluble human IL-21).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 78).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTG ATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCA GAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGG CCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATC AATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACA GAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCC AAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGC ATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC (SEQ ID NO: 94).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 79).

In some embodiments of these multi-chain chimeric polypeptides, the sequence of soluble human IL-7 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 11).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGG TCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCT GAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAA GGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAA TAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAA TACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGG TGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACA GAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAA ACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACAC (SEQ ID NO: 95).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQ VYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVEST GSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLS LRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRT VNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHP SCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECE ELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 96).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTG ATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCA GAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGG

CCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATC AATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACA GAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCC AAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGC ATCTGTCCTCTAGAACACACGGAAGTGAAGATTCCTCAGGCACTACAAATAC TGTGGCAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTGG AGTGGGAACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAA GTCAGGAGATTGGAAAAGCAAATGCTTTTACACAACAGACACAGAGTGTGAC CTCACCGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCT TCTCCTACCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCCTCT GTATGAGAACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAG CCAACAATTCAGAGTTTTGAACAGGTGGGAACAAAAGTGAATGTGACCGTAG AAGATGAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGA TGTTTTTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTC AGGAAAGAAAACAGCCAAAACAAACACTAATGAGTTTTTGATTGATGTGGAT AAAGGAGAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAG TTAACCGGAAGAGTACAGACAGCCCGGTAGAGTGTATGGGCCAGGAGAAAG GGGAATTCAGAGAAAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCG AAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGAC GTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCA AGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAAT TTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTC CGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCT GCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 97).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MGVKVLFALICIAVAEAQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED VETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLT CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSSGTTNTVAAYNLTW KSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQ TYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVN VTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDV DKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIED LIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAN NSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 98).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCCCA AGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATC AGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAA GATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCA ACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATT AAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAA CACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAG AATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTG TCCTCTAGAACACACGGAAGTGAAGATTCCTCAGGCACTACAAATACTGTGG CAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTGGAGTGG GAACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAG GAGATTGGAAAAGCAAATGCTTTTACACAACAGACACAGAGTGTGACCTCAC CGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCTTCTCC TACCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCCTCTGTATG AGAACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAGCCAAC AATTCAGAGTTTTGAACAGGTGGGAACAAAAGTGAATGTGACCGTAGAAGAT GAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGATGTTT TTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTCAGGA AAGAAAACAGCCAAAACAAACACTAATGAGTTTTTGATTGATGTGGATAAAG GAGAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTAAC CGGAAGAGTACAGACAGCCCGGTAGAGTGTATGGGCCAGGAGAAAGGGGAA TTCAGAGAAAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATT TAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCAC CCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTAT CTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATC ATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCT GCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAAT CCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 99)

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHITCPPPMSVEH ADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 100)

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGG TCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCT GAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAA GGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAA TAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAA TACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGG TGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACA GAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAA ACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACACATCACGTGCCCTC CCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTA CTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACG TCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGA CAACCCCCAGTCTCAAATGCATTAGA (SEQ ID NO: 101).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: MGVKVLFALICIAVAEADCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNN EFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNC TGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL MGTKEHITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNK ATNVAHWTTPSLKCIR (SEQ ID NO: 102).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCCGA TTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTC AGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGA ATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGT ATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAG CACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATAC TGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGA AGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAA AAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACT TGTTGGAATAAAATTTTGATGGGCACTAAAGAACACATCACGTGCCCTCCCC CCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTC CAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCC AGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAA CCCCCAGTCTCAAATGCATTAGA (SEQ ID NO: 103).

Exemplary Multi-Chain Chimeric Polypeptides- Type D

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each independently bind specifically to a receptor of IL-7 or a receptor of IL-21. In some examples of these multi-chain chimeric polypeptides, the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target-binding domain and the second target-binding domain is a soluble IL-21 (e.g., a soluble human IL-21 polypeptide) or a soluble IL-7 (e.g., a soluble human IL-7 polypeptide). In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain are each independently a soluble IL-21 or a soluble IL-7. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain both bind specifically to a receptor of IL-21 or a receptor of IL-7. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain include the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain binds specifically to a receptor for IL-21, and the second target-binding domain binds specifically to a receptor for IL-7. In some embodiments of these multichain chimeric polypeptides, the first target-binding domain binds specifically to a receptor for IL-7, and the second target-binding domain binds specifically to a receptor for IL-21.

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 78).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTG ATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCA GAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGG CCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATC AATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACA GAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCC AAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGC ATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC (SEQ ID NO: 94).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 79).

In some embodiments of these multi-chain chimeric polypeptides, the sequence of soluble human IL-7 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least

90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 11).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGG TCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCT GAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAA GGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAA TAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAA TACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGG TGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACA GAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAA ACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACAC (SEQ ID NO: 95).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAY NLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVK DVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVG TKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNE FLIDVDKGENYCF S VQ AVIPSRTVNRKSTD SPVECMGQEKGEFRENWVNVISDL KKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVEN LIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 104).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATC AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACA ACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAA CCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGAT CAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACC GAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCG CCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGG CGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATT TAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGT GACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGC CTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTC CTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATC GACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCT CCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCA AGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAA GAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACA GAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACT GGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACC GTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGT GACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAA GGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 105).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNN EFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNC TGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL MGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKS KCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP YLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYY WKS S S SGKKTAKTNTNEFLIDVDKGENYCF S VQ AVIPSRTVNRKSTDSPVECMG QEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLEL QVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV HIVQMFINTS (SEQ ID NO: 106).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC CGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGAT GGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGC CTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGG AGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGAT GAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACC ACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTC TGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGG AGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGAT CAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCAC AACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAA ACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGA TCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACAC CGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTC GCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTG GCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAA TTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAAT GTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCA GCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAG TCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAA TCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCC CTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGC CAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTA AAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACA CAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTA CTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACA

CCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAAC GTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATC AAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTC C (SEQ ID NO: 107).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGF KRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 108).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTC CCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAG CCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAG CAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACA ACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 109).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED VETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLT CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIW VKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 110).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC CCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTC GACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC CCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAA GGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGT GAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAG GCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCC CCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATC AGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCT CCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACA GCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCA GCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC AACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 111).

Exemplary Multi-Chain Chimeric Polypeptides- Type E

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each independently bind specifically to a receptor for IL-18 (e.g., a soluble human IL-18), a receptor for IL-12 (e.g., a soluble human IL-12), or CD16 (e.g., an anti-CD16 scFv). In some embodiments of these multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target-binding domain binds specifically to CD16 or a receptor for IL-12.

In some examples of these multi-chain chimeric polypeptides, the first targetbinding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second domain of the pair of affinity domains directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second target-binding domain and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or more of the first target-binding domain, the second target-binding domain and the additional antigen-binding domain is an agonistic antigen-binding domain. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain, the second target-binding domain, and the additional antigen-binding domain are each agonistic antigen-binding domains. In some embodiments of these multi-chain chimeric polypeptides, the antigen-binding domain includes a scFv or single-domain antibody.

In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target-binding domain and the second target-binding domain is a soluble IL-15 or a soluble IL-18. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain are each independently a soluble IL-15 or a soluble IL-18. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain both bind specifically to a receptor of IL-18 or a receptor of IL-12. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain include the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain binds specifically to a receptor for IL-12, and the second target-binding domain binds specifically to a receptor for IL-18. In some embodiments of these multi chain chimeric polypeptides, the first target-binding domain binds specifically to a receptor for IL-18, and the second target-binding domain bind specifically to a receptor for IL-12. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain binds specifically to CD 16, and the second target-binding domain binds specifically to a receptor for IL-18. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain binds specifically to a receptor for IL-18, and the second target-binding domain bind specifically to CD 16.

In some embodiments of these multi-chain chimeric polypeptides, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain includes a soluble IL-18 (e.g., a soluble human IL-18).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-18 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQ PRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKM QFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 16).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-18 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACC AAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGAC

TCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAA GGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAA AATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACC CCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCC GTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCT ACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAA GGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT (SEQ ID NO: 65).

In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain includes a soluble IL-12 (e.g., a soluble human IL-12). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-15 includes a sequence of soluble human ΙΕ-12β (p40) and a sequence of soluble human IL12a (p35). In some embodiments of these multi-chain chimeric polypeptides, the soluble IL-15 (e.g., soluble human IL-15) further includes a linker sequence (e.g., any of the exemplary linker sequences described herein) between the sequence of soluble ΙΕ-12β (p40) and the sequence of soluble human IL-12a (p35). In some examples of these multichain chimeric polypeptides, the linker sequence comprises GGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, the sequence of soluble human ΙΕ-12β (p40) comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLT IQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLR CEAKNYSGRFTCWWLTTISTDLTF S VKS SRGS SDPQGVTCGAATLS AERVRGDN KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKN LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKT SATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 66).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human ΙΕ-12β (p40) is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCG ATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGG CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACC CTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACA AGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGA CGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAG ACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTG GCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGA AGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGA GGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAG ATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGAC GCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGA CATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATA GCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCA CAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGG AGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCG GAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGC TGGTCCGAGTGGGCCAGCGTGCCTTGTTCC (SEQ ID NO: 67).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-12a (p35) includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKD KTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKM YQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPD FYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 68).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-12a (p35) is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCA CAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAG ACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCA CCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAA GAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTT GTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCT

ACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCT GCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTG ATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGA AGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCAT TTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCT ATTTAAACGCCAGC (SEQ ID NO: 69).

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain includes an scFv that specifically binds to CD 16 (e.g., an antiCD 16 scFv).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 includes a light chain variable domain that includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGH (SEQ ID NO: 112).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 is encoded by a light chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGA GGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA GCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGG CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA CCGTGGGCCAT (SEQ ID NO: 113).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 includes a heavy chain variable domain that includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINW NGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDY WGQGTLVTVSR (SEQ ID NO: 114).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 is encoded by a heavy chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCC CTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTC CTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAAC TGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCA TCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAG GGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTC GACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG (SEQ ID NO: 115).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQ PRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKM QFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDSGTTNTVAAYN LTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKD VKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGT KVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEF LIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLK KIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLI ILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 116).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACC AAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGAC TCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAA GGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAA AATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACC CCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCC GTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCT ACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAA GGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCA ACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACAC CGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACC ACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGA CCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGG TTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCG AGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAA GGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACAC CTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATT ACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGA GTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCT GTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGT GCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCA GCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCAC TTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAAT GTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATC CACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAA CGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAA GAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCA ATACCTCC (SEQ ID NO: 117).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD CRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKD

TKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIM FTVQNEDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKS KCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP YLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYY WKS S S SGKKTAKTNTNEFLIDVDKGENYCF S VQ AVIPSRTVNRKSTDSPVECMG QEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLEL QVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV HIVQMFINTS (SEQ ID NO: 118).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACC AAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGAC TCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAA GGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAA AATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACC CCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCC GTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCT ACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAA GGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCA ACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACAC CGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACC ACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGA CCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGG TTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCG AGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAA GGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACAC CTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATT ACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGA GTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCT GTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGT GCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCA GCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCAC TTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAAT GTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATC CACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAA CGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAA

GAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCA ATACCTCC (SEQ ID NO: 119).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLT IQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLR CEAKNYSGRFTCWWLTTISTDLTF S VKS SRGS SDPQGVTCGAATLS AERVRGDN KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKN LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKT SATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPV ATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTV EACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEF KTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTK IKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHADIWVKSYSLYSRERYICN SGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSELTQDPAVSVALGQTVRIT CQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITG AQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQL VESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGG STGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQ GTLVTVSR (SEQ ID NO: 120).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCG ATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGG CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACC CTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACA AGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGA CGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAG ACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTG GCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGA AGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGA GGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAG

ATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGAC GCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGA CATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATA GCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCA CAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGG AGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCG GAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGC TGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAG GTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATCC CGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGC AACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCG AGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGG CTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAA ACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTAT GATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGG AGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTT TTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACT TCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTAC AAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCG TGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGCATTACATGCCCCCCT CCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACA GCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCA GCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC AACACCCTCTTTAAAGTGCATCCGGTCCGAGCTGACCCAGGACCCTGCTGTGT CCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAG GTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTG GTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCG GATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGA GGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTG GTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCG GAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCG GAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTC CGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGA AAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCT ACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGA ACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTA CTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACC CTGGTGACCGTGTCCAGG (SEQ ID NO: 121).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKW VTFISLLFLF S S AYSIWELKKD VYVVELDW YPD APGEMVVLTCDTPEEDGI TWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIW STDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTF S VKS SRGS SDPQG VTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYE NYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQV QGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGG GSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYP CTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMM ALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNS ETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHA DIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSE LTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGI PDRF SGS S SGNTASLTITGAQ AEDEAD YYCNSRDS SGNHVVFGGGTKLT VGHGG GGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQA PGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV YYCARGRSLLFDYWGQGTLVTVSR (SEQ ID NO: 122).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCG ATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGG CATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACC CTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACA AGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGA CGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAG ACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTG GCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGA AGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGA GGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAG ATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGAC GCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGA CATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATA GCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCA CAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGG AGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCG GAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGC

TGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAG GTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATCC CGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGC AACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCG AGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGG CTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAA ACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTAT GATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGG AGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTT TTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACT TCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTAC AAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCG TGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGCATTACATGCCCCCCT CCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACA GCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCA GCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC AACACCCTCTTTAAAGTGCATCCGGTCCGAGCTGACCCAGGACCCTGCTGTGT CCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAG GTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTG GTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCG GATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGA GGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTG GTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCG GAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCG GAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTC CGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGA AAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCT ACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGA ACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTA CTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACC CTGGTGACCGTGTCCAGG (SEQ ID NO: 123).

Exemplary Multi-Chain Chimeric Polypeptides- Type F

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each independently bind specifically to a receptor for IL-7 (e.g., a soluble human IL-7), CD 16 (e.g., an anti-CD16 scFv), or a receptor for IL-21 (e.g., a soluble human IL-21). In some embodiments of these multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target-binding domain binds specifically to CD 16 or a receptor for IL-21.

In some examples of these multi-chain chimeric polypeptides, the first targetbinding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second domain of the pair of affinity domains directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second target-binding domain and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or more of the first target-binding domain, the second target-binding domain and the additional antigen-binding domain is an agonistic antigen-binding domain. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain, the second target-binding domain, and the additional antigen-binding domain are each agonistic antigen-binding domains. In some embodiments of these multi-chain chimeric polypeptides, the antigen-binding domain includes a scFv or single-domain antibody.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain binds specifically to a receptor IL-7 and the second target-binding domain binds specifically to CD 16 or a receptor for IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain includes a soluble IL-7 protein. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble IL-7 protein is a soluble human IL-7. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second antigen-binding domain includes a targetbinding domain that binds specifically to CD 16. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target-binding domain includes an scFv that binds specifically to CD 16. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target-binding domain binds specifically to a receptor for IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target-binding domain includes a soluble IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble IL-21 is a soluble human IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes an additional target-binding domain that binds specifically to a receptor for IL-2L In some embodiments of any of the multi-chain chimeric polypeptides described herein, the additional target-binding domain includes a soluble IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble IL-21 is a soluble human IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes an additional target-binding domain that binds specifically to CD 16.

In some embodiments of these multi-chain chimeric polypeptides, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain includes a soluble IL-7 (e.g., a soluble human IL-7).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 11).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGG TCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCT GAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAA GGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAA TAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAA TACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGG TGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACA GAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAA ACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACAC (SEQ ID NO: 95).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATC AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (SEQ ID NO: 124).

In some embodiments of these multi-chain chimeric polypeptides, the sequence of soluble human IL-21 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 78).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTG ATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCA GAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGG CCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATC AATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACA GAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCC AAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGC ATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC (SEQ ID NO: 94).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 79)

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain includes an scFv that specifically binds to CD 16 (e.g., an antiCD 16 scFv).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 includes a light chain variable domain that includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGH (SEQ ID NO: 112).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 is encoded by a light chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGA GGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA GCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGG CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA CCGTGGGCCAT (SEQ ID NO: 113).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 includes a heavy chain variable domain that includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINW NGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDY WGQGTLVTVSR (SEQ ID NO: 114).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 is encoded by a heavy chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCC CTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTC CTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAAC TGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCA TCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAG GGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTC GACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG (SEQ ID NO: 115).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAY NLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVK DVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVG TKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNE FLIDVDKGENYCF S VQ AVIPSRTVNRKSTD SPVECMGQEKGEFRENWVNVISDL KKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVEN LIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 125).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATC AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACA ACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAA CCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGAT

CAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACC GAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCG CCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGG CGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATT TAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGT GACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGC CTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTC CTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATC GACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCT CCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCA AGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAA GAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACA GAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACT GGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACC GTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGT GACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAA GGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 126).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNN EFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNC TGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL MGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKS KCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP YLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYY WKS S S SGKKTAKTNTNEFLIDVDKGENYCF S VQ AVIPSRTVNRKSTDSPVECMG QEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLEL QVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV HIVQMFINTS (SEQ ID NO: 127).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC CGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGAT GGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGC CTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGG AGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGAT GAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACC ACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTC TGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGG AGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGAT CAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCAC AACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAA ACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGA TCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACAC CGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTC GCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTG GCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAA TTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAAT GTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCA GCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAG TCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAA TCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCC CTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGC CAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTA AAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACA CAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTA CTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACA CCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAAC GTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATC AAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTC C (SEQ ID NO: 128).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHG GGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQ APGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTA VYYCARGRSLLFDYWGQGTLVTVSRITCPPPMSVEHADIWVKSYSLYSRERYIC NSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVD

QLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKK LKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTH GSEDS (SEQ ID NO: 129).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGA GGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA GCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGG CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA CCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAG GATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAG GCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGC ATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCA TCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTT CACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCC CTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGC TGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGGATTACATG CCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGC CTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCG GCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCA CTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACATG ATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGA ACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTG CGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAAC ACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGG AAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGC CCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGT TCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCA CGGCTCCGAGGACTCC (SEQ ID NO: 130).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQ KPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSS GNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSC AASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNA KNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRITCPPPMSVEH ADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQ GQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQL KSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERF KSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 131).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC CTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTG AGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACC AGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAG GCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCT CCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAA CTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTG ACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGA GGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGA GGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGG CATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGC ATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGT TCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTC CCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTG CTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGGATTACAT GCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAG CCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCC GGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTC ACTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACAT GATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTG AACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACT GCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAA CACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCG GAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTG CCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGG TTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCC ACGGCTCCGAGGACTCC (SEQ ID NO: 132).

Exemplary Multi-Chain Chimeric Polypeptides- Type G

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each independently bind specifically to ΤΘΡβ (e.g., a human ΤΘΡβΚΙΙ receptor), CD 16 (e.g., an anti-CD16 scFv), or a receptor for IL-21 (e.g., a soluble human IL-21). In some embodiments of these multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target-binding domain binds specifically to CD 16 or a receptor for IL-21.

In some examples of these multi-chain chimeric polypeptides, the first targetbinding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second domain of the pair of affinity domains directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second target-binding domain and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or more of the first target-binding domain, the second target-binding domain and the additional antigen-binding domain is an agonistic antigen-binding domain. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain, the second target-binding domain, and the additional antigen-binding domain are each agonistic antigen-binding domains. In some embodiments of these multi-chain chimeric polypeptides, the antigen-binding domain includes a scFv or single-domain antibody.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each independently bind specifically to TGF-β, CD 16, or a receptor for IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain binds specifically to a TGF-β and the second target-binding domain binds specifically to CD 16 or a receptor of IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain is a soluble TGF-β receptor. In some embodiments of any of the multi-chain chimeric polypeptides described herein, soluble TGF-β receptor is a soluble ΤΟΡβΜΙ receptor. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target-binding domain binds specifically to CD 16. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second antigen-binding domain includes an antigen-binding domain that binds specifically to CD 16. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second antigen-binding domain includes an scFv that binds specifically to CD 16. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target-binding domain binds specifically to a receptor for IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target-binding domain includes a soluble IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target-binding domain includes a soluble human IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes an additional target-binding domain that binds specifically to a receptor for IL-2L In some embodiments of any of the multi-chain chimeric polypeptides described herein, the additional target-binding domain includes a soluble IL-2L In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble IL-21 is a soluble human IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes an additional targetbinding domain that binds specifically to CD 16.

In some embodiments of these multi-chain chimeric polypeptides, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope. In some embodiments, two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain includes a TGFpRII receptor (e.g., a soluble human TGFpRII receptor). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRpRII includes a first sequence of soluble human TGFRpRII and a second sequence of soluble human TGFRpRII. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRpRII includes a linker disposed between the first sequence of soluble human TGFRpRII and the second sequence of soluble human TGFRpRII. In some examples of these multi-chain chimeric polypeptides, the linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGFRpRII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 80).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGFRpRII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 81).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGFRpRII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 82)

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGFRpRII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT CTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAG AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGAC AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 83).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-β receptor includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPD (SEQ ID NO: 85).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human ΤΟΡβΕΙΙ receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 84).

In some embodiments of these multi-chain chimeric polypeptides, the sequence of soluble human IL-21 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 78).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTG ATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCA GAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGG CCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATC

AATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACA GAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCC AAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGC ATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC (SEQ ID NO: 94).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 79).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 includes a light chain variable domain that includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGH (SEQ ID NO: 112).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 is encoded by a light chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGA GGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA GCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGG

CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA CCGTGGGCCAT (SEQ ID NO: 113).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 includes a heavy chain variable domain that includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINW NGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDY WGQGTLVTVSR (SEQ ID NO: 114).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 is encoded by a heavy chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCC CTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTC CTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAAC TGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCA TCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAG GGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTC GACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG (SEQ ID NO: 115).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK

NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKS GDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYEN SPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLI YTLYYWKS S S SGKKTAKTNTNEFLIDVDKGENYCF S VQ AVIPSRTVNRKSTDSP V ECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKC FLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS (SEQ ID NO: 133).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGC TGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGG GAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCG GCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCAC CGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGC TACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACG AGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCAC CATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGAC GAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGT TCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGC AAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAA GGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGA

ATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCG AGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGA TTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGC ACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTT ATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAA TCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGC TGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAA TCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 134).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLF S S AYSIPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCDVRF ST CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEW EPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYP AGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLV RRNNTFLSLRD VFGKDLIYTL YYWKS S S SGKKTAKTNTNEFLIDVDKGENYCF SV QAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATL YTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVT ESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 135).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC CATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGAC AACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT CAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCC ATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGAC GAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACG ACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAA GAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAAC

GACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTG GCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGT GCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTG AAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAA CCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGC TGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGG GAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCG GCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCAC CGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGC TACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACG AGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCAC CATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGAC GAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGT TCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGC AAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAA GGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGA ATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCG AGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGA TTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGC ACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTT ATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAA TCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGC TGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAA TCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 136).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHG GGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQ APGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTA VYYCARGRSLLFDYWGQGTLVTVSRITCPPPMSVEHADIWVKSYSLYSRERYIC NSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVD QLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKK

LKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTH GSEDS (SEQ ID NO: 137).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGA GGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA GCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGG CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA CCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAG GATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAG GCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGC ATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCA TCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTT CACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCC CTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGC TGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGGATTACATG CCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGC CTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCG GCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCA CTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACATG ATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGA ACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTG CGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAAC ACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGG AAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGC CCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGT TCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCA CGGCTCCGAGGACTCC (SEQ ID NO: 138).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQ KPGQ AP VL VIYGKNNRP SGIPDRF SGS S SGNT ASLTITGAQ AEDE AD YYCNSRD S S GNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSC AASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNA KNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRITCPPPMSVEH ADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQ GQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQL KSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERF KSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 139).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC CTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTG AGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACC AGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAG GCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCT CCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAA CTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTG ACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGA GGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGA GGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGG CATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGC ATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGT TCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTC CCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTG CTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGGATTACAT GCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAG CCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCC GGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTC ACTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACAT GATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTG AACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACT GCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAA CACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCG GAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTG CCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGG TTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCC ACGGCTCCGAGGACTCC (SEQ ID NO: 140).

Exemplary Multi-Chain Chimeric Polypeptides- Type H

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each independently bind specifically to a receptor of IL-7. In some examples of these multichain chimeric polypeptides, the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain and the second target-binding domain each independently bind specifically to a receptor for IL-7. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain include the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain and the second target-binding domain include a soluble IL-7 (e.g., a soluble human IL-7). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 11).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATC AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (SEQ ID NO: 95).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAY NLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVK DVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVG TKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNE FLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDL KKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVEN LIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 141).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATC AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACA ACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAA CCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGAT CAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACC GAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCG CCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGG CGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATT TAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGT GACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGC CTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTC CTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATC GACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCT CCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCA AGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAA GAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACA

GAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACT GGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACC GTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGT GACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAA GGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 142).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNN EFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNC TGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL MGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKS KCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP YLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYY WKS S S SGKKTAKTNTNEFLID VDKGENYCF S VQ AVIP SRTVNRK STD SP VECMG QEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLEL QVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV HIVQMFINTS (SEQ ID NO: 143).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC CGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGAT GGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGC CTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGG AGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGAT GAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACC ACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTC TGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGG AGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGAT CAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCAC AACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAA ACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGA

TCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACAC CGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTC GCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTG GCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAA TTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAAT GTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCA GCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAG TCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAA TCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCC CTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGC CAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTA AAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACA CAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTA CTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACA CCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAAC GTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATC AAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTC C (SEQ ID NO: 144).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHITCPPPMSVEH ADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 145).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGG TCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCT GAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAA GGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAA TAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAA

TACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGG TGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACA GAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAA ACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACACATCACGTGCCCTC CCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTA CTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACG TCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGA CAACCCCCAGTCTCAAATGCATTAGA (SEQ ID NO: 146).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNN EFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNC TGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL MGTKEHITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNK ATNVAHWTTPSLKCIR (SEQ ID NO: 147).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC CGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGAT GGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGC CTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGG AGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGAT GAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACC ACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTC TGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGG AGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGAT CAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATATTACATGC CCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCC TCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGG CACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCAC TGGACAACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 148).

Exemplary Multi-Chain Chimeric Polypeptides- Type I

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each independently bind specifically to TGF-β. In some examples of these multi-chain chimeric polypeptides, the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain and the second target-binding domain each independently bind specifically to TGF-β. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain bind specifically to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain include the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain is a soluble TGF-β receptor (e.g., a soluble ΤΟΡβΙΜΙ receptor, e.g., a soluble human ΤΟΡβΙΜΙ). In some embodiments of these multi-chain chimeric polypeptides, the soluble human ΤΟΡΚβΚΙΙ includes a first sequence of soluble human ΤΟΡΚβΚΙΙ and a second sequence of soluble human ΤΟΡΚβΚΙΙ. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR^RII includes a linker disposed between the first sequence of soluble human TGFRβRII and the second sequence of soluble human TGFRβRII. In some examples of these multi-chain chimeric polypeptides, the linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGFR^RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 80).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGFR^RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: c (SEQ ID NO: 81).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human ΤΟΡΕβΕΙΙ receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 82).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGFRpRII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT CTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAG AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGAC AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 83).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-β receptor includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPD (SEQ ID NO: 85).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-β receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGAC (SEQ ID NO: 84).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKS GDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYEN SPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLI YTLYYWKS S S SGKKTAKTNTNEFLIDVDKGENYCF S VQ AVIPSRTVNRKSTDSP V ECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKC FLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS (SEQ ID NO: 149).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGC TGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGG GAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCG GCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCAC CGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGC TACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACG AGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCAC CATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGAC GAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGT TCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGC AAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAA GGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGA ATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCG AGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGA TTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGC ACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTT ATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAA TCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGC TGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAA TCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 150).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLF S S AYSIPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCDVRF ST CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEW EPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYP AGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLV RRNNTFLSLRD VFGKDLIYTL YYWKS S S SGKKTAKTNTNEFLIDVDKGENYCF SV QAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATL YTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVT ESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 151).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC CATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGAC AACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT CAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCC ATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGAC GAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACG ACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAA GAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAAC GACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTG GCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGT GCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTG AAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAA CCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG

CGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGC TGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGG GAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCG GCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCAC CGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGC TACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACG AGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCAC CATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGAC GAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGT TCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGC AAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAA GGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGA ATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCG AGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGA TTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGC ACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTT ATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAA TCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGC TGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAA TCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 152).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLT ECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 153).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGC GTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGA GGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCAC CGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCT TTAAAGTGCATCCGG (SEQ ID NO: 154).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLF S S AYSIPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCDVRF ST CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRER YICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 155).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC CATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGAC AACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTT CAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCC ATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGAC GAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACG ACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAA GAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAAC GACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTG GCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGT GCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTG AAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAA CCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGC GTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGA GGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCAC CGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCT TTAAAGTGCATCCGG (SEQ ID NO: 156).

Exemplary Multi-Chain Chimeric Polypeptides- Type J

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each independently bind specifically to a receptor of IL-7, a receptor of IL-21, or a receptor of CD137L. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target-binding domain binds specifically to a receptor for IL-21 (e.g., a soluble IL-21, e.g., a soluble human IL-21) or a receptor for CD137L (e.g., a soluble CD137L, e.g., a soluble human CD137L).

In some examples of these multi-chain chimeric polypeptides, the first targetbinding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second domain of the pair of affinity domains directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second target-binding domain and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments, the second chimeric polypeptide can include an additional target-binding domain. In some embodiments, the additional target-binding domain and the

In some embodiments of these multi-chain chimeric polypeptides, one or more of the first target-binding domain, the second target-binding domain and the additional target-binding domain is an agonistic antigen-binding domain. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain, the second target-binding domain, and the additional target-binding domain are each agonistic antigen-binding domains. In some embodiments of these multi-chain chimeric polypeptides, the antigen-binding domain includes a scFv or single-domain antibody.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain binds specifically to a receptor for IL-7, and the second target-binding domain binds specifically to a receptor for IL-21 or a receptor for CD137L. In some embodiments, the additional target-binding domain binds specifically to a receptor for IL21 or a receptor for CD137L.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain is a soluble IL-7 (e.g., a soluble human IL-7). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 11).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATC AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (SEQ ID NO: 124).

In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain or the additional target-binding domain binds specifically to a receptor for IL-21. In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain or the additional target-binding domain is a soluble IL-21 (e.g., a soluble human IL-21).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human IL-21 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 78).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 79).

In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain binds specifically to a receptor for CD137L. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises an additional target-binding domain that binds specifically to a receptor for CD137L. In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain and/or the additional target-binding domain is a soluble CD137L (e.g., a soluble human CD137L).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGL SYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQ GATVLGLFRVTPEIPAGLPSPRSE (SEQ ID NO: 157).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGC GGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGG GCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGG GGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGA GTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGG CTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTG

GGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCT CGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCC AGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCA GCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC CCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 158).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKEL VVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLP PASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFR VTPEI (SEQ ID NO: 159).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: GATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGG CCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGG CCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAA GGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGC TGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCA CCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGG ACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGC CGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGA GGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGA CTCTTCCGGGTGACCCCCGAAATC (SEQ ID NO: 160).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ

PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAY NLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVK DVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVG TKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNE FLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDL KKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVEN LIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 161).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATC AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACA ACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAA CCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGAT CAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACC GAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCG CCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGG CGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATT TAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGT GACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGC CTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTC CTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATC GACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCT CCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCA AGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAA GAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACA GAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACT GGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACC GTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGT GACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAA

GGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 162).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNN EFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNC TGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL MGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKS KCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP YLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYY WKS S S SGKKTAKTNTNEFLIDVDKGENYCF S VQ AVIPSRTVNRKSTDSPVECMG QEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLEL QVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV HIVQMFINTS (SEQ ID NO: 163).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC CGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGAT GGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGC CTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGG AGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGAT GAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACC ACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTC TGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGG AGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGAT CAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCAC AACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAA ACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGA TCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACAC CGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTC GCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTG GCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAA

TTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAAT GTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCA GCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAG TCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAA TCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCC CTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGC CAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTA AAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACA CAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTA CTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACA CCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAAC GTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATC AAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTC C (SEQ ID NO: 164).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGF KRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELS PDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTK ELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVD LPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLF RVTPEIPAGLPSPRSE (SEQ ID NO: 165).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC

CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTC CCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAG CCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAG CAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACA ACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCT CCGGCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGC CGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAAT GTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAG GCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGG TGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGC GTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGC CACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCA CCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCT GCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGG GCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCC GGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 166).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED VETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLT CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIW VKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGS GGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPG LAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHL QPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEAR ARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE (SEQ ID NO: 167).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC CCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTC

GACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC CCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAA GGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGT GAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAG GCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCC CCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATC AGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCT CCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACA GCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCA GCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC AACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGC TCCGGCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCG CCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAA TGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCA GGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTG GTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCG CGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAG CCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCC ACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGC TGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAG GGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTC CGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 168).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGF KRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSDPAGLL DLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKA GVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEA RNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI (SEQ ID NO: 169).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTC CCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAG CCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAG CAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACA ACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCT CCGGCGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCAT GTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCT GGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTA CAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTC TTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCG TTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCC CTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGC CTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGC GTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGG GCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC (SEQ ID NO: 170).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED VETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLT CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIW VKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGS GGGGSGGGGSDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTG GLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAG AAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQL TQGATVLGLFRVTPEI (SEQ ID NO: 171).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC CCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTC GACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC CCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAA GGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGT GAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAG GCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCC CCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATC AGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCT CCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACA GCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCA GCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGAC AACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGC TCCGGCGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCA TGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGC TGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCT ACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATG TCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTC CGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCG CCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCG GCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGG GCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCA GGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC (SEQ ID NO: 172).

Exemplary Multi-Chain Chimeric Polypeptides- Type K

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each independently bind specifically to a receptor of IL-7 or TGF-β. In some examples of these multi-chain chimeric polypeptides, the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain binds specifically to a receptor for IL-7, and the second target-binding domain binds specifically to TGF-β. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain binds specifically to TGF-β, and the second target-binding domain binds specifically to a receptor for IL-7.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain includes a soluble IL-7 protein (e.g., a soluble human IL-7 protein). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 protein includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 11).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATC AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (SEQ ID NO: 124).

In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain comprises a target-binding domain that binds specifically to TGFβ. In some embodiments of these multi-chain chimeric polypeptides, the second targetbinding domain is a soluble TGF-β receptor (e.g., a soluble ΤΟΡβΚΙΙ receptor, e.g., a soluble human ΤΟΡβΙΜΙ receptor). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR^RII includes a first sequence of soluble human ΤΟΡΕβΕΙΙ and a second sequence of soluble human ΤΟΡΕβΙΜΙ. In some embodiments of these multi-chain chimeric polypeptides, the soluble human ΤΟΡΕβΕΙΙ includes a linker disposed between the first sequence of soluble human TGFR^RII and the second sequence of soluble human ΤΟΡΕβΕΙΙ. In some examples of these multi-chain chimeric polypeptides, the linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human ΤΟΕΕβΕΙΙ receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 80).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGFRpRII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 81).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGFRpRII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 82).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGFRpRII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT CTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAG AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGAC AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 83).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-β receptor includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPD (SEQ ID NO: 85).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-β receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG

CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGAC (SEQ ID NO: 84).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYN LTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKD VKQTYLARVF S YPAGNVESTGS AGEPLYENSPEFTPYLETNLGQPTIQ SFEQ VGTK VNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKS S S SGKKTAKTNTNEFLI DVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIE DLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILA NNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 173).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGG TGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTC AACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGG GCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAA CTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACC ATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGG GAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGC AGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAA GACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACC AACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCAT CCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCA CCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGC GATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGT GTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTT TATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAG

CCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGG AGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGA TGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTC CGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGAT AAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGT GAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGC GAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAG ATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGC ACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTA TCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATC ATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTG CAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCC TTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 174).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNN EFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNC TGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL MGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSK CFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWK SSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEK GEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS (SEQ ID NO: 175).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGG TGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTC AACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGG GCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAA

CTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACC ATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGG GAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGC AGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAA GACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACC AACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCAT CCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCA CCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGC GATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGT GTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTT TATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAG CCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGG AGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGA TGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTC CGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGAT AAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGT GAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGC GAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAG ATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGC ACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTA TCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATC ATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTG CAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCC TTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 176).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMI VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI IFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTEC VLNKATNVAHWTTPSLKCIR (SEQ ID NO: 177).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCG ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTG AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG G (SEQ ID NO: 178).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLF S S AYSIPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCDVRF ST CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKP GETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERY ICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 179).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCG ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTG AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG G (SEQ ID NO: 180).

Exemplary Multi-Chain Chimeric Polypeptides- Type L

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each independently bind specifically to TGF-β, a receptor of IL-21, or a receptor of CD137L. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target-binding domain binds specifically to a receptor for IL-21 (e.g., a soluble IL-21, e.g., a soluble human IL-21) or a receptor for CD137L (e.g., a soluble CD137L, e.g., a soluble human CD137L).

In some examples of these multi-chain chimeric polypeptides, the first targetbinding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second domain of the pair of affinity domains directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second target-binding domain and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or more of the first target-binding domain, the second target-binding domain and the additional target-binding domain is an agonistic antigen-binding domain. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain, the second target-binding domain, and the additional target-binding domain are each agonistic antigen-binding domains. In some embodiments of these multi-chain chimeric polypeptides, the antigen-binding domain includes a scFv or single-domain antibody.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain binds specifically to TGF-β and the second target-binding domain binds specifically to a receptor for IL-21 or a receptor for CD137L.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain is a soluble TGF-β receptor (e.g., a soluble ΤΟΡβΚΙΙ receptor, e.g., a soluble human ΤΟΡβΚΙΙ receptor). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR^RII includes a first sequence of soluble human TGFRβRII and a second sequence of soluble human TGFRβRIL In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR^RII includes a linker disposed between the first sequence of soluble human TGFRβRII and the second sequence of soluble human TGFRβRII. In some examples of these multi-chain chimeric polypeptides, the linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGFRβRII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 80).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGFRpRII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 81).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGFRpRII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 82)

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGFRpRII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT CTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAG

AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGAC AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 83).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-β receptor includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPD (SEQ ID NO: 85).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-β receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGAC (SEQ ID NO: 84).

In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain or the additional target-binding domain binds specifically to a receptor for IL-21. In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain or the additional target-binding domain includes a soluble IL-21 (e.g., a soluble human IL-21).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human IL-21 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 78).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 79).

In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain or the additional target-binding domain binds specifically to a receptor for CD137L. In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain and/or the additional target-binding domain includes a soluble CD137L (e.g., a soluble human CD137L).

In some embodiments of these multi-chain chimeric polypeptides, a soluble CD137L includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGL SYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQ GATVLGLFRVTPEIPAGLPSPRSE (SEQ ID NO: 157).

In some embodiments of these multi-chain chimeric polypeptides, a soluble CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGC GGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGG GCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGG GGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGA GTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGG CTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTG GGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCT CGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCC AGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCA GCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC CCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 158).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L includes a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to: DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKEL VVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLP PASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFR VTPEI (SEQ ID NO: 159).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to:

GATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGG CCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGG CCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAA GGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGC TGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCA CCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGG ACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGC CGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGA GGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGA CTCTTCCGGGTGACCCCCGAAATC (SEQ ID NO: 160).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMI VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI IFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGD WKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPE FTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTL YYWKS S S SGKKTAKTNTNEFLID VDKGENYCF S VQ AVIPSRTVNRKSTDSPVECM GQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLE LQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSF VHIVQMFINTS (SEQ ID NO: 181).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC

CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC ACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCG TTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGA AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATT TACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGC AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACC AACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC CCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGA ACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCG ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCC ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT CATCAATACCTCC (SEQ ID NO: 182).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLF S S AYSIPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCDVRF ST CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKP GETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWE

PKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPA GNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRR NNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQA VIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTE SDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESG CKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 183).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC ACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCG TTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGA AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATT TACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGC AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACC AACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC CCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGA

ACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCG ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCC ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT CATCAATACCTCC (SEQ ID NO: 184).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGF KRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSP DDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKE LVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPP ASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVT PEIPAGLPSPRSE (SEQ ID NO: 185).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGA GCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGC AGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCC CAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAG CACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCC CATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCC GGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAG CCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACA CCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCG GCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGG

CCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTC TGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGT GTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGT GGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGG TGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACT GCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCG CCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCAC CTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCAC GCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTG ACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 186).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED VETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLT CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIW VKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGS GGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPG LAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQ PLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARA RHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE (SEQ ID NO: 187).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGA GCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGC AGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCC CAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAG CACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCC

CATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCC GGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAG CCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACA CCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCG GCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGG CCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTC TGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGT GTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGT GGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGG TGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACT GCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCG CCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCAC CTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCAC GCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTG ACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 188).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGF KRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSDPAGLLD LRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGV YYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNS AFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI (SEQ ID NO: 189).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGA

GCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGC AGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCC CAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAG CACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCC CATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCC GGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAG CCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACA CCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCG GCGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTT GCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTA CAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAA GAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCT TTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTC ACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGG CTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTC GGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCC ATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCC ACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC (SEQ ID NO: 190).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED VETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLT CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIW VKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGS GGGGSGGGGSDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGG LSYKEDTKELWAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQG ATVLGLFRVTPEI (SEQ ID NO: 191).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG

ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGA GCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGC AGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCC CAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAG CACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCC CATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCC GGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAG CCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACA CCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCG GCGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTT GCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTA CAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAA GAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCT TTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTC ACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGG CTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTC GGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCC ATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCC ACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC (SEQ ID NO: 192).

Exemplary Multi-Chain Chimeric Polypeptides- Type M

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each independently bind specifically to TGF-β or a receptor of IL-21. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target-binding domain binds specifically to a receptor for IL-21 (e.g., a soluble IL-21, e.g., a soluble human IL-21) or a TGF-β (e.g., a soluble TGF-β receptor, e.g., a soluble ΤΟΡβΜΙ receptor).

In some examples of these multi-chain chimeric polypeptides, the first targetbinding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second domain of the pair of affinity domains directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second target-binding domain and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain binds specifically to TGF-β, and the second target-binding domain binds specifically to TGF-β or a receptor for IL-21. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain is a soluble TGF-β receptor (e.g., a soluble ΤΟΡβΚΙΙ receptor, e.g., a soluble human ΤΟΡβΚΙΙ receptor). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR^RH includes a first sequence of soluble human ΤΟΡΚβΚΙΙ and a second sequence of soluble human TGFR^RII. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRΠ includes a linker disposed between the first sequence of soluble human TGFRβRII and the second sequence of soluble human TGFRβRII. In some examples of these multi-chain chimeric polypeptides, the linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGFRβRΠ receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 80).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGFRβRΠ receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 81).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGFRpRII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 82).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGFRpRII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT CTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAG AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGAC AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 83).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-β receptor includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPD (SEQ ID NO: 85).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-β receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 84).

In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain binds specifically to a receptor for IL-21. In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain includes a soluble IL-21 (e.g., a human soluble IL-21). In some embodiments of these multi-chain chimeric polypeptides, the soluble IL-21 includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 78).

In some embodiments of these multi-chain chimeric polypeptides, the soluble IL21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 79).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMI VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI IFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGD WKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPE FTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTL YYWKS S S SGKKTAKTNTNEFLID VDKGENYCF S VQ AVIPSRTVNRKSTDSPVECM GQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLE LQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSF VHIVQMFINTS (SEQ ID NO: 193).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or

100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC ACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCG TTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGA AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATT TACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGC AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACC AACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC CCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGA ACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCG ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCC ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT CATCAATACCTCC (SEQ ID NO: 194).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLF S S AYSIPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCDVRF ST CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKP GETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWE PKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPA GNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRR NNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQA VIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTE SDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESG CKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 195).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC ACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCG TTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGA

AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATT TACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGC AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACC AACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC CCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGA ACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCG ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCC ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT CATCAATACCTCC (SEQ ID NO: 196).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMI VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI IFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTEC VLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAP EDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHR LTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 197).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT

GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCG ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTG AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG GCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTC GACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC CCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAA GGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 198).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLF S S AYSIPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCDVRF ST CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKP GETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERY ICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIV DQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIK KLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRT HGSEDS (SEQ ID NO: 199).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCG ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTG AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG GCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTC GACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCC CCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAA GGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 200).

Exemplary Multi-Chain Chimeric Polypeptides- Type N

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each independently bind specifically to TGF-β or CD16. In some embodiments of these multi chain chimeric polypeptides described herein, the second chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multichain chimeric polypeptides described herein, the additional target-binding domain binds specifically to CD16 (e.g., an anti-CD16 scFv) or a TGF- β (e.g., a soluble TGF-β receptor, e.g., a soluble ΤΟΡβΚΙΙ receptor).

In some examples of these multi-chain chimeric polypeptides, the first targetbinding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second domain of the pair of affinity domains directly abut each other in the second chimeric polypeptide. In some embodiments of these multi chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second target-binding domain and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain binds specifically to TGF-β, and the second target-binding domain binds specifically to TGF-β or CD16. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain is a soluble TGF-β receptor (e.g., a soluble ΤΟΡβΚΙΙ receptor, e.g., a soluble human ΤΟΡβΚΙΙ receptor). In some embodiments of these multi-chain chimeric polypeptides, the soluble human ΤΟΡΚβΚΙΙ includes a first sequence of soluble human TGFR^RII and a second sequence of soluble human TGFRβRII. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII includes a linker disposed between the first sequence of soluble human TGFRβRII and the second sequence of soluble human TGFRβRII. In some examples of these multi-chain chimeric polypeptides, the linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGFR^RII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 80).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGFRpRII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 81).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGFRpRII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 82).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGFRpRII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT

TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT CTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAG AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGAC AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 83).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-β receptor includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPD (SEQ ID NO: 85).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-β receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC

TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 84).

In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain binds specifically to CD 16. In some embodiments of these multichain chimeric polypeptides, the second target-binding domain includes an anti-CD16 scFv. In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 includes a light chain variable domain that includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGH (SEQ ID NO: 112).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 is encoded by a light chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGA GGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA GCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGG CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA CCGTGGGCCAT (SEQ ID NO: 113).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 includes a heavy chain variable domain that includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINW NGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDY WGQGTLVTVSR (SEQ ID NO: 114).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that binds specifically to CD 16 is encoded by a heavy chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCC CTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTC CTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAAC TGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCA TCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAG GGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTC GACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG (SEQ ID NO: 115).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMI VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI IFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGD WKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPE FTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTL YYWKS S S SGKKTAKTNTNEFLID VDKGENYCF S VQ AVIPSRTVNRKSTDSPVECM GQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLE LQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSF VHIVQMFINTS (SEQ ID NO: 201).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC ACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCG TTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGA AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATT TACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGC AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACC AACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC CCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGA ACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCG ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCC ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT CATCAATACCTCC (SEQ ID NO: 202).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLF S S AYSIPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCDVRF ST CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKP GETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWE PKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPA GNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRR NNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQA VIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTE SDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESG CKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 203).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA

TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC ACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCG TTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGA AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATT TACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGC AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACC AACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC CCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGA ACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCG ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCC ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT CATCAATACCTCC (SEQ ID NO: 204).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMI VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI IFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTEC VLNKATNVAHWTTPSLKCIRSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQ QKPGQ APVLVIYGKNNRP SGIPDRF SGS S SGNTASLTITGAQ AEDEAD YYCNSRDS SGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLS CAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDN AKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR (SEQ ID NO: 205).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least

86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCG ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTG AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG GTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTG AGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA GCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGG CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA CCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAG GATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAG GCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGC ATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCA TCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTT CACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCC CTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGC TGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG (SEQ ID NO: 206).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFST CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKP GETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERY ICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSELTQDPAVSVALGQTVR ITCQGDSLRS YYASWYQQKPGQ APVLVIYGKNNRPSGIPDRF SGS S SGNTASLTIT GAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQ LVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGG STGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQG TLVTVSR (SEQ ID NO: 207).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCG ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTG AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG GTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTG

AGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCA GCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGG CCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTC CCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAAC TCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA CCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAG GATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAG GCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGC ATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCA TCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTT CACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCC CTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGC TGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG (SEQ ID NO: 208).

Exemplary Multi-Chain Chimeric Polypeptides- Type O

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target-binding domain and the second targeting-binding domain each independently bind specifically to TGF-β or a receptor of CD137L. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further includes the additional target-binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target-binding domain binds specifically to a receptor to TGF- β (e.g., a soluble TGF-β receptor, e.g., a soluble ΤΟΕβΚΙΙ receptor) or CD137L.

In some examples of these multi-chain chimeric polypeptides, the first targetbinding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second domain of the pair of affinity domains directly abut each other in the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target-binding domain and the second target-binding domain directly abut each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linkers described herein) between the second target-binding domain and the additional target-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first targetbinding domain binds specifically to TGF-β, and the second target-binding domain binds specifically to CD137L. In some embodiments of these multi-chain chimeric polypeptides, the first target-binding domain or the additional target-binding domain is a soluble TGF-β receptor (e.g., a soluble ΤΟΡβΙΠΙ receptor, e.g., a soluble human ΤΟΡβΙΠΙ receptor).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR^RII includes a first sequence of soluble human TGFR)3RII and a second sequence of soluble human TGFRβRII. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII includes a linker disposed between the first sequence of soluble human TGFRβRII and the second sequence of soluble human TGFRβRII. In some examples of these multi-chain chimeric polypeptides, the linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human TGFRβRII receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 80).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human ΤΟΡΕβΕΙΙ receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 81).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of soluble human ΤΟΡΕβΕΙΙ receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 82).

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of soluble human TGFRpRII receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATA ACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTT TCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCAT CTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAG AATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTT CATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAA GCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGAC AATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 83).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-β receptor includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGET FFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDM IVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND NIIFSEEYNTSNPD (SEQ ID NO: 85).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-β receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACA ACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTC AGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCA TCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACG AGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGA CTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAG AAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACG ACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGG CGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTG CAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGA AATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAAC CAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTC AGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGA AACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGAC GCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACC TTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAG CGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 84).

In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain includes a soluble CD137L protein (e.g., a soluble human CD137L protein). In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L includes a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to: REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGL SYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQ GATVLGLFRVTPEIPAGLPSPRSE (SEQ ID NO: 157).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to:

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGC GGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGG GCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGG GGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGA GTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGG CTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTG GGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCT CGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCC AGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCA GCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC CCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 158).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L includes a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to:

DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKEL VVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLP PASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFR VTPEI (SEQ ID NO: 159).

In some embodiments of these multi-chain chimeric polypeptides, a soluble human CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to:

GATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGG CCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGG CCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAA GGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGC TGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCA CCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGG ACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGC CGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGA GGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGA CTCTTCCGGGTGACCCCCGAAATC (SEQ ID NO: 160).

In some embodiments, the first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least

94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMI VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI IFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGD WKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPE FTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTL YYWKS S S SGKKTAKTNTNEFLIDVDKGENYCF S VQ AVIPSRTVNRKSTDSPVECM GQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLE LQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSF VHIVQMFINTS (SEQ ID NO: 209).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC ACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCG TTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGA AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT

GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATT TACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGC AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACC AACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC CCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGA ACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCG ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCC ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT CATCAATACCTCC (SEQ ID NO: 210).

In some embodiments, a first chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFST CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKP GETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWE PKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPA GNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRR NNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQA VIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTE SDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESG CKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 211).

In some embodiments, a first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTC ACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCG TTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTC CAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGA AAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAAT GTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATT TACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGC AAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCG GCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCT ACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACC AACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCC CCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGA ACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCG ACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCC ATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCC AGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAG GAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTT CATCAATACCTCC (SEQ ID NO: 212).

In some embodiments, the second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF FMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMI VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI IFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTEC VLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLRQ GMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYV FFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFG FQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRS E (SEQ ID NO: 213).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCG ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTG AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG GGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGA GGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAG

GGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCT GAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTG AGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACT ATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGC TCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGC CGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACT CGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTG GGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCA GGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGA CTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 214).

In some embodiments, a second chimeric polypeptide can include a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFST CDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSG GGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT SICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKP GETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERY ICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSRE GPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYK EDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALAL TVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVL GLFRVTPEIPAGLPSPRSE (SEQ ID NO: 215).

In some embodiments, a second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAA CAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCT GCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGA ACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTC ATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGC CCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAAC

ATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATC CGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAG AGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGT CCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTG TGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTG CCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCC CTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGC TCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAA TACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCG ACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAAC AGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTG AATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCG GGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGA GGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAG GGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCT GAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTG AGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACT ATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGC TCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGC CGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACT CGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTG GGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCA GGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGA CTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 216).

Compositions/Kits

Also provided herein are compositions (e.g., pharmaceutical compositions) that include at least one of any multi-chain chimeric polypeptides, any of the cells, or any of the nucleic acids described herein. In some embodiments, the compositions include at least one of any of the multi-chain chimeric polypeptides described herein. In some embodiments, the compositions include any of the immune cells (e.g., any of the immune cells described herein, e.g., any of the immune cells produced using any of the methods described herein).

In some embodiments, the pharmaceutical compositions are formulated for different routes of administration (e.g., intravenous, subcutaneous). In some embodiments, the pharmaceutical compositions can include a pharmaceutically acceptable carrier (e.g., phosphate buffered saline).

Single or multiple administrations of pharmaceutical compositions can be given to a subject in need thereof depending on for example: the dosage and frequency as required and tolerated by the subject. The formulation should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate conditions, diseases or symptoms.

Also provided herein are kits that include any of the multi-chain chimeric polypeptides, compositions, nucleic acids, or cells (e.g., immune cells) described herein. In some embodiments, the kits can include instructions for performing any of the methods described herein. In some embodiments, the kits can include at least one dose of any of the pharmaceutical compositions described herein.

Nucleic Acids/Vectors

Also provided herein are nucleic acids that encode any of the multi-chain chimeric polypeptides described herein. In some embodiments, a first nucleic acid can encode the first chimeric polypeptide and a second nucleic acid can encode the second chimeric polypeptide. In some embodiments, a single nucleic acid can encode both the first chimeric polypeptide and the second chimeric polypeptide.

Also provided herein are vectors that include any of the nucleic acids encoding any of the multi-chain chimeric polypeptides described herein. In some embodiments, a first vector can include a nucleic acid encoding the first chimeric polypeptide and a second vector can include a nucleic acid encoding the second chimeric polypeptide. In some embodiments, a single vector can include a first nucleic acid encoding the first chimeric polypeptide and a second nucleic acid encoding the second chimeric polypeptide.

Any of the vectors described herein can be an expression vector. For example, an expression vector can include a promoter sequence operably linked to the sequence encoding the first chimeric polypeptide and the second chimeric polypeptide.

Non-limiting examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vectors (e.g., pSV or pCMV vectors), adeno-associated virus (AAV) vectors, lentivirus vectors, and retroviral vectors), and any Gateway® vectors. A vector can, e.g., include sufficient cis-acting elements for expression; other elements for expression can be supplied by the host mammalian cell or in an in vitro expression system. Skilled practitioners will be capable of selecting suitable vectors and mammalian cells for making any of the multi-chain chimeric polypeptides described herein.

Cells

Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the nucleic acids described herein that encode any of the multi-chain chimeric polypeptides described herein (e.g., encoding both the first and second chimeric polypeptides). Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the nucleic acids described herein that encode any of the first chimeric polypeptides described herein. Also provided are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the nucleic acids described herein that encode any of the second chimeric polypeptides described herein.

Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) that include any of the vectors described herein that encode any of the multi-chain chimeric polypeptides described herein (e.g., encoding both the first and second chimeric polypeptides). Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) that include any of the vectors described herein that encode any of the first chimeric polypeptides described herein. Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) that include any of the vectors described herein that encode any of the second chimeric polypeptides described herein).

In some embodiments of any of the methods described herein, the cell can be a eukaryotic cell. As used herein, the term “eukaryotic cell” refers to a cell having a distinct, membrane-bound nucleus. Such cells may include, for example, mammalian (e.g., rodent, non-human primate, or human), insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryote, such as mammalian, avian, plant, or insect cells. Non-limiting examples of mammalian cells include Chinese hamster ovary cells and human embryonic kidney cells (e.g., HEK293 cells).

Methods of introducing nucleic acids and expression vectors into a cell (e.g., a eukaryotic cell) are known in the art. Non-limiting examples of methods that can be used to introduce a nucleic acid into a cell include lipofection, transfection, electroporation, microinjection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, impalefection, hydrodynamic delivery, magnetofection, viral transduction (e.g., adenoviral and lentiviral transduction), and nanoparticle transfection.

Methods of Producing Multi-Chain Chimeric Polypeptides

Also provided herein are methods of producing any of the multi-chain chimeric polypeptides described herein that include culturing any of the cells described herein in a culture medium under conditions sufficient to result in the production of the multi-chain chimeric polypeptide; and recovering the multi-chain chimeric polypeptide from the cell and/or the culture medium.

Also provided herein are method of producing any of the multi-chain chimeric polypeptides described herein that include: culturing any of cells described herein in a first culture medium under conditions sufficient to result in the production of the first chimeric polypeptide; recovering the first chimeric polypeptide from the cell and/or the first culture medium; culturing any of the cells described herein in a second culture medium under conditions sufficient to result in the production of the second chimeric polypeptide; recovering the second chimeric polypeptide from the cell and/or the second culture medium; and combining (e.g., mixing) the recovered first chimeric polypeptide and the recovered second chimeric polypeptide to form the multi-chain chimeric polypeptide (e.g., any of the multi-chain chimeric polypeptides described herein).

The recovery of the multi-chain chimeric polypeptide, the first chimeric polypeptide, or the second chimeric polypeptide from a cell (e.g., a eukaryotic cell) can be performed using techniques well-known in the art (e.g., ammonium sulfate precipitation, polyethylene glycol precipitation, ion-exchange chromatography (anion or cation), chromatography based on hydrophobic interaction, metal-affinity chromatography, ligand-affinity chromatography, and size exclusion chromatography).

Methods of culturing cells are well known in the art. Cells can be maintained in vitro under conditions that favor proliferation, differentiation and growth. Briefly, cells can be cultured by contacting a cell (e.g., any cell) with a cell culture medium that includes the necessary growth factors and supplements to support cell viability and growth.

Also provided herein are multi-chain chimeric polypeptides (e.g., any of the multi-chain chimeric polypeptides described herein), first chimeric polypeptides (e.g., any of the first chimeric polypeptides), or second chimeric polypeptides (e.g., any of the second chimeric polypeptides described herein) produced by any of the methods described herein.

Methods of Stimulating an Immune Cell

Also provided herein are methods of stimulating an immune cell (e.g., any of the exemplary immune cells described herein or known in the art) that include contacting an immune cell with an effective amount of any of the multi-chain chimeric polypeptides described herein or any of the compositions (e.g., pharmaceutical compositions) described herein. In some examples, the immune cell is contacted in vitro (e.g., in a suitable liquid culture medium under conditions sufficient to result in stimulation of the immune cell).

In some examples, the immune cell has been previously obtained from a subject (e.g., a mammal, e.g., a human). Some embodiments of these methods further include obtaining the immune cell from the subject prior to the contacting step.

In some examples, the immune cell is contacted in vivo. In such embodiments, the multi-chain chimeric polypeptide is administered to a subject (e.g., a mammal, e.g., a human) in an amount sufficient to result in stimulation of an immune cell in the subject.

In some examples of any of the methods described herein, the immune cell can be an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, a Th 17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, γδ T cell, an αβ T cell, a tumorinfiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, or a natural killer cell, or a combination thereof.

In some examples, the immune cell has previously been genetically-modified to express a chimeric antigen receptor or a recombinant T-cell receptor. In some examples, the immune cell (e.g., any of the immune cells described herein) has previously been genetically-modified to express a co-stimulatory molecule (e.g., CD28).

Some embodiments of these methods can further include, after the contacting step, introducing into the immune cell (e.g., any of the immune cells described herein) a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. Some embodiments of these methods can further include, after the contacting step, introducing into the immune cell (e.g., any of the immune cells described herein) a nucleic acid encoding a co-stimulatory molecule (e.g., CD28).

Some embodiments of these methods can further include administering a therapeutically effective amount of the immune cell to a subject in need thereof (e.g., any of the exemplary subjects described herein).

In some examples, the subject can be a subject identified or diagnosed as having an age-related disease or condition. Non-limiting examples of age-related diseases or disorders include: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some examples, the subject can be a subject that has been identified or diagnosed as having a cancer. Non-limiting examples of cancers include: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma,

B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some examples, the subject can be a subject that has been diagnosed or identified as having an infectious disease. Non-limiting examples of infectious disease include infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, or influenza virus.

Activation of an immune cell can be determined using methods known in the art. For example, activation of an immune cell can be determined by detecting the levels of cytokines and chemokines that are secreted or cytotoxicity granules and regulatory molecules that are upregulated upon activation of an immune cell. Non-limiting examples of cytokines, chemokines, cytotoxicity granules, and regulatory molecules that are secreted or upregulated upon activation of an immune cell include: IL-2, IFN-γ, IL-1, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-22, IL-33, leukotriene B4, CCL5, TNFa, granzymes, perforin, TGF3, STAT3, STAT4, STAT5, RORyT, FOXP3, STAT6, and GATA3. The detection of these cytokines, chemokines, cytotoxicity granules, or regulatory molecules can be performed using an immunoassay (e.g., an enzyme-linked immunosorbent assay) and quantitative PCR. For example, activation of an immune cell can result in an increase of about 1% to about 800% (e.g., about 1% to about 750%, about 1% to about 700%, about 1% to about 650%, about 1% to about 600%, about 1% to about 550%, about 1% to about 500%, about 1% to about 450%, about 1% to about 400%, about 1% to about 350%, about 1% to about 300%, about 1% to about 280%, about 1% to about 260%, about 1% to about 240%, about 1% to about 220%, about 1% to about 200%, about 1% to about 180%, about 1% to about 160%, about 1% to about 140%, about 1% to about 120%, about 1% to about 100%, about 1% to about 90%, about 1% to about 80%, about 1% to about 70%, about 1% to about 60%, about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 800%, about 5% to about 750%, about 5% to about 700%, about 5% to about 650%, about 5% to about 600%, about 5% to about 550%, about 5% to about 500%, about 5% to about 450%, about 5% to about 400%, about 5% to about 350%, about 5% to about 300%, about 5% to about 280%, about 5% to about 260%, about 5% to about 240%, about 5% to about 220%, about 5% to about 200%, about 5% to about 180%, about 5% to about 160%, about 5% to about 140%, about 5% to about 120%, about 5% to about 100%, about 5% to about 90%, about 5% to about 80%, about 5% to about 70%, about 5% to about 60%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 800%, about 10% to about 750%, about 10% to about 700%, about 10% to about 650%, about 10% to about 600%, about 10% to about 550%, about 10% to about 500%, about 10% to about 450%, about 10% to about 400%, about 10% to about 350%, about 10% to about 300%, about 10% to about 280%, about 10% to about 260%, about 10% to about 240%, about 10% to about 220%, about 10% to about 200%, about 10% to about 180%, about 10% to about 160%, about 10% to about 140%, about 10% to about 120%, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 15% to about 800%, about 15% to about 750%, about 15% to about 700%, about 15% to about 650%, about 15% to about 600%, about 15% to about 550%, about 15% to about 500%, about 15% to about 450%, about 15% to about 400%, about 15% to about 350%, about 15% to about 300%, about 15% to about 280%, about 15% to about 260%, about 15% to about 240%, about 15% to about 220%, about 15% to about 200%, about 15% to about 180%, about 15% to about 160%, about 15% to about 140%, about 15% to about 120%, about 15% to about 100%, about 15% to about 90%, about 15% to about

80%, about 15% to about 70%, about 15% to about 60%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 15% to about 20%, about 20% to about 800%, about 20% to about 750%, about 20% to about 700%, about 20% to about 650%, about 20% to about 600%, about 20% to about 550%, about 20% to about 500%, about 20% to about 450%, about 20% to about 400%, about 20% to about 350%, about 20% to about 300%, about 20% to about 280%, about 20% to about 260%, about 20% to about 240%, about 20% to about 220%, about 20% to about 200%, about 20% to about 180%, about 20% to about 160%, about 20% to about 140%, about 20% to about 120%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 800%, about 25% to about 750%, about 25% to about 700%, about 25% to about 650%, about 25% to about 600%, about 25% to about 550%, about 25% to about 500%, about 25% to about 450%, about 25% to about 400%, about 25% to about 350%, about 25% to about 300%, about 25% to about 280%, about 25% to about 260%, about 25% to about 240%, about 25% to about 220%, about 25% to about 200%, about 25% to about 180%, about 25% to about 160%, about 25% to about 140%, about 25% to about 120%, about 25% to about 100%, about 25% to about 90%, about 25% to about 80%, about 25% to about 70%, about 25% to about 60%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 35% to about 800%, about 35% to about 750%, about 35% to about 700%, about 35% to about 650%, about 35% to about 600%, about 35% to about 550%, about 35% to about 500%, about 35% to about 450%, about 35% to about 400%, about 35% to about 350%, about 35% to about 300%, about 35% to about 280%, about 35% to about 260%, about 35% to about 240%, about 35% to about 220%, about 35% to about 200%, about 35% to about 180%, about 35% to about 160%, about 35% to about 140%, about 35% to about 120%, about 35% to about 100%, about 35% to about 90%, about 35% to about 80%, about 35% to about 70%, about 35% to about 60%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 800%, about 40% to about 750%, about 40% to about 700%, about 40% to about 650%, about 40% to about

600%, about 40% to about 550%, about 40% to about 500%, about 40% to about 450%, about 40% to about 400%, about 40% to about 350%, about 40% to about 300%, about 40% to about 280%, about 40% to about 260%, about 40% to about 240%, about 40% to about 220%, about 40% to about 200%, about 40% to about 180%, about 40% to about 160%, about 40% to about 140%, about 40% to about 120%, about 40% to about 100%, about 40% to about 90%, about 40% to about 80%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 40% to about 45%, about 45% to about 800%, about 45% to about 750%, about 45% to about 700%, about 45% to about 650%, about 45% to about 600%, about 45% to about 550%, about 45% to about 500%, about 45% to about 450%, about 45% to about 400%, about 45% to about 350%, about 45% to about 300%, about 45% to about 280%, about 45% to about 260%, about 45% to about 240%, about 45% to about 220%, about 45% to about 200%, about 45% to about 180%, about 45% to about 160%, about 45% to about 140%, about 45% to about 120%, about 45% to about 100%, about 45% to about 90%, about 45% to about 80%, about 45% to about 70%, about 45% to about 60%, about 45% to about 50%, about 50% to about 800%, about 50% to about 750%, about 50% to about 700%, about 50% to about 650%, about 50% to about 600%, about 50% to about 550%, about 50% to about 500%, about 50% to about 450%, about 50% to about 400%, about 50% to about 350%, about 50% to about 300%, about 50% to about 280%, about 50% to about 260%, about 50% to about 240%, about 50% to about 220%, about 50% to about 200%, about 50% to about 180%, about 50% to about 160%, about 50% to about 140%, about 50% to about 120%, about 50% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 800%, about 60% to about 750%, about 60% to about 700%, about 60% to about 650%, about 60% to about 600%, about 60% to about 550%, about 60% to about 500%, about 60% to about 450%, about 60% to about 400%, about 60% to about 350%, about 60% to about 300%, about 60% to about 280%, about 60% to about 260%, about 60% to about 240%, about 60% to about 220%, about 60% to about 200%, about 60% to about 180%, about 60% to about 160%, about 60% to about 140%, about 60% to about 120%, about 60% to about 100%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 800%, about 70% to about 750%, about 70% to about 700%, about 70% to about

650%, about 70% to about 600%, about 70% to about 550%, about 70% to about 500%, about 70% to about 450%, about 70% to about 400%, about 70% to about 350%, about 70% to about 300%, about 70% to about 280%, about 70% to about 260%, about 70% to about 240%, about 70% to about 220%, about 70% to about 200%, about 70% to about 180%, about 70% to about 160%, about 70% to about 140%, about 70% to about 120%, about 70% to about 100%, about 70% to about 90%, about 70% to about 80%, about 80% to about 800%, about 80% to about 750%, about 80% to about 700%, about 80% to about 650%, about 80% to about 600%, about 80% to about 550%, about 80% to about 500%, about 80% to about 450%, about 80% to about 400%, about 80% to about 350%, about 80% to about 300%, about 80% to about 280%, about 80% to about 260%, about 80% to about 240%, about 80% to about 220%, about 80% to about 200%, about 80% to about 180%, about 80% to about 160%, about 80% to about 140%, about 80% to about 120%, about 80% to about 100%, about 80% to about 90%, about 90% to about 800%, about 90% to about 750%, about 90% to about 700%, about 90% to about 650%, about 90% to about 600%, about 90% to about 550%, about 90% to about 500%, about 90% to about 450%, about 90% to about 400%, about 90% to about 350%, about 90% to about 300%, about 90% to about 280%, about 90% to about 260%, about 90% to about 240%, about 90% to about 220%, about 90% to about 200%, about 90% to about 180%, about 90% to about 160%, about 90% to about 140%, about 90% to about 120%, about 90% to about 100%, about 100% to about 800%, about 100% to about 750%, about 100% to about 700%, about 100% to about 650%, about 100% to about 600%, about 100% to about 550%, about 100% to about 500%, about 100% to about 450%, about 100% to about 400%, about 100% to about 350%, about 100% to about 300%, about 100% to about 280%, about 100% to about 260%, about 100% to about 240%, about 100% to about 220%, about 100% to about 200%, about 100% to about 180%, about 100% to about 160%, about 100% to about 140%, about 100% to about 120%, about 120% to about 800%, about 120% to about 750%, about 120% to about 700%, about 120% to about 650%, about 120% to about 600%, about 120% to about 550%, about 120% to about 500%, about 120% to about 450%, about 120% to about 400%, about 120% to about 350%, about 120% to about 300%, about 120% to about 280%, about 120% to about 260%, about 120% to about 240%, about 120% to about 220%, about 120% to about

200%, about 120% to about 140%, about 140% to about 700%, about 140% to about 550%, about 140% to about 400%, about 140% to about 280%, about 140% to about 220%, about 140% to about 160%, about 160% to about 700%, about 160% to about 550%, about 160% to about 400%, about 160% to about 280%, about 160% to about 220%, about 160% to about 800%, about 180% to about 650%, about 180% to about 500%, about 180% to about 350%, about 180% to about 260%, about 180% to about 200%, about 200% to about 700%, about 200% to about 550%, about 200% to about 400%, about 200% to about 280%, about 200% to about 220%, about 220% to about 700%, about 220% to about 550%, about 220% to about 400%, about 220% to about 280%, about 220% to about 800%, about 240% to about 650%, about 240% to about 500%, about 240% to about

180%, about 120% to about 800%, about 140% to about 650%, about 140% to about 500%, about 140% to about 350%, about 140% to about 260%, about 140% to about 200%, about 140% to about 800%, about 160% to about 650%, about 160% to about 500%, about 160% to about 350%, about 160% to about 260%, about 160% to about 200%, about 160% to about 750%, about 180% to about 600%, about 180% to about 450%, about 180% to about 300%, about 180% to about 240%, about 180% to about 800%, about 200% to about 650%, about 200% to about 500%, about 200% to about 350%, about 200% to about 260%, about 200% to about 800%, about 220% to about 650%, about 220% to about 500%, about 220% to about 350%, about 220% to about 260%, about 220% to about 750%, about 240% to about 600%, about 240% to about 450%, about 240% to about

160%, about 120% to about 750%, about 140% to about 600%, about 140% to about 450%, about 140% to about 300%, about 140% to about 240%, about 140% to about 180%, about 140% to about 750%, about 160% to about 600%, about 160% to about 450%, about 160% to about 300%, about 160% to about 240%, about 160% to about 180%, about 180% to about 700%, about 180% to about 550%, about 180% to about 400%, about 180% to about 280%, about 180% to about 220%, about 180% to about 750%, about 200% to about 600%, about 200% to about 450%, about 200% to about 300%, about 200% to about 240%, about 200% to about 750%, about 220% to about 600%, about 220% to about 450%, about 220% to about 300%, about 220% to about 240%, about 240% to about 700%, about 240% to about 550%, about 240% to about 400%, about 240% to about

350%, about 240% to about 300%, about 240% to about 280%, about 240% to about 260%, about 260% to about 800%, about 260% to about 750%, about 260% to about 700%, about 260% to about 650%, about 260% to about 600%, about 260% to about 550%, about 260% to about 500%, about 260% to about 450%, about 260% to about 400%, about 260% to about 350%, about 260% to about 300%, about 260% to about 280%, about 280% to about 800%, about 280% to about 750%, about 280% to about 700%, about 280% to about 650%, about 280% to about 600%, about 280% to about 550%, about 280% to about 500%, about 280% to about 450%, about 280% to about 400%, about 280% to about 350%, about 280% to about 300%, about 300% to about 800%, about 300% to about 750%, about 300% to about 700%, about 300% to about 650%, about 300% to about 600%, about 300% to about 550%, about 300% to about 500%, about 300% to about 450%, about 300% to about 400%, about 300% to about 350%, about 350% to about 800%, about 350% to about 750%, about 350% to about 700%, about 350% to about 650%, about 350% to about 600%, about 350% to about 550%, about 350% to about 500%, about 350% to about 450%, about 350% to about 400%, about 400% to about 800%, about 400% to about 750%, about 400% to about 700%, about 400% to about 650%, about 400% to about 600%, about 400% to about 550%, about 400% to about 500%, about 400% to about 450%, about 450% to about 800%, about 450% to about 750%, about 450% to about 700%, about 450% to about 650%, about 450% to about 600%, about 450% to about 550%, about 450% to about 500%, about 500% to about 800%, about 500% to about 750%, about 500% to about 700%, about 500% to about 650%, about 500% to about 600%, about 500% to about 550%, about 550% to about 800%, about 550% to about 750%, about 550% to about 700%, about 550% to about 650%, about 550% to about 600%, about 600% to about 800%, about 600% to about 750%, about 600% to about 700%, about 600% to about 650%, about 650% to about 800%, about 650% to about 750%, about 650% to about 700%, about 700% to about 800%, about 700% to about 750%, or about 750% to about 800%) of one or more of any of the cytokines or chemokines or cytotoxicity granules or regulatory molecules described herein (e.g., one or more of any of IL-2, IFN-γ, IL-1, IL4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-22, IL-33, leukotriene B4, CCL5, TNFa, granzymes, perforin, ΤΘΡβ, STAT3, STAT4, STAT5,

RORyT, FOXP3, and GATA3) (e.g., as compared to the level of the one or more cytokines, chemokines, cytotoxicity granules, and regulatory molecules in a control not contacted with any of the multi-chain chimeric polypeptides described herein).

Methods of Inducing or Increasing Proliferation of an Immune Cell

Also provided herein are methods of inducing or increasing proliferation of an immune cell (e.g., any of the exemplary immune cells described herein or known in the art) that include contacting an immune cell with an effective amount of any of the multichain chimeric polypeptides described herein or any of the compositions (e.g., pharmaceutical compositions) described herein. In some examples, the immune cell is contacted in vitro (e.g., in a suitable liquid culture medium under conditions sufficient to result in stimulation of the immune cell).

In some examples, the immune cell has been previously obtained from a subject (e.g., a mammal, e.g., a human). Some embodiments of these methods further include obtaining the immune cell from the subject prior to the contacting step.

In some examples, the immune cell is contacted in vivo. In such embodiments, the multi-chain chimeric polypeptide is administered to a subject (e.g., a mammal, e.g., a human) in an amount sufficient to result in stimulation of an immune cell in the subject.

In some examples of any of the methods described herein, the immune cell can be an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, a Th 17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, γδ T cell, an αβ T cell, a tumorinfiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, or a natural killer cell, or a combination thereof.

In some examples, the immune cell has previously been genetically-modified to express a chimeric antigen receptor or a recombinant T-cell receptor. In some examples, the immune cell (e.g., any of the immune cells described herein) has previously been genetically-modified to express a co-stimulatory molecule (e.g., CD28).

Some embodiments of these methods can further include, after the contacting step, introducing into the immune cell (e.g., any of the immune cells described herein) a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. Some embodiments of these methods can further include, after the contacting step, introducing into the immune cell (e.g., any of the immune cells described herein) a nucleic acid encoding a co-stimulatory molecule (e.g., CD28).

Some embodiments of these methods can further include administering a therapeutically effective amount of the immune cell to a subject in need thereof (e.g., any of the exemplary subjects described herein).

In some examples, the subject can be a subject identified or diagnosed as having an age-related disease or condition. Non-limiting examples of age-related diseases or disorders include: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some examples, the subject can be a subject that has been identified or diagnosed as having a cancer. Non-limiting examples of cancers include: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some examples, the subject can be a subject that has been diagnosed or identified as having an infectious disease. Non-limiting examples of infectious disease include infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, or influenza virus.

Detection of the proliferation of an immune cell can be performed using methods known in the art, e.g., cytometry (e.g., fluorescence-assisted flow cytometry), microscopy, and immunofluorescence microscopy, e.g., by comparing the rate of increase in the concentration of the immune cell in a sample not contacted with a multi-chain chimeric polypeptide to the rate of increase in the concentration of the immune cell in a similar sample contacted with any of the multi-chain chimeric polypeptides described herein).

In other examples, the proliferation of an immune cell can be indirectly detected by detecting an increase in the level of one or more cytokines or chemokines or cytotoxicity granules or regulatory molecules secreted or upregulated by proliferating immune cells (e.g., one or more of IL-2, IFN-γ, IL-1, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-22, IL-33, leukotriene B4, CCL5, TNFa, granzymes, perforin, TGFp, STAT3, STAT4, STAT5, RORyT, FOXP3, and GATA3) (e.g., as compared to the level of the one or more cytokines, chemokines, cytotoxicity granules, and regulatory molecules in a control not contacted with any of the multi-chain chimeric polypeptides described herein).

In some embodiments, the methods provided herein can result in an increase (e.g., about 1% to about 800% increase, or any of the subranges of this range described herein) in the rate of increase in the concentration of the immune cell in a sample contacted with any of the multi-chain chimeric polypeptides described herein as compared to the rate of increase in a similar control sample not contacted with any of the multi-chain chimeric polypeptides described herein.

Methods of Inducing Differentiation of an Immune Cell

Also provided herein are method of inducing differentiation of an immune cell (e.g., any of the exemplary immune cells described herein or known in the art) into a memory or memory-like immune cell that include contacting an immune cell with an effective amount of any of the multi-chain chimeric polypeptides described herein or any of the compositions (e.g., pharmaceutical compositions) described herein. In some examples, the immune cell is contacted in vitro (e.g., in a suitable liquid culture medium under conditions sufficient to result in stimulation of the immune cell).

In some examples, the immune cell has been previously obtained from a subject (e.g., a mammal, e.g., a human). Some embodiments of these methods further include obtaining the immune cell from the subject prior to the contacting step.

In some examples, the immune cell is contacted in vivo. In such embodiments, the multi-chain chimeric polypeptide is administered to a subject (e.g., a mammal, e.g., a human) in an amount sufficient to result in stimulation of an immune cell in the subject.

In some examples of any of the methods described herein, the immune cell can be an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a Th 17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, or a natural killer cell, or a combination thereof.

In some examples, the immune cell has previously been genetically-modified to express a chimeric antigen receptor or a recombinant T-cell receptor. In some examples, the immune cell (e.g., any of the immune cells described herein) has previously been genetically-modified to express a co-stimulatory molecule (e.g., CD28).

In some examples, an effective amount of any of the multi-chain chimeric polypeptides described herein or any of the compositions (e.g., pharmaceutical compositions) described herein is combined with an anti-TF IgGl antibody to create a memory or memory like immune cell.

Some embodiments of these methods can further include, after the contacting step, introducing into the immune cell (e.g., any of the immune cells described herein) a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor. Some embodiments of these methods can further include, after the contacting step, introducing into the immune cell (e.g., any of the immune cells described herein) a nucleic acid encoding a co-stimulatory molecule (e.g., CD28).

Some embodiments of these methods can further include administering a therapeutically effective amount of the immune cell to a subject in need thereof (e.g., any of the exemplary subjects described herein).

In some examples, the subject can be a subject identified or diagnosed as having an age-related disease or condition. Non-limiting examples of age-related diseases or disorders include: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some examples, the subject can be a subject that has been identified or diagnosed as having a cancer. Non-limiting examples of cancers include: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some examples, the subject can be a subject that has been diagnosed or identified as having an infectious disease. Non-limiting examples of infectious disease include infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, or influenza virus.

In some examples, the immune cell is a NK cell, and the detection of a memory NK cell can include, e.g., the detection of the level of one or more of IL-12, IL-18, IL-33, CD25, CD69, CD62L, STAT4, Zbtb32, DNAM-1, NKp30, NKp44, NKp46, BIM, Noxa, SOCS1, BNIP3, BNIP3L, IFN-γ, CXCL16, CXCR6, NKG2D, TRAIL, CD49, Ly49D, CD49b, and Ly79H. A description of NK memory cells and methods of detecting the same is described in O’Sullivan et al., Immunity 43:634-645, 2015.

In some examples, the immune cell is a T cell, and the detection of memory T cells can include, e.g., the detection of the level of expression of one or more of CD45RO, CCR7, L-selectin (CD62L), CD44, CD45RA, integrin αεβ7, CD43, CD27, CD28, IL-7Ra, CD95, ΙΕ-2Κβ, CXCR3, and LFA-1. In some examples, the immune cell is a B cell and the detection of memory B cells can include, e.g., the detection of the level of expression of CD27. Other types and markers of memory or memory-like immune cells are known in the art.

Methods of Treatment

Also provided herein are methods of treating a subject in need thereof (e.g., any of the exemplary subjects described herein or known in the art) that include administering to the subject a therapeutically effective amount of any of the multi-chain chimeric polypeptides described herein or any of the compositions (e.g., pharmaceutical compositions) described herein.

In some embodiments of these methods, the subject has been identified or diagnosed as having a cancer. Non-limiting examples of cancer include: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, these methods can result in a reduction in the number, severity, or frequency of one or more symptoms of the cancer in the subject (e.g., as compared to the number, severity, or frequency of the one or more symptoms of the cancer in the subject prior to treatment). In some embodiments, these methods can result in a reduction (e.g., about 1% reduction to about 99% reduction, about 1% reduction to about 95% reduction, about 1% reduction to about 90% reduction, about 1% reduction to about 85% reduction, about 1% reduction to about 80% reduction, about 1% reduction to about 75% reduction, about 1% reduction to about 70% reduction, about 1% reduction to about 65% reduction, about 1% reduction to about 60% reduction, about 1% reduction to about 55% reduction, about 1% reduction to about 50% reduction, about 1% reduction to about 45% reduction, about 1% reduction to about 40% reduction, about 1% reduction to about 35% reduction, about 1% reduction to about 30% reduction, about 1% reduction to about 25% reduction, about 1% reduction to about 20% reduction, about 1% reduction to about 15% reduction, about 1% reduction to about 10% reduction, about 1% reduction to about 5% reduction, about 5% reduction to about 99% reduction, about 5% reduction to about 95% reduction, about 5% reduction to about 90% reduction, about 5% reduction to about 85% reduction, about 5% reduction to about 80% reduction, about 5% reduction to about 75% reduction, about 5% reduction to about 70% reduction, about 5% reduction to about 65% reduction, about 5% reduction to about 60% reduction, about 5% reduction to about 55% reduction, about 5% reduction to about 50% reduction, about 5% reduction to about 45% reduction, about 5% reduction to about 40% reduction, about 5% reduction to about 35% reduction, about 5% reduction to about 30% reduction, about 5% reduction to about 25% reduction, about 5% reduction to about 20% reduction, about 5% reduction to about 15% reduction, about 5% reduction to about 10% reduction, about 10% reduction to about 99% reduction, about 10% reduction to about 95% reduction, about 10% reduction to about 90% reduction, about 10% reduction to about 85% reduction, about 10% reduction to about 80% reduction, about 10% reduction to about 75% reduction, about 10% reduction to about 70% reduction, about 10% reduction to about 65% reduction, about 10% reduction reduction to about 55% reduction, about 10% 10% reduction to about 45% reduction, about about 10% reduction to about 35% reduction, reduction, about 10% reduction to about 25% 20% reduction, about 10% reduction to about about 99% reduction, about 15% reduction to to about 90% reduction, about 15% reduction reduction to about 80% reduction, about 15% 15% reduction to about 70% reduction, about about 15% reduction to about 60% reduction, reduction, about 15% reduction to about 50% 45% reduction, about 15% reduction to about about 35% reduction, about 15% reduction to to about 25% reduction, about 15% reduction reduction to about 99% reduction, about 20% 20% reduction to about 90% reduction, about about 20% reduction to about 80% reduction, reduction, about 20% reduction to about 70% 65% reduction, about 20% reduction to about about 55% reduction, about 20% reduction to to about 45% reduction, about 20% reduction reduction to about 35% reduction, about 20% 20% reduction to about 25% reduction, about about 25% reduction to about 95% reduction, reduction, about 25% reduction to about 85% 80% reduction, about 25% reduction to about about 70% reduction, about 25% reduction to to about 60% reduction, about 25% reduction reduction to about 50% reduction, about 25% 25% reduction to about 40% reduction, about to about 60% reduction, about 10% reduction to about 50% reduction, about 10% reduction to about 40% reduction, about 10% reduction to about 30% reduction, about 10% reduction to about 15% reduction, about 15% reduction to about 95% reduction, about 15% reduction to about 85% reduction, about 15% reduction to about 75% reduction, about 15% reduction to about 65% reduction, about 15% reduction to about 55% reduction, about 15% reduction to about 40% reduction, about 15% reduction to about 30% reduction, about 15% reduction to about 20% reduction, about 20% reduction to about 95% reduction, about 20% reduction to about 85% reduction, about 20% reduction to about 75% reduction, about 20% reduction to about 60% reduction, about 20% reduction to about 50% reduction, about 20% reduction to about 40% reduction, about 20% reduction to about 30% reduction, about 25% reduction to about 99% reduction, about 25% reduction to about 90% reduction, about 25% reduction to about 75% reduction, about 25% reduction to about 65% reduction, about 25% reduction to about 55% reduction, about 25% reduction to about 45% reduction, about 25% reduction to about 35% reduction, about 25% reduction to about 30% reduction, reduction, about 30% reduction to about 95% 90% reduction, about 30% reduction to about about 80% reduction, about 30% reduction to to about 70% reduction, about 30% reduction reduction to about 60% reduction, about 30% 30% reduction to about 50% reduction, about about 30% reduction to about 40% reduction, reduction, about 35% reduction to about 99% 95% reduction, about 35% reduction to about about 85% reduction, about 35% reduction to to about 75% reduction, about 35% reduction reduction to about 65% reduction, about 35% 35% reduction to about 55% reduction, about about 35% reduction to about 45% reduction, reduction, about 40% reduction to about 99% 95% reduction, about 40% reduction to about about 85% reduction, about 40% reduction to to about 75% reduction, about 40% reduction reduction to about 65% reduction, about 40% 40% reduction to about 55% reduction, about about 40% reduction to about 45% reduction, reduction, about 45% reduction to about 95% 90% reduction, about 45% reduction to about about 80% reduction, about 45% reduction to to about 70% reduction, about 45% reduction reduction to about 60% reduction, about 45% 45% reduction to about 50% reduction, about about 50% reduction to about 95% reduction, reduction, about 50% reduction to about 85% 80% reduction, about 50% reduction to about about 30% reduction to about 99% reduction, about 30% reduction to about 85% reduction, about 30% reduction to about 75% reduction, about 30% reduction to about 65% reduction, about 30% reduction to about 55% reduction, about 30% reduction to about 45% reduction, about 30% reduction to about 35% reduction, about 35% reduction to about 90% reduction, about 35% reduction to about 80% reduction, about 35% reduction to about 70% reduction, about 35% reduction to about 60% reduction, about 35% reduction to about 50% reduction, about 35% reduction to about 40% reduction, about 40% reduction to about 90% reduction, about 40% reduction to about 80% reduction, about 40% reduction to about 70% reduction, about 40% reduction to about 60% reduction, about 40% reduction to about 50% reduction, about 45% reduction to about 99% reduction, about 45% reduction to about 85% reduction, about 45% reduction to about 75% reduction, about 45% reduction to about 65% reduction, about 45% reduction to about 55% reduction, about 50% reduction to about 99% reduction, about 50% reduction to about 90% reduction, about 50% reduction to about 75% reduction, about 50% reduction to about 70% reduction, about 50% reduction to about 65% reduction, about 50% reduction to about 60% reduction, about 50% reduction to about 55% reduction, about 55% reduction to about 99% reduction, about 55% reduction to about 95% reduction, about 55% reduction to about 90% reduction, about 55% reduction to about 85% reduction, about 55% reduction to about 80% reduction, about 55% reduction to about 75% reduction, about 55% reduction to about 70% reduction, about 55% reduction to about 65% reduction, about 55% reduction to about 60% reduction, about 60% reduction to about 99% reduction, about 60% reduction to about 95% reduction, about 60% reduction to about 90% reduction, about 60% reduction to about 85% reduction, about 60% reduction to about 80% reduction, about 60% reduction to about 75% reduction, about 60% reduction to about 70% reduction, about 60% reduction to about 65% reduction, about 65% reduction to about 99% reduction, about 65% reduction to about 95% reduction, about 65% reduction to about 90% reduction, about 65% reduction to about 85% reduction, about 65% reduction to about 80% reduction, about 65% reduction to about 75% reduction, about 65% reduction to about 70% reduction, about 70% reduction to about 99% reduction, about 70% reduction to about 95% reduction, about 70% reduction to about 90% reduction, about 70% reduction to about 85% reduction, about 70% reduction to about 80% reduction, about 70% reduction to about 75% reduction, about 75% reduction to about 99% reduction, about 75% reduction to about 95% reduction, about 75% reduction to about 90% reduction, about 75% reduction to about 85% reduction, about 75% reduction to about 80% reduction, about 80% reduction to about 99% reduction, about 80% reduction to about 95% reduction, about 80% reduction to about 90% reduction, about 80% reduction to about 85% reduction, about 85% reduction to about 99% reduction, about 85% reduction to about 95% reduction, about 85% reduction to about 90% reduction, about 90% reduction to about 99% reduction, about 90% reduction to about 95% reduction, or about 95% reduction to about 99% reduction) in the volume of one or more solid tumors in the subject (e.g., as compared to the volume of the one or more solid tumors prior to treatment or at the start of treatment). In some embodiments, the these methods can reduce (e.g., about 1% reduction to about 99% reduction, or any of the subranges of this range described herein) the risk of developing a metastasis or developing one or more additional metastasis in a subject (e.g., as compared to the risk of developing a metastasis or developing one or more additional metastasis in a subject prior to treatment or in a similar subject or a population of subjects administered a different treatment).

In some examples of these methods, the subject has been identified or diagnosed as having an aging-related disease or condition. Non-limiting examples of aging-related diseases and conditions include Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. In some examples, these methods can result in a reduction in the number, severity, or frequency of one or more symptoms of the agingrelated disease or condition in the subject (e.g., as compared to the number, severity, or frequency of the one or more symptoms of the aging-related disease or condition in the subject prior to treatment). In some examples, the methods can result in a decrease (e.g., about 1% decrease to about 99% decrease, an about 1% decrease to about 95% decrease, about 1% decrease to about 90% decrease, about 1% decrease to about 85% decrease, about 1% decrease to about 80% decrease, about 1% decrease to about 75% decrease, about 1% to about 70% decrease, about 1% decrease to about 65% decrease, about 1% decrease to about 60% decrease, about 1% decrease to about 55% decrease, about 1% decrease to about 50% decrease, about 1% decrease to about 45% decrease, about 1% decrease to about 40% decrease, about 1% decrease to about 35% decrease, about 1% decrease to about 30% decrease, about 1% decrease to about 25% decrease, about 1% decrease to about 20% decrease, about 1% decrease to about 15% decrease, about 1% decrease to about 10% decrease, about 1% decrease to about 5% decrease, about 5% decrease to about 99% decrease, an about 5% decrease to about 95% decrease, about 5% decrease to about 90% decrease, about 5% decrease to about 85% decrease, about 5% decrease to about 80% decrease, about 5% decrease to about 75% decrease, about 5% to about 70% decrease, about 5% decrease to about 65% decrease, about 5% decrease to about 60% decrease, about 5% decrease to about 55% decrease, about 5% decrease to about 50% decrease, about 5% decrease to about 45% decrease, about 5% decrease to about 40% decrease, about 5% decrease to about 35% decrease, about 5% decrease to about 30% decrease, about 5% decrease to about 25% decrease, about 5% decrease to about 20% decrease, about 5% decrease to about 15% decrease, about 5% decrease to about 10% decrease, about 10% decrease to about 99% decrease, an about 10% decrease to about 95% decrease, about 10% decrease to about 90% decrease, about 10% decrease to about 85% decrease, about 10% decrease to about 80% decrease, about 10% decrease to about 75% decrease, about 10% to about 70% decrease, about 10% decrease to about 65% decrease, about 10% decrease to about 60% decrease, about 10% decrease to about 55% decrease, about 10% decrease to about 50% decrease, about 10% decrease to about 45% decrease, about 10% decrease to about 40% decrease, about 10% decrease to about 35% decrease, about 10% decrease to about 30% decrease, about 10% decrease to about 25% decrease, about 10% decrease to about 20% decrease, about 10% decrease to about 15% decrease, about 15% decrease to about 99% decrease, an about 15% decrease to about 95% decrease, about 15% decrease to about 90% decrease, about 15% decrease to about 85% decrease, about 15% decrease to about 80% decrease, about 15% decrease to about 75% decrease, about 15% to about 70% decrease, about 15% decrease to about 65% decrease, about 15% decrease to about 60% decrease, about 15% decrease to about 55% decrease, about 15% decrease to about 50% decrease, about 15% decrease to about 45% decrease, about 15% decrease to about 40% decrease, about 15% decrease to about 35% decrease, about 15% decrease to about 30% decrease, about 15% decrease to about 25% decrease, about 15% decrease to about 20% decrease, about 20% decrease to about 99% decrease, an about 20% decrease to about 95% decrease, about 20% decrease to about 90% decrease, about 20% decrease to about 85% decrease, about 20% decrease to about 80% decrease, about 20% decrease to about 75% decrease, about 20% to about 70% decrease, about 20% decrease to about 65% decrease, about 20% decrease to about 60% decrease, about 20% decrease to about 55% decrease, about 20% decrease to about 50% decrease, about 20% decrease to about 45% decrease, about 20% decrease to about

40% decrease, about 20% decrease to about 35% decrease, about 20% decrease to about 30% decrease, about 20% decrease to about 25% decrease, about 25% decrease to about 99% decrease, an about 25% decrease to about 95% decrease, about 25% decrease to about 90% decrease, about 25% decrease to about 85% decrease, about 25% decrease to about 80% decrease, about 25% decrease to about 75% decrease, about 25% to about 70% decrease, about 25% decrease to about 65% decrease, about 25% decrease to about 60% decrease, about 25% decrease to about 55% decrease, about 25% decrease to about 50% decrease, about 25% decrease to about 45% decrease, about 25% decrease to about 40% decrease, about 25% decrease to about 35% decrease, about 25% decrease to about 30% decrease, about 30% decrease to about 99% decrease, an about 30% decrease to about 95% decrease, about 30% decrease to about 90% decrease, about 30% decrease to about 85% decrease, about 30% decrease to about 80% decrease, about 30% decrease to about 75% decrease, about 30% to about 70% decrease, about 30% decrease to about 65% decrease, about 30% decrease to about 60% decrease, about 30% decrease to about 55% decrease, about 30% decrease to about 50% decrease, about 30% decrease to about 45% decrease, about 30% decrease to about 40% decrease, about 30% decrease to about 35% decrease, about 35% decrease to about 99% decrease, an about 35% decrease to about 95% decrease, about 35% decrease to about 90% decrease, about 35% decrease to about 85% decrease, about 35% decrease to about 80% decrease, about 35% decrease to about 75% decrease, about 35% to about 70% decrease, about 35% decrease to about 65% decrease, about 35% decrease to about 60% decrease, about 35% decrease to about 55% decrease, about 35% decrease to about 50% decrease, about 35% decrease to about 45% decrease, about 35% decrease to about 40% decrease, about 40% decrease to about 99% decrease, an about 40% decrease to about 95% decrease, about 40% decrease to about 90% decrease, about 40% decrease to about 85% decrease, about 40% decrease to about 80% decrease, about 40% decrease to about 75% decrease, about 40% to about 70% decrease, about 40% decrease to about 65% decrease, about 40% decrease to about 60% decrease, about 40% decrease to about 55% decrease, about 40% decrease to about 50% decrease, about 40% decrease to about 45% decrease, about 45% decrease to about 99% decrease, an about 45% decrease to about 95% decrease, about 45% decrease to about 90% decrease, about 45% decrease to about 85% decrease, about 45% decrease to about 80% decrease, about 45% decrease to about 75% decrease, about 45% to about 70% decrease, about 45% decrease to about 65% decrease, about 45% decrease to about 60% decrease, about 45% decrease to about 55% decrease, about 45% decrease to about 50% decrease, about 50% decrease to about 99% decrease, an about 50% decrease to about 95% decrease, about 50% decrease to about 90% decrease, about 50% decrease to about 85% decrease, about 50% decrease to about 80% decrease, about 50% decrease to about 75% decrease, about 50% to about 70% decrease, about 50% decrease to about 65% decrease, about 50% decrease to about 60% decrease, about 50% decrease to about 55% decrease, about 55% decrease to about 99% decrease, an about 55% decrease to about 95% decrease, about 55% decrease to about 90% decrease, about 55% decrease to about 85% decrease, about 55% decrease to about 80% decrease, about 55% decrease to about 75% decrease, about 55% to about 70% decrease, about 55% decrease to about 65% decrease, about 55% decrease to about 60% decrease, about 60% decrease to about 99% decrease, an about 60% decrease to about 95% decrease, about 60% decrease to about 90% decrease, about 60% decrease to about 85% decrease, about 60% decrease to about 80% decrease, about 60% decrease to about 75% decrease, about 60% to about 70% decrease, about 60% decrease to about 65% decrease, about 65% decrease to about 99% decrease, an about 65% decrease to about 95% decrease, about 65% decrease to about 90% decrease, about 65% decrease to about 85% decrease, about 65% decrease to about 80% decrease, about 65% decrease to about 75% decrease, about 65% to about 70% decrease, about 70% decrease to about 99% decrease, an about 70% decrease to about 95% decrease, about 70% decrease to about 90% decrease, about 70% decrease to about 85% decrease, about 70% decrease to about 80% decrease, about 70% decrease to about 75% decrease, about 75% decrease to about 99% decrease, an about 75% decrease to about 95% decrease, about 75% decrease to about 90% decrease, about 75% decrease to about 85% decrease, about 75% decrease to about 80% decrease, about 80% decrease to about 99% decrease, an about 80% decrease to about 95% decrease, about 80% decrease to about 90% decrease, about 80% decrease to about 85% decrease, about 85% decrease to about 99% decrease, an about 85% decrease to about 95% decrease, about 85% decrease to about 90% decrease, about 90% decrease to about 99% decrease, an about 90% decrease to about 95% decrease, or about 95% decrease to about 99% decrease) in the number of senescent cells in the subject (e.g., a decrease in the number of senescent cells in one or more specific tissues involved and/or implicated in the agingrelated disease or disorder in the subject), e.g., as compared to the number of senescent cells in the subject prior to treatment.

In some examples of these methods, the subject has been diagnosed or identified as having an infectious disease. Non-limiting examples of infectious disease include infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus. In some embodiments, these methods can result in a decrease in the infectious titer (e.g., viral titer) in a subject (e.g., as compared to the infectious titer in the subject prior to treatment). In some embodiments, these methods can result in a reduction in the number, severity, or frequency of one or more symptoms of the infectious disease (e.g., viral infection) in the subject (e.g., as compared to the number, severity, or frequency of the one or more symptoms of the infectious disease in the subject prior to treatment).

The term “subject” refers to any mammal. In some embodiments, the subject or “subject in need of treatment” may be a canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), ovine, bovine, porcine, caprine, primate, e.g., a simian (e.g., a monkey (e.g., marmoset, baboon), or an ape (e.g., a gorilla, chimpanzee, orangutan, or gibbon) or a human; or rodent (e.g., a mouse, a guinea pig, a hamster, or a rat). In some embodiments, the subject or “subject in need of treatment” may be a non-human mammal, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g., murine, lapine, porcine, canine or primate animals) may be employed.

Methods of Killing a Cancer Cell, an Infected Cell, or a Senescent Cell

Also provided herein are methods of killing a cancer cell (e.g., any of the exemplary types of cancer described herein or known in the art), an infected cell (e.g., a cell infected with any of the exemplary viruses described herein or known in the art), or a senescent cell (e.g., a senescent cancer cell, a senescent fibroblast, or a senescent endothelial cell) in a subject in need thereof (e.g., any of the exemplary subjects described herein or known in the art) that include administering to the subject a therapeutically effective amount of any of the multi-chain chimeric polypeptides described herein or any of the compositions (e.g., pharmaceutical compositions) described herein.

In some embodiments of these methods, the subject has been identified or diagnosed as having a cancer. Non-limiting examples of cancer include: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some examples of these methods, the subject has been identified or diagnosed as having an aging-related disease or condition. Non-limiting examples of aging-related diseases and conditions include Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

In some examples of these methods, the subject has been diagnosed or identified as having an infectious disease. Non-limiting examples of an infectious disease include infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Senescent Cells

Senescence is a form of irreversible growth arrest accompanied by phenotypic changes, resistance to apoptosis and activation of damage-sensing signaling pathways. Cellular senescence was first described in cultured human fibroblast cells that lost their ability to proliferate, reaching permanent arrest after about 50 population doublings (referred to as the Hayflick limit). Senescence is considered a stress response that can be induced by a wide range of intrinsic and extrinsic insults, including oxidative and genotoxic stress, DNA damage, telomere attrition, oncogenic activation, mitochondrial dysfunction, or chemotherapeutic agents.

Senescent cells remain metabolically active and can influence the tissue hemostasis, disease and aging through their secretory phenotype. Senescence is considered as a physiologic process and is important in promoting wound healing, tissue homeostasis, regeneration, and fibrosis regulation. For instance, transient induction of senescent cells is observed during would healing and contributes to wound resolution. Perhaps one of the most important roles of senescence is its role in tumor suppression. However, the accumulation of senescent cells also drives aging- and aging-related diseases and conditions. The senescent phenotype also can trigger chronic inflammatory responses and consequently augment chronic inflammatory conditions to promote tumor growth. The connection between senescence and aging was initially based on observations that senescent cells accumulate in aged tissue. The use of transgenic models has enabled the detection of senescent cells systematically in many age-related pathologies. Strategies to selectively eliminate senescent cells has demonstrated that senescent cells can indeed play a causal role in aging and related pathologies.

Senescent cells display important and unique properties which include changes in morphology, chromatin organization, gene expression, and metabolism. There are several biochemical and functional properties associated with cellular senescence, such as (i) increased expression of pl6 and p21, inhibitors of cyclin-dependent kinases, (ii) presence of senescence-associated β-galactosidase, a marker of lysosomal activity, (iii) appearance of senescence-associated heterochromatin foci and downregulation of lamin Bl levels, (iv) resistance to apoptosis caused by an increased expression of anti-apoptotic BCL-family protein, and (v) upregulation of CD26 (DPP4), CD36 (Scavenger receptor), forkhead box 4 (FOXO4), and secretory carrier membrane protein 4 (SCAMP4). Senescent cells also express an inflammatory signature, the so-called senescenceassociated secretory phenotype (SASP). Through SASP, the senescent cells produce a wide range of inflammatory cytokines (IL-6, IL-8), growth factors (TGF-β), chemokines (CCL-2), and matrix metalloproteinases (MMP-3, MMP-9) that operate in a cellautonomous manner to reinforce senescence (autocrine effects) and communicate with and modify the microenvironment (paracrine effects). SASP factors can contribute to tumor suppression by triggering senescence surveillance, an immune-mediated clearance of senescent cells. However, chronic inflammation is also a known driver of tumorigenesis, and accumulating evidence indicates that chronic SASP can also boost cancer and aging-related diseases.

The secretion profile of senescent cells is context dependent. For instance, the mitochondrial dysfunction-associated senescence (MiDAS), induced by different mitochondrial dysfunction in human fibroblasts, led to the appearance of a SASP that was deficient in IL dependent inflammatory factors. A decrease in the NAD+/NADH ratio activated AMPK signaling which induced MiDAS through the activation of p53. As a result, p53 inhibited NF-κΒ signaling which is a crucial inducer of pro-inflammatory SASP. In contrast, the cellular senescence caused by persistent DNA damage in human cells induced an inflammatory SASP, which was dependent on the activation of ataxiatelangiectasia mutated (ATM) kinase but not on that of p53. In particular, the expression and secretion levels of IL-6 and IL-8 were increased. It was also demonstrated that cellular senescence caused by the ectopic expression pl6INK4a and p21CIPl induced the senescent phenotype in human fibroblasts without an inflammatory SASP indicating that the growth arrest itself did not stimulate SASP.

One of the most defining characteristics of senescence is stable growth arrest. This is achieved by two important pathways, the pl6/Rb and the p53/p21, both of which are central in tumor suppression. DNA damage results in: (1) high deposition of γΗ2Αχ (histone coding gene) and 53BP1 (involved in DNA damage response) in chromatin: this leads to activation of a kinase cascade eventually resulting in p53 activation, and (2) activation of pl6INK4a and ARF (both encoded by CDKN2A) and P15INK4b (encoded by CDKN2B): p53 induces transcription of cyclin-dependent kinase inhibitor (p21) and along with both pl6INK4a and pl5INK4b block genes for cell cycle progression (CDK4 and CDK6). This eventually leads to hypophosphorylation of Retinoblastoma protein (Rb) and cell cycle arrest at the G1 phase.

Selectively killing senescent cells has been shown to significantly improve the health span of mice in the context of normal aging and ameliorates the consequences of age-related disease or cancer therapy (Ovadya, J Clin Invest. 128(4):1247-1254, 2018). In nature, the senescent cells are normally removed by the innate immune cells. Induction of senescence not only prevents the potential proliferation and transformation of damaged/altered cells, but also favors tissue repair through the production of SASP factors that function as chemoattractants mainly for Natural Killer (NK) cells (such as IL15 and CCL2) and macrophages (such as CFS-1 and CCL2). These innate immune cells mediate the immunosurveillance mechanism for eliminating stressed cells. Senescent cells usually up-regulate the NK-cell activating receptor NKG2D and DNAM-1 ligands, which belong to a family of stress-inducible ligands: an important component of the frontline immune defense against infectious diseases and malignancies. Upon receptor activation, NK cells can then specifically induce the death of senescent cells through their cytolytic machinery. A role for NK cells in the immune surveillance of senescent cells has been pointed out in liver fibrosis (Sagiv, Oncogene 32(15): 1971-1977, 2013), hepatocellular carcinoma (lannello, J Exp Med 210(10): 2057-2069, 2013), multiple myeloma (Soriani, Blood 113(15): 3503-3511, 2009), and glioma cells stressed by dysfunction of the mevalonate pathway (Ciaglia, Int J Cancer 142(1): 176-190, 2018). Endometrial cells undergo acute cellular senescence and do not differentiate into decidual cells. The differentiated decidual cells secrete IL-15 and thereby recruit uterine NK cells to target and eliminate the undifferentiated senescent cells thus helping to re-model and rejuvenate the endometrium (Brighton, Elife 6: e31274, 2017). With a similar mechanism, during liver fibrosis, p53-expressing senescent liver satellite cells skewed the polarization of resident Kupfer macrophages and freshly infiltrated macrophages toward the pro-inflammatory Ml phenotype, which display senolytic activity. F4/80+ macrophages have been shown to play a key role in the clearance of mouse uterine senescent cells to maintain postpartum uterine function.

Senescent cells recruit NK cells by mainly upregulating ligands to NKG2D (expressed on NK cells), chemokines, and other SASP factors. In vivo models of liver fibrosis have shown effective clearance of senescent cells by activated NK cells (Krizhanovsky, Cell 134(4): 657-667, 2008). Studies have described various models to study senescence including liver fibrosis (Krizhanovsky, Cell 134(4): 657-667, 2008), osteoarthritis (Xu, J Gerontol A Biol SciMed Sci 72(6): 780-785, 2017), and Parkinson’s disease (Chinta, Cell Rep 22(4): 930-940, 2018). Animal models for studying senescent cells are described in: Krizhanovsky, Cell 134(4): 657-667, 2008; Baker, Nature 479(7372): 232-236, 2011; Farr, NatMed23(9\ 1072-1079, 2017; Bourgeois, FEBSLett 592(12): 2083-2097, 2018; Xu, W Med 24(8): 1246-1256, 2018).

Additional Therapeutic Agents

Some embodiments of any of the methods described herein can further include administering to a subject (e.g., any of the subjects described herein) a therapeutically effective amount of one or more additional therapeutic agents. The one or more additional therapeutic agents can be administered to the subject at substantially the same time as the multi-chain chimeric polypeptide (e.g., any of the multi-chain chimeric polypeptides described herein) or immune cell (e.g., administered as a single formulation or two or more formulations to the subject). In some embodiments, one or more additional therapeutic agents can be administered to the subject prior to administration of the multi-chain chimeric polypeptide (e.g., any of the multi-chain chimeric polypeptides described herein) or immune cell. In some embodiments, one or more additional therapeutic agents can be administered to the subject after administration of the multichain chimeric polypeptide (e.g., any of the multi-chain chimeric polypeptides described herein) or immune cell to the subject.

Non-limiting examples of additional therapeutic agents include: anti-cancer drugs, activating receptor agonists, immune checkpoint inhibitors, agents for blocking HLAspecific inhibitory receptors, Glucogen Synthase Kinase (GSK) 3 inhibitors, and antibodies.

Non-limiting examples of anticancer drugs include antimetabolic drugs (e.g., 5fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxycarbamide, methotrexate, 6-thioguanine, cladribine, nelarabine, pentostatin, or pemetrexed), plant alkaloids (e.g., vinblastine, vincristine, vindesine, camptothecin, 9-methoxycamptothecin, coronaridine, taxol, naucleaorals, diprenylated indole alkaloid, montamine, schischkiniin, protoberberine, berberine, sanguinarine, chelerythrine, chelidonine, liriodenine, clivorine, β-carboline, antofine, tylophorine, cryptolepine, neocryptolepine, corynoline, sampangine, carbazole, crinamine, montanine, ellipticine, paclitaxel, docetaxel, etoposide, tenisopide, irinotecan, topotecan, or acridone alkaloids), proteasome inhibitors (e.g., lactacystin, disulfiram, epigallocatechin gallate, marizomib (salinosporamide A), oprozomib (ONX-0912), delanzomib (CEP-18770), epoxomicin, MG132, beta-hydroxy beta-methylbutyrate, bortezomib, carfilzomib, or ixazomib), antitumor antibiotics (e.g., doxorubicin, daunorubicin, epirubicin, mitoxantrone, idarubicin, actinomycin, plicamycin, mitomycin, or bleomycin), histone deacetylase inhibitors (e.g., vorinostat, panobinostat, belinostat, givinostat, abexinostat, depsipeptide, entinostat, phenyl butyrate, valproic acid, trichostatin A, dacinostat, mocetinostat, pracinostat, nicotinamide, cambinol, tenovin 1, tenovin 6, sirtinol, ricolinostat, tefinostat, kevetrin, quisinostat, resminostat, tacedinaline, chidamide, or selisistat), tyrosine kinase inhibitors (e.g., axitinib, dasatinib, encorafinib, erlotinib, imatinib, nilotinib, pazopanib, and sunitinib), and chemotherapeutic agents (e.g., all-trans retinoic acid, azacitidine, azathioprine, doxifluridine, epothilone, hydroxyurea, imatinib, teniposide, tioguanine, valrubicin, vemurafenib, and lenalidomide). Additional examples of chemotherapeutic agents include alkylating agents, e.g., mechlorethamine, cyclophosphamide, chlorambucil, melphalan, ifosfamide, thiotepa, hexamethylmelamine, busulfan, altretamine, procarbazine, dacarbazine, temozolomide, carmustine, lumustine, streptozocin, carboplatin, cisplatin, and oxaliplatin.

Non-limiting examples of activating receptor agonists include any agonists for activating receptors which activate and enhance the cytotoxicity of NK cells, including anti-CD16 antibodies (e.g., anti-CD16/CD30 bispecific monoclonal antibody (BiMAb)) and Fc-based fusion proteins. Non-limiting examples of checkpoint inhibitors include anti-PD-1 antibodies (e.g., MEDI0680), anti-PD-Ll antibodies (e.g., BCD-135, BGBA333, CBT-502, CK-301, CS1001, FAZ053, KN035, MDX-1105, MSB2311, SHR1316, anti-PD-Ll/CTLA-4 bispecific antibody KN046, anti-PD-Ll/TGF3RII fusion protein M7824, anti-PD-Ll/TIM-3 bispecific antibody LY3415244, atezolizumab, or avelumab), anti-TIM3 antibodies (e.g., TSR-022, Sym023, 0rMBG453) and anti-CTLA4 antibodies (e.g., AGEN1884, MK-1308, or an anti-CTLA-4/OX40 bispecific antibody ATOR-1015). Non-limiting examples of agents for blocking HLA-specific inhibitory receptors include monalizumab (e.g., an anti-HLA-E NKG2A inhibitory receptor monoclonal antibody). Non-limiting examples of GSK3 inhibitor include tideglusib or CHIR99021. Non-limiting examples of antibodies that can be used as additional therapeutic agents include anti-CD26 antibodies (e.g., YS110), anti-CD36 antibodies, and any other antibody or antibody construct that can bind to and activate an Fc receptor (e.g., CD 16) on a NK cell. In some embodiments, an additional therapeutic agent can be insulin or metformin.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1. Construction of exemplary multi-chain chimeric polypeptides and evaluation of properties thereof

Two multi-chain chimeric polypeptides were generated and their properties were evaluated. Each of the two multi-chain chimeric polypeptides includes a first chimeric polypeptide that includes a soluble tissue factor domain covalently linked a first targetbinding domain and a first domain of an affinity pair of domains. The second chimeric polypeptide in each of the two multi-chain chimeric polypeptides includes a second domain of the affinity pair of domains, and a second target-binding domain.

Description of logic underlying construction of multi-chain chimeric polypeptides

Tissue Factor (TF) is a stable, transmembrane protein containing 236 amino acid residues. The truncated, recombinant 219-amino-acid extracellular domain of tissue factor is soluble and is known to be expressed at high levels in bacteria or mammalian cells. Without wishing to be bound to a particular theory, the applicants speculated that the 219-aa tissue factor could be used as a connector linker for creation of unique multichain chimeric polypeptides.

First chimeric polypeptides including soluble tissue factor domain were produced at high levels by CHO cells grown in fermentation broth. These first chimeric polypeptides were purified by an anti-tissue factor monoclonal antibody (mAb) coupled on a solid matrix. Notably, tissue factor contains binding sites for FVIIa and FX. The catalytic activity of the tissue factor-FVIIa complex for FX is approximately 1 millionfold lower when tissue factor is not anchored to a phospholipid bilayer. Thus, without wishing to be bound to a particular theory, applicants speculated that using the 219-aa extracellular domain of tissue factor without the transmembrane in construction of the first chimeric polypeptides may eliminate the pro-coagulation activity of tissue factor in the first chimeric polypeptides. In an effort to further reduce or eliminate the procoagulation activity of the 219-aa tissue factor, select mutations in tissue factor can be made, specifically at seven amino acid residues that are known to contribute to binding energy of the FVIIa binding site.

Characterization of binding interactions for described chimeric polypeptides

To determine if the first and second chimeric polypeptides bind to each other to form multi-chain chimeric polypeptides, in vitro binding assays were performed. To determine if the first chimeric polypeptide comprising soluble tissue factor domain are recognized and bound by anti-TF mAb, in vitro binding assays were performed. Notably, the data indicated that the mutated tissue factor proteins are still recognized and selectively bound by the anti-TF mAb which is known to bind to the FX binding site on tissue factor. To determine if the first chimeric polypeptides comprising soluble tissue factor domain covalently linked to scFvs or cytokines (see Figure 1 and Figure 2) possess functional scFvs or cytokines, in vitro binding assays were performed. The data from the aforementioned assays were consistent with the purified first chimeric polypeptides having the expected biological activities (e.g. scFvs selectively bind expected target antigens or cytokines selectively bind expected receptors or binding proteins).

In addition, experiments performed using the two multi-chain chimeric polypeptides including a first and second chimeric polypeptide bound to each other demonstrate the expected target binding activity (e.g., the multi-chain chimeric polypeptide binds specifically to the target specifically recognized by the first targetbinding domain and the target specifically recognized by the second target-binding domain).

Based on the aforementioned results, applicants concluded that the soluble tissue factor connecter linker provided or enabled appropriate display of the polypeptides encoding either scFvs, interleukins, cytokines, interleukin receptors, or cytokine receptors in three-dimensional space relative to soluble tissue factor domain and relative to one another such that each retained expected biological properties and activities.

When both the first and second chimeric polypeptides were co-expressed, the heterodimeric complexes were secreted into the fermentation broths at high levels. The complexes were captured and readily purified by anti-TF mAb conjugated to a solid matrix using affinity chromatography. The first and second target-binding domains of these multi-chain chimeric polypeptides retained their expected biological activities as assayed by in vitro binding assays. Thus, the assembly of the multi-chain chimeric polypeptides provides the appropriate spatial display and folding of the domains for biological activities. Importantly, the spatial arrangement of the multi-chain chimeric polypeptides does not interfere with the FX binding site on tissue factor which enables the use of anti-TF mAb for affinity purification.

Characterization of stability for described chimeric polypeptides

Both purified multi-chain chimeric polypeptides are stable. These multi-chain chimeric polypeptides are structurally intact and fully biologically active when they are incubated in human serum at 37 °C for 72 hours.

Characterization ofpropensity of described chimeric polypeptides to aggregate

Both purified multi-chain chimeric polypeptides developed do not form aggregates when stored at 4 °C in PBS.

Characterization of viscosity of described chimeric polypeptides

There is no viscosity issue when the multi-chain chimeric polypeptides are formulated at a concentration as high as 50 mg/mL in PBS.

Addditional applications of the multi-chain chimeric polypeptide platform

The data from these studies show that the platform technologies described herein can be utilized to create molecules that could be fused to target-binding domains derived from antibodies, in any of the formats as described herein including, without limitation, adhesion molecules, receptors, cytokines, ligands, and chemokines. With the appropriate target-binding domain, the resulting multi-chain chimeric polypeptides could promote conjugation of various immune effector cells and mediate destruction of target cells, including cancer cells, virally-infected cells, or senescent cells. Other domains in the multi-chain chimeric polypeptides stimulate, activate, and attract the immune system for enhancing cytotoxicity of effector cells for the targeted cells.

Example 2: Creation of an IL-12/IL-15RaSu DNA construct

In a non-limiting example, an IL-12/IL-15RaSu DNA construct was created (Figure 3). The human IL-12 subunit sequences, human IL-15RaSu sequence, human IL-15 sequence, human tissue factor 219 sequence, and human IL-18 sequence were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. A DNA construct was made linking the IL-12 subunit beta (p40) to IL-12 subunit alpha (p35) with a GS (3) linker to generate a single chain version of IL-12 and then directly linking the IL-12 sequence to the IL-15RaSu sequence. The final IL-12/IL15RaSu DNA construct sequence was synthesized by Genewiz.

The nucleic acid sequence of the IL12/IL-15RaSu construct (including signal peptide sequence) is as follows (SEQ ID NO: 77):

(Signal peptide)

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCT ACTCC (Human IL-12 subunit beta (p40))

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTAT CCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAG ACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAA GACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGC CACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGG AAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGA ATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGT TGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCG GGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCT GAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAA GAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGG TGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATC CGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCA AAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCAC ACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCA AGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCA TCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCC AGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCC (Linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human IL-12 subunit alpha (p35j)

CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTAC ACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAG GCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGAT ATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGA CAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGG CTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTC CATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCC AAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGG CTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCT

CAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGT GCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATG AGCTATTTAAACGCCAGC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

Example 3: Creation of an IL-18/TF/IL-15 DNA construct

In a non-limiting example, an IL-18/TF/IL-15 construct was made (Figure 4) linking the IL-18 sequence to the N-terminus coding region of tissue factor 219, and further linking the IL-18/TF construct with the N-terminus coding region of IL-15. The nucleic acid sequence of the IL-18/TF/IL-15 construct (including leader sequence), synthesized by Genewiz, is as follows (SEQ ID NO: 73):

(Signal peptide)

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCT ACAGC (Human IL-18)

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAAC GACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGAC CGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGT ACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGA GAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATG AACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGC GGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGA GGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCA AGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGA GGAT (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

Example 4: Secretion of IL-12/IL-15RaSu and IL-18/TF/IL-15 fusion proteins

The IL-12/IL-15RaSu and IL-18/TF/IL-15 DNA constructs were cloned into a pMSGV-1 modified retrovirus expression vector (as described by Hughes, Hum Gene Ther 16:457-72, 2005, hereby incorporated by reference), and the expression vector was transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of a soluble IL-18/TF/IL-15:IL-12/IL-15RaSu protein complex (referred to as 18tl5-12s; Figure 5 and Figure 6). The 18tl5-12s protein was purified from CHO-K1 cell culture supernatant using anti-TF antibody affinity chromatography and size exclusion chromatography resulting in soluble (non-aggregated) protein complexes consisting of IL-12/IL-15RaSu and IL-18/TF/IL-15 fusion proteins.

The amino acid sequence of the IL12/IL-15RaSu fusion protein (including signal peptide sequence) is as follows (SEQ ID NO: 76):

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL-12 subunit beta (p40))

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGS GKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKN KTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERV RGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPD PPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVF TDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (Linker)

GGGGSGGGGSGGGGS (Human IL-12 subunit alpha (p35))

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHE DITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYE DLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSS LEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR

The amino acid sequence of the IL-18/TF/IL-15 fusion protein (including signal peptide sequence) is as follows (SEQ ID NO: 72):

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL-18)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISM YKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPG HDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

In some cases, the leader (signal sequence) peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

Example 5: Purification of 18tl5-12s by immunoaffinity chromatography

An anti-TF antibody affinity column was connected to a GE Healthcare™ AKTA Avant protein purification system. The flow rate was 4 mL/min for all steps except the elution step, which was 2 mL/min.

Cell culture harvest of 18tl5-12s was adjusted to pH 7.4 with IM Tris base and loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes PBS, followed by elution with 6 column volumes 0. IM acetic acid, pH 2.9. Absorbance at 280 nm was collected and then the sample was neutralized to pH 7.5-8.0 by adding IM Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon® centrifugal filters with a 30 KDa molecular weight cutoff. Figure 7 shows that the 18tl512s complex binds the anti-TF antibody affinity column, wherein TF is an 18tl5-12s binding partner. The buffer-exchanged protein sample is stored at 2-8°C for further biochemical analysis and biological activity testing.

After each elution, the anti-TF antibody affinity column was then stripped using 6 column volumes 0. IM glycine, pH 2.5. The column was then neutralized using 10 column volumes PBS, 0.05% sodium azide and stored at 2-8°C.

Example 6: Size exclusion chromatography of 18tl5-12s

A GE Healthcare Superdex® 200 Increase 10/300 GL gel filtration column was connected to a GE Healthcare AKTA™ Avant protein purification system. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.8 mL/min. A capillary loop was used to inject 200pL of 1 mg/mL of 18tl5-12s complex onto the column. The injection was chased with 1.25 column volumes of PBS. The SEC chromatograph is shown in Figure 8. There is a main 18tl5-12s protein peak with a minor high molecular weight peak, likely due to differing degrees of glycosylation of 18tl5-12s dimers or aggregates.

Example 7: SDS-PAGE of 18tl5-12s

To determine the purity and protein molecular weight, the purified 18tl5-12s protein sample was analyzed using 4-12% NuPage Bis-Tris protein gel SDS-PAGE. The gel was stained with InstantBlue™ for about 30 min, followed by destaining overnight in purified water. Figure 9 shows an example SDS gel of anti-TF antibody affinity purified 18tl5-12s, with bands at the expected molecular weights (66 kDa and 56 kDa).

Example 8: Glycosylation of 18tl5-12s in CHO-K1 cells

Glycosylation of 18tl5-12s in CHO-K1 cells was confirmed using the Protein Deglycosylation Mix II kit (New England Biolabs), according to the manufacturer’s instructions. Figure 10 shows an example SDS PAGE of deglycosylated and nondeglycosylated 18tl5-12s. Deglycosylation reduces the molecular weight of 18tl5-12s as seen in Figure 10, lane 4.

Example 9: Recombinant protein quantitation of 18tl5-12s complexes

The 18tl5-12s complex was detected and quantified using standard sandwich ELISA methods (Figures 11-14). Anti-human tissue factor antibody served as the capture antibody and biotinylated anti-human IL-12, IL-15, or IL-18 antibody (BAF 219, BAM 247, D045-6, all R&D Systems) served as the detection antibody. Tissue factor in purified 18tl5-12s protein complexes was also detected using an anti-human tissue factor capture antibody (143), and anti-human tissue factor antibody detection antibody. The 143/ anti-TF antibody ELISA was compared to purified tissue factor at similar concentrations.

Example 10: Immunostimulatory capacity of the 18tl5-12s complex

To assess the IL-15 immunostimulatory activity of the 18tl5-12s complex, increasing concentrations of 18tl5-12s was added to 32ϋβ cells (104 cell/well) in 200 pL IMDM: 10% FBS media. The 32ϋβ cells were incubated for 3 days at 37°C. On the fourth day, WST-1 proliferation reagent (10 pL/well) was added and after 4 hours, absorbance was measured at 450 nm to determine cell proliferation based on cleavage of WST-1 to a soluble formazan dye. Bioactivity of human recombinant IL-15 was assessed as a positive control. As shown in Figure 15, 18tl5-12s demonstrated IL dependent cell proliferation of 32ϋβ cells. The 18tl5-12s complex demonstrated reduced activity compared to human recombinant IL-15, possibly due to the linkage of IL-18 and tissue factor to the IL-15 domain.

In order to assess the individual activities of IL-12 and IL-18 in the 18tl5-12s complex, 18tl5-12s was added to HEK-Blue IL-12 and HEK-Blue IL-18 reporter cells (5xl04 cell/well; hkb-ill2 and hkb-hmill8, InvivoGen) in 200 pL IMDM: 10% heatinactivated FBS media. Cells were incubated for overnight at 37°C. 20 pl of induced HEK-Blue IL-12 and HEK-Blue IL-18 reporter cell supernatant was added to 180 pl of QUANTI-Blue (InvivoGen), and incubated for 1-3 hours at 37°C. IL-12 or IL-18 activity was assessed by measuring absorbance at 620 nm. Human recombinant IL-12 or IL-18 was assessed as a positive or negative control. As shown in Figure 16 and Figure 17, each of the cytokine domains of the 18tl5-12s complex retain specific biological activity. The activity of 18tl5-12s was reduced compared to that of human recombinant IL-18 or IL-12, possibly due to linkage of IL-15 and tissue factor to the IL-18 domain and linkage of IL-12 to the IL-15R0C sushi domain.

Example 11: Induction of cytokine-induced memory-like NK cells by the 18tl5-12s complex

Cytokine-induced memory-like NK cells can be induced ex vivo following overnight stimulation of purified NK cells with saturating amounts of IL-12 (10 ng/mL), IL-15 (50 ng/mL), and IL-18 (50 ng/mL). These memory-like properties have been measured through expression of IL-2 receptor a (IL-2Ra, CD25), CD69 (and other activation markers), and increased IFN-γ production. To evaluate the ability of 18tl5-12s complexes to promote generation of cytokine-induced memory-like NK cells, purified human NK cells (>95% CD56+) were stimulated for 14-18 hours with O.OlnM to 10000 nM of the 18tl5-12s complex or a combination of individual cytokines (recombinant IL12 (10 ng/mL), IL-18 (50 ng/mL), and IL-15 (50 ng/mL)). Cell-surface CD25 and CD 69 expression and intracellular IFN-γ levels were assessed by antibody-staining and flow cytometry.

Fresh human leukocytes were obtained from a blood bank and CD56+ NK cells were isolated with the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >70% and confirmed by staining with antibodies specific to CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 (BioLegend). Cells were counted and resuspended in 0.2 x 106/mL in a 96 well flat bottom plate in 0.2 mL of complete media (RPMI 1640 (Gibco), supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). Cells were stimulated with either a mixture of cytokines hIL-12 (10 ng/mL) (Biolegend), hIL-18 (50 ng/mL) (R&D Systems) and hIL15 (50 ng/mL) (NCI) or with 0.01 nM to lOOOOnM of the 18tl5-12s at 37°C, 5% CO2 for 14-18 hrs. The cells were then harvested and surface stained with antibodies specific to CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 (BioLegend) for 30 minutes. After staining, cells were washed (1500 RPM for 5 minutes at room temperature) in FACS buffer (IX PBS (Hyclone), with 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)). After two washes, cells were analyzed using a BD FACSCelesta™ flow cytometer (Plotted Data-Mean Fluorescence Intensity; Figure. 18A and Figure 18B).

Fresh human leukocytes were obtained from a blood bank and CD56+ NK cells were isolated with the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16BV510, CD25-PE, CD69-APCFire750 specific antibodies (BioLegend). Cells were counted and resuspended in 0.2 x 106/mL in a 96 well flat bottom plate in 0.2 mL of complete media (RPMI 1640 (Gibco), supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). Cells were stimulated with either a cytokine mix of hIL-12 (10 ng/mL) (Biolegend), hIL-18 (50 ng/mL) (R&D), and hIL-15 (50 ng/mL) (NCI), or 0.01 nM to 10000 nM of the 18tl5-12s complex at 37°C, 5% CO2 for 14-18 hrs. The cells were then treated with 10 pg/mL of Brefeldin A (Sigma) and IX of Monensin (eBioscience) for 4 hrs before harvesting and staining with antibodies specific to CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 for 30 minutes. After staining, cells were washed (1500 RPM for 5 minutes in room temperature) in FACS buffer (IX PBS (Hyclone), with 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) and fixed for 10 minutes at room temperature. After fixation, cells were washed (1500 RPM for 5 minutes in room temperature) in lx permeabilized buffer (eBioscience) and stained with IFN-γ- PE Ab (Biolegend) for 30 minutes at room temperature. Cells were washed once again with lx permeabilized buffer and then washed with FACS buffer. Cell pellets were resuspended in 300 pls of FACS buffer and analyzed using a BD FACSCelesta™ flow cytometer (Plotted % of IFN-γ Positive Cells; Figure 19).

Example 12: In vitro cytotoxicity of NK cells against human tumor cells

Human myelogenous leukemia cells, K562 (CellTrace violet labelled), were incubated with purified human NK cells in the presence of increasing concentrations of the 18tl5-12s complex or a mixture of cytokines as a control. After 20 hours, the cultures were harvested, stained with propidium iodide (PI), and assessed by flow cytometry. As shown in Figure 20, the 18tl5-12s complex induced human NK cytotoxicity against K562, at levels similar or greater than the cytokine mixture, wherein both the 18tl5-12s complex and the cytokine mixture induced greater cytotoxicity than the medium control.

Example 13: Creation of IL-12/IL-15RaSu/aCD16scFv and IL-18/TF/IL-15 DNA constructs

In a non-limiting example, IL-12/IL-15RaSu/aCD16scFv and IL-18/TF/IL-15 DNA constructs were created (Figure 21 and Figure 22). The human IL-12 subunit sequences, human IL-15RaSu sequence, human IL-15 sequence, human tissue factor 219 sequence, and human IL-18 sequence were synthesized by Genewiz. A DNA construct was made linking the IL-12 subunit beta (p40) to IL-12 subunit alpha (p35) with a GS (3) linker to generate a single chain version of IL-12, directly linking the IL-12 sequence to the IL-15RaSu sequence, and directly linking the IL-12/ IL-15RaSu construct to the Nterminus coding region of aCD16scFv.

The nucleic acid sequence of the IL-12/IL-15RaSu/aCD16scFv construct is as follows (SEQ ID NO: 123):

(Signal peptide)

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (Human IL-12 subunit beta (p40)) ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTAT

CCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAG ACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAA GACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGC CACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGG AAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGA ATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGT TGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCG GGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCT GAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAA GAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGG TGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATC CGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCA AAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCAC ACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCA

AGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCA TCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCC AGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCC (Linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human IL-12 subunit alpha (p35))

CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTAC ACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAG GCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGAT ATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGA CAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGG CTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTC CATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCC AAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGG CTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCT CAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGT GCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATG AGCTATTTAAACGCCAGC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (anti-Human CD16 light chain variable domain)

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACC GTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGT ACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAA CAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCG CCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTG CAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAG CTGACCGTGGGCCAT (Linker)

GGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCC (anti-Human CD16 heavy chain variable domain)

GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGG CTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCA TGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCAT CAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTC ACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCC TGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCT GTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG

Constructs were also made linking the IL-18 sequence to the N-terminus coding region of tissue factor 219, and linking the IL-18/TF construct with the N-terminus coding region of IL-15 (Figure 22). The nucleic acid sequence of the IL-18/TF/IL-15 construct (including leader sequence) is as follows (SEQ ID NO: 73):

(Signal peptide)

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCT ACAGC (Human IL-18)

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAAC GACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGAC CGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGT ACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGA GAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATG AACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGC GGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGA GGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCA AGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGA GGAT (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT

TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

Example 14: Secretion of IL-12/IL-15RaSu/aCD16scFv and IL-18/TF/IL-15 fusion proteins

The IL-12/IL-15RaSu/aCD16scFv and IL-18/TF/IL-15 constructs were cloned into a pMSGV-1 modified retrovirus expression vector (Hughes, Hum Gene Ther 16:457-72, 2005, herein incorporated by reference), and the expression vector was transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells resulted in secretion of a soluble IL-18/TF/IL-15:IL-12/IL-15RaSu/aCD16scFv protein complex (referred to as 18tl5-12s/aCD16; Figure 23 and 24). Co-expression of the two constructs in CHO-K1 cells resulted in secretion of the soluble IL-18/TF/IL-15:IL-12/IL15RaSu/aCD16scFv protein complex (referred to as 18tl5-12s/aCD16; Figure 23 and Figure 24), which can be purified by anti-TF Ab affinity and other chromatography methods. In some cases, the signal peptide is cleaved from the intact polypeptide to generate the mature form.

The amino acid sequence of the IL-12/IL-15RaSu/aCD16scFv fusion protein (including signal peptide sequence) is as follows (SEQ ID NO: 122):

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL-12 subunit beta (p40))

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGS GKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKN KTFLRCEAKNYSGRFTCWWLTTISTDLTF S VKS SRGS SDPQGVTCGAATLS AERV RGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPD PPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVF TDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (Linker)

GGGGSGGGGSGGGGS (Human IL-12 subunit alpha (p35))

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHE DITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYE DLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSS LEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR (anti-Human CD16 light chain variable domain)

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKL TVGH (Linker)

GGGGSGGGGSGGGGS (anti-Human CD16 heavy chain variable domain)

EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWV SGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRS LLFDYWGQGTLVTVSR

The amino acid sequence of the IL-18/TF/IL-15 fusion protein (including leader sequence) is as follows (SEQ ID NO: 72):

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL-18)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISM YKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPG HDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

Example 15: Creation of IL-18/IL-15RaSu and IL-12/TF/IL-15 DNA constructs

In a non-limiting example, IL-18/IL-15RaSu and IL-12/TF/IL-15 DNA constructs were created. The human IL-18 subunit sequences, human IL-15RaSu sequence, human IL-12 sequence, human tissue factor 219 sequence, and human IL-15 sequence were synthesized by Genewiz. A DNA construct was made linking IL-18 directly to IL15RaSu. An additional construct was also made linking IL-12 sequence to the Nterminus coding region of human tissue factor 219 form, and further linking the IL-12/TF construct to the N-terminus coding region of IL-15. As described above, a single-chain version of IL-12 (p40-linker-p35) was used.

The nucleic acid sequence of the IL-18/IL-15RaSu construct (including signal peptide sequence) is as follows (SEQ ID NO: 217):

(Signal peptide)

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCT ACAGC (Human IL-18)

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAAC GACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGAC CGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGT ACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGA GAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATG AACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGC GGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGA GGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCA AGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGA GGAT (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The nucleic acid sequence of the IL-12/TF/IL-15 construct (including leader sequence) is as follows (SEQ ID NO: 218):

(Signal peptide)

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCT ACTCC (Human IL-12 subunit beta (p40))

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTAT CCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAG ACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAA GACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGC CACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGG AAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGA ATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGT TGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCG GGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCT GAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAA GAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGG TGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATC CGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCA AAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCAC ACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCA AGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCA TCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCC AGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCC (Linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human IL-12 subunit alpha (p35))

CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTAC ACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAG GCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGAT ATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGA CAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGG CTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTC CATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCC AAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGG CTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCT CAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGT

GCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATG AGCTATTTAAACGCCAGC (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

Example 16: Secretion of IL-18/IL-15RaSu and IL-12/TF/IL-15 fusion proteins

The IL-18/IL-15RaSu and IL-12/TF/IL-15 constructs were cloned into a pMSGV1 modified retrovirus expression vector (Hughes, Hum Gene Ther 16:457-72, 2005 herein incorporated by reference), and the expression vector was transfected into CHOKI cells. Co-expression of the two constructs in CHO-K1 cells resulted in secretion of a soluble IL-12/TF/IL-15:IL-18/IL-15RaSu protein complex (referred to as 12tl5/sl8), which can be purified by anti-TF Ab affinity and other chromatography methods.

The amino acid sequence of the IL-18/IL-15RaSu fusion protein (including signal peptide sequence) is as follows (SEQ ID NO: 219):

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL-18)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISM YKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPG HDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR

The amino acid sequence of the IL-12/TF/IL-15 fusion protein (including leader sequence) is as follows (SEQ ID NO: 220):

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL-12 subunit beta (p40))

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGS GKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKN KTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERV RGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPD PPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVF TDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (Linker)

GGGGSGGGGSGGGGS (Human IL-12 subunit alpha (p35))

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHE DITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYE

DLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSS LEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

Example 17: Recombinant protein quantitation of the 18tl5-12sl6 complex

The 18tl5-12sl6 complex (comprising IL-12/IL-15RaSu/aCD16scFv;IL18/TF/IL-15) was detected and quantified using standard sandwich ELISA methods (Figure 25). Anti-human tissue factor antibody /IL-2 or anti-TF Ab/IL-18 served as the capture antibody and biotinylated anti-human IL-12 or IL-18 antibody (BAF 219, D0456, both R&D Systems) served as the detection antibody. Tissue factor was also detected using an anti-human tissue factor antibody (143), and anti-human tissue factor antibody detection antibody.

Example 18: Creation of TGFBRII/IL-15RaSu and IL-21/TF/IL-15 DNA constructs

In a non-limiting example, a TGF3RII/IL-15RaSu DNA construct was created (Figure 26). The human TGFPRII dimer and human IL-21 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. A DNA construct was made linking the TGFPRII to another TGFPRII with a linker to generate a single chain version of TGFPRII and then directly linking the TGFPRII single chain dimer sequence to the N-terminal coding region of IL-15RaSu.

The nucleic acid sequences of the TGF3RII/IL-15RaSu construct (including signal sequence) is as follows (SEQ ID NO: 93):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human TGFpRH-1stfragment)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (Linker)

GGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGT (Human TGF01UI-2ndfragment) ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACC

GATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGA GGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCAC CTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAAT GACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACC ACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAA AAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGC AACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (Human IL-15R a sushi domain)

ATCACGTGTCCTCCTCCTATGTCCGTGGAACACGCAGACATCTGGGTC AAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAA GCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACG AATGTCGCCCACTGGACAACCCCCAGTCTCAAATGTATTAGA

Additionally, an IL-21/TF/IL-15 construct was made linking the IL-21 sequence to the N-terminus coding region of tissue factor 219, and further linking the IL-21/TF construct to the N-terminus coding region of IL-15 (Figure 27). The nucleic acid sequence of the IL-21/TF/IL-15 construct (including leader sequence) is as follows (SEQ ID NO: 89):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human Tissue Factor 219)

TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGC ACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTT ACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTA CACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAG CAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCAC CGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATT TAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCAC CAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAAC ACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTA CTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAAC GAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAG CCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGA GTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

Example 19: Secretion of TGFBRII/IL-15RaSu and IL-21/TF/IL-15 fusion proteins

The TGF3RII/IL-15RaSu and IL-21/TF/IL-15 DNA constructs were cloned into a pMSGV-1 modified retrovirus expression vector (as described in Hughes et al., Hum Gene Ther 16:457-72, 2005, herein incorporated by reference), and the expression vector was transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells resulted in secretion of the soluble IL-21/TF/IL-15:TGF3RII/IL-15RaSu protein complex (referred to as 21tl5-TGFRs; Figure 28 and Figure 29). The 21tl5-TGFRs complex was purified from CHO-K1 cell culture supernatant using anti-TF antibody affinity chromatography and other chromatography methods.

The amino acid sequence of the TGF3RII/IL-15RaSu construct (including signal peptide sequence) is as follows (SEQ ID NO: 92):

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human TGF0RH-1stfragment)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Linker)

GGGGSGGGGSGGGGS (Human TGF0RH-2ndfragment)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR

The amino acid sequence of the mature IL-21/TF/IL-15 fusion protein (including signal peptide sequence) is as follows (SEQ ID NO: 88):

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL-21)

Q GQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLP APED VETNCEWSAF SCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

In some cases, the leader peptide is cleaved from the intact polypeptide to generate a mature form that may be soluble or secreted.

Example 20: Purification of 21tl5-TGFRs by immunoaffinity chromatography

An anti-TF antibody affinity column was connected to a GE Healthcare AKTA™ Avant protein purification system. The flow rate was 4 mL/min for all steps except the elution step, which was 2 mL/min.

Cell culture harvest of 21tl5-TGFRs was adjusted to pH 7.4 with IM Tris base and loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes PBS, followed by elution with 6 column volumes 0.1M acetic acid, pH 2.9. Absorbance at 280 nm was collected and then the sample was then neutralized to pH 7.5-8.0 by adding IM Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon® centrifugal filters with a 30 KDa molecular weight cutoff. Figure 30 shows that the 21tl5-TGFRs complex binds anti-TF antibody affinity column, wherein TF is a 21tl5TGFRs binding partner. The buffer-exchanged protein sample is stored at 2-8°C for further biochemical analysis and biological activity testing.

After each elution, the anti-TF antibody affinity column was then stripped using 6 column volumes 0. IM glycine, pH 2.5. The column was then neutralized using 10 column volumes PBS, 0.05% sodium azide, and stored at 2-8°C.

Example 21: Size exclusion chromatography of 21tl5-TGFRs

A GE Healthcare Superdex® 200 Increase 10/300 GL gel filtration column was connected to a GE Healthcare AKTA™ Avant protein purification system. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.8 mL/min. A capillary loop was used to inject 200pL of 1 mg/mL of 21tl5-TGFRs complex onto the column. The injection was then chased with 1.25 column volumes of PBS. The SEC chromatograph was shown in Figure 31. There were two protein peaks, likely representing a monomer and dimer forms of 21tl5-TGFRs.

Example 22: SDS-PAGE of 21tl5-TGFRs

To determine the purity and protein molecular weight, the purified 21tl5-TGFRs complex protein sample was analyzed using 4-12% NuPage Bis-Tris protein gel SDSPAGE under reduced conditions. The gel was stained with InstantBlue™ for about 30 min, followed by destaining overnight in purified water. Figure 32 shows an example SDS gel of anti-TF antibody affinity purified 21tl5-TGFRs, with bands at 39.08 kDa and 53 kDa

Glycosylation of 21tl5-TGFRs in CHO cells was confirmed using the Protein Deglycosylation Mix II kit (New England Biolabs) and the manufacturer’s instructions. Deglycosylation reduces the molecular weight of 21tl5-TGFRs, as seen in lane 4 of Figure 32.

Example 23: Recombinant protein quantitation of 21tl5-TGFRs complexes

The 21tl5-TGFRs complex was detected and quantified using standard sandwich ELISA methods (Figures. 33-37). Anti-human tissue factor antibody served as the capture antibody and biotinylated anti-human IL-21, IL-15, or TGFPRII served as the detection antibody. Tissue factor was also detected using an anti-human tissue factor capture antibody (143), and anti-human tissue factor antibody detection antibody. The I43/anti-TF antibody ELISA was compared to purified tissue factor at similar concentrations.

Example 24: Immunostimulatory capacity of the 21tl5-TGFRs complex

To assess the IL-15 immunostimulatory activity of the 21tl5-TGFRs complexes, increasing concentrations of 21tl5-TGFRs was added to 32ϋβ cells (104 cell/well) in 200 pL IMDM: 10% FBS media and cells were incubated for 3 days at 37°C. On the fourth day, WST-1 proliferation reagent (10 pL/well) then was added and after 4 hours, absorbance was measured at 450 nm to determine cell proliferation based on cleavage of WST-1 to a soluble formazan dye. Bioactivity of the human recombinant IL-15 was assessed as a positive control. As shown in Figure 37, 21tl5-TGFRs demonstrated IL15-dependent 32ϋβ cell proliferation. The 21tl5-TGFRs complex was reduced compared to that of human recombinant IL-15, possibly due to the linkage of IL-21 and tissue factor to the IL-15 domain.

Additionally, HEK-Blue ΤΟΡβ reporter cells (hkb-tgfb, InvivoGen) were used to measure the ability of 21tl5-TGFRs to block ΤΟΡβΙ activity (Figure 38). Increasing concentrations of 21tl5-TGFRs were mixed with 0.1 nM of ΤΟΕβΙ and added to HEK-Blue TGF3 reporter cells (2.5xl04 cell/well) in 200 pL IMDM: 10% heat-inactivated FBS media. Cells were incubated overnight at 37°C. The next day, 20 pl of induced HEK-Blue TGF3 reporter cell supernatant was added to 180 μΐ of QUANTI-Blue (InvivoGen) and incubated for 1-3 hours at 37°C. 21tl5-TGFRs activity was assessed by measuring absorbance at 620 nm. Human recombinant TGF3RII/Fc activity was assessed as a positive control.

These results demonstrate that TGFpRII domain of the 21tl5-TGFRs complex retains its ability to trap TGFpi. The ability of 21tl5-TGFRs to block TGFpi activity was reduced compared to that of human recombinant TGF3RII/Fc, possibly due to the linkage of TGFPRII to the IL-15R0C sushi domain.

Example 25: Induction of cytokine-induced memory-like NK cells by the 21tl5TGFRs complex

Cytokine-induced memory-like NK cells can be induced ex vivo following overnight stimulation of purified NK cells with saturating amounts of cytokines. These memory-like properties can be measured through expression of IL-2 receptor a (IL-2Ra, CD25), CD69 (and other activation markers), and increased IFN-γ production. To evaluate the ability of 21tl5-TGFRs complexes to promote generation of cytokineinduced memory-like NK cells, purified human NK cells (>95% CD56+) were stimulated for 14-18 hours with 1 nM to 100 nM of the 21tl5-TGFRs complex. Cell-surface CD25 and CD 69 expression and intracellular IFN-γ levels were assessed by antibody-staining and flow cytometry.

Fresh human leukocytes were obtained from a blood bank and CD56+ NK cells were isolated with the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16BV510, CD25-PE, CD69-APCFire750 specific antibodies (BioLegend). Cells were counted and resuspended in 0.2 x 106/mL in a 96 well flat bottom plate in 0.2 mL of complete media (RPMI 1640 (Gibco), supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). Cells were stimulated with either mixcytokines of hIL-21 (50 ng/mL) (Biolegend) and hIL-15 (50 ng/mL) (NCI) or with 1 nM, nM, or 100 nM 21tl5-TGFRs complex overnight at 37°C, 5% CO2 for 14-18 hrs. The cells were then harvested and surface stained with CD56-BV421, CD16-BV510, CD25PE, CD69-APCFire750 specific antibodies for 30 minutes. After staining, cells were washed (1500 RPM for 5 minutes at room temperature) in FACS buffer (IX PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)). After two washes, cells were analyzed using a BD FACSCelesta™ flow cytometer. (Plotted Data-Mean Fluorescence Intensity; Figure 39 and Figure 40).

Fresh human leukocytes were obtained from a blood bank and CD56+ NK cells were isolated with the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16BV510, CD25-PE, CD69-APCFire750 specific antibodies (BioLegend). Cells were counted and resuspended in 0.2 x 106/mL in a 96 well flat bottom plate in 0.2 mL of complete media (RPMI 1640 (Gibco), supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). Cells were stimulated with either mixcytokines of hIL-21 (50 ng/mL) (Biolegend) and hIL-15 (50 ng/mL) (NCI) or with 1 nM, 10 nM, or 100 nM 21tl5-TGFRs complex overnight at 37°C, 5% CO2 for 14-18 hrs. The cells were then treated with 10 pg/mL of Brefeldin A (Sigma) and IX of Monensin (eBioscience) for 4 hrs. Cells were harvested and surface stained with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies for 30 minutes. After staining, cells were washed (1500 RPM for 5 minutes at room temperature) in FACS buffer (IX PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) and fixed for 10 minutes at room temperature. After fixation, cells were washed (1500 RPM for 5 minutes at room temperature) with lx permeabilized buffer (eBioscience) and stained for intracellular IFN-γ- PE Ab (Biolegend) for 30 minutes at room temperature. Cells were washed once again with lx permeabilized buffer and then washed with FACS buffer. Cell pellets were resuspended in 300 pls of FACS Buffer and analyzed using a BD FACSCelesta™ flow cytometer. (Plotted % of IFN-γ Positive Cells; Figure 41).

Example 26: In vitro cytotoxicity of NK cells against human tumor cells

K562 (CellTrace violet labelled), human myelogenous leukemia cells, were incubated with purified human NK cells (using StemCell human NK cell purification kit (E:T ratio; 2:1)) in the presence of increasing concentrations of the 21tl5-TGFRs complex. After 20 hours, the cultures were harvested, stained with propidium iodide (PI), and assessed by flow cytometry. As shown in Figure 42, the 21tl5-TGFRs complex induced human NK cytotoxicity against K562, as compared to control.

Example 27: Creation of an IL-21/TF mutant/IL-15 DNA construct and resulting fusion protein complex with TGFBRII /IL-15RaSu

In a non-limiting example, an IL-21/TF mutant/IL-15 DNA construct was made by linking IL-21 directly to the N-terminus coding region of a tissue factor 219 mutant, and further linking the IL-21/TF mutant to the N-terminus coding region of IL-15.

The nucleic acid sequence of the IL-21/TF mutant/IL-15 construct (including signal peptide sequence) is as follows (SEQ ID NO: 221, shaded nucleotides are mutant and the mutant codons are underlined):

(Signal sequence)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human Tissue Factor 219 mutants)

TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGC accaacttcMgacaMtctggaatgggaacccaagcccgtcaatcaagttt ACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTA CACAACAGACACCGAGTGTGiTTTAACCGACGAAATCGTCAAGGACGTCAAG CAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCAC CGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATT TAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCAC caaggtgaacgtcaccgtcgaggatgaaaggactttagtgMgcggaataa CACA^TTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGT ACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAA CGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAA GCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTG AGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of the IL-21/TF mutant/IL-15 construct (including signal peptide sequence) is as follows (SEQ ID NO: 222, substituted residues are shaded):

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS (Human Tissue Factor 219) sgttntvaaynltwkstnfItIlewepkpvnqvytvqistksgdwkskc fyttdtec||ltdeivkdvkqtylarvfsypagnvestgsageplyenspeftpyl ETNLGQPTIQSFEQVGTKVNVTVEDERTLX^RNNTiLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

In some cases, the leader peptide is cleaved from the intact polypeptide to generate a mature form that may be soluble or secreted.

In some embodiments, the IL-21/TF mutant/IL-15 DNA construct may be combined with an TGF3RII /IL-15RaSu DNA construct, transfected into cells using a retroviral vector as described above, and expressed as IL-21/TF mutant/IL-15 and TGF3RIEIL-15RaSu fusion proteins. The IL-15RaSu domain of the TGFPRIEIL15RaSu fusion protein binds to the IL-15 domain of the IL-21/TF mutant/IL-15 fusion protein to create an IL-21/TF mutant/IL-15:TGF3RII /IL-15RaSu complex.

Example 28: Creation of IL-21/IL-15RaSu and TGFBRII/TF/IL-15 DNA constructs and the resulting fusion protein complex

In a non-limiting example, an IL-21/IL-15RaSu DNA construct was made by linking IL-21 directly to the IL-15RaSu subunit sequence. The nucleic acid sequence of the IL-21/IL-15RaSu construct (including signal sequence) is as follows (SEQ ID NO: 111):

(Signal sequence)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The amino acid sequence of the IL-21/IL-15RaSu construct (including signal peptide sequence) is as follows (SEQ ID NO: 110):

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL-21)

Q GQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLP APED VETNCEWSAF SCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR

In some cases, the leader peptide is cleaved from the intact polypeptide to generate a mature form that may be soluble or secreted.

In some embodiments, the IL-21/IL-15RaSu DNA construct may be combined with a TGFpRII/TF/IL- l 5 DNA construct, transfected into a retroviral vector as described above, and expressed as IL-21/IL-15RaSu and TGF3RII/TF/IL-15 fusion proteins. The IL-15RaSu domain of the IL-21/IL-15RaSu fusion protein binds to the IL15 domain of the TGFpRII/TF/IL-l 5 fusion protein to create a TGF3RII/TF/IL-15:IL21/IL-15RaSu complex.

The TGF3RII/TF/IL-15RaSu DNA construct was created by linking the TGFpRII sequence to the N-terminus coding region of human tissue factor 219 form, and then linking the TGF3RIETF construct to the N-terminus coding region of IL-15. As described above, a single-chain version of TGFPRII (TGF3RII-linker-TGF3RII) was used. The nucleic acid sequence of the TGF3RII/TF/IL-15 construct (including leader sequence) is as follows (SEQ ID NO: 136):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human TGFpRH-1stfragment)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (Linker)

GGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGT (Human TGF0RH-2ndfragment) ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACC

GATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGA GGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCAC CTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAAT GACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACC ACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAA

AAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGC AACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (Human Tissue Factor 219)

TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGC ACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTT ACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTA CACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAG CAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCAC CGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATT TAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCAC CAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAAC ACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTA CTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAAC GAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAG CCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGA GTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of the TGF3RIVTF/IL-15 fusion protein (including signal peptide) is as follows (SEQ ID NO: 135):

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human TGFpRH-1stfragment)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Linker)

GGGGSGGGGSGGGGS (Human TGF01UI-2ndfragment)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

Example 29: Creation of an IL-7/IL-15RaSu DNA construct

In a non-limiting example, an IL-7/IL-15RaSu DNA construct was created (see Figure 43). The human IL-7 sequence, human IL-15RaSu sequence, human IL-15 sequence, and human tissue factor 219 sequence were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. A DNA construct was made linking the IL-7 sequence to the IL-15RaSu sequence. The final IL-7/IL-15RaSu DNA construct sequence was synthesized by Genewiz.

The nucleic acid sequence encoding the second chimeric polypeptide of IL-7/IL15RaSu construct (including signal peptide sequence) is as follows (SEQ ID NO: 103):

(Signal peptide)

ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGG CC (Human IL-7)

GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTA ATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATT GCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAG GAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAAT GAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAA CAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCT GGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGA ACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATA AAAACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACAC (Human IL-15R a sushi domain)

ATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTC AAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAA GCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACG AATGTCGCCCACTGGACAACCCCCAGTCTCAAATGCATTAGA

The second chimeric polypeptide of IL-7/IL-15RaSu construct (including signal peptide sequence) is as follows (SEQ ID NO: 102):

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL-7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN KEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAAL GEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL

ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

Example 30: Creation of an IL-21/TF/IL-15 DNA construct

In a non-limiting example, an IL-21/TF/IL-15 construct was made (Figure 44) by linking the IL-21 sequence to the N-terminus coding region of tissue factor 219, and further linking the IL-21/TF construct with the N-terminus coding region of IL-15.

The nucleic acid sequence encoding the first chimeric polypeptide of IL21/TF/IL-15 construct (including leader sequence), synthesized by Genewiz, is as follows (SEQ ID NO: 89):

(Signal peptide)

ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGG CC (Human IL-21 fragment)

CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATT GTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGC TCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGA AGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATG TATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAA GACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACC ACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCAT CAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC (Human Tissue Factor 219)

TCAGGCACTACAAATACTGTGGCAGCATATAATTTAACTTGGAAATCA ACTAATTTCAAGACAATTTTGGAGTGGGAACCCAAACCCGTCAATCAAGTCT

ACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTA CACAACAGACACAGAGTGTGACCTCACCGACGAGATTGTGAAGGATGTGAA GCAGACGTACTTGGCACGGGTCTTCTCCTACCCGGCAGGGAATGTGGAGAGC ACCGGTTCTGCTGGGGAGCCTCTGTATGAGAACTCCCCAGAGTTCACACCTTA CCTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTGGGA ACAAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAAC AACACTTTCCTAAGCCTCCGGGATGTTTTTGGCAAGGACTTAATTTATACACT TTATTATTGGAAATCTTCAAGTTCAGGAAAGAAAACAGCCAAAACAAACACT AATGAGTTTTTGATTGATGTGGATAAAGGAGAAAACTACTGTTTCAGTGTTCA AGCAGTGATTCCCTCCCGAACAGTTAACCGGAAGAGTACAGACAGCCCGGTA GAGTGTATGGGCCAGGAGAAAGGGGAATTCAGAGAA (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The first chimeric polypeptide of IL-21/TF/IL-15 construct including leader sequence is SEQ ID NO: 88:

(Signal peptide)

MGVKVLFALICIAVAEA (SEQ ID NO: 223) (Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTD TECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQ VGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTL YYWKS S S SG KKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFR E (Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS

Example 31: Secretion of IL-7/IL-15RaSu and IL-21/TF/IL-15 fusion proteins

The IL-7/IL-15RaSu and IL-21/TF/IL-15 DNA constructs were cloned into a pMSGV-1 modified retrovirus expression vector (as described by Hughes, Hum Gene Ther 16:457-72, 2005, hereby incorporated by reference), and the expression vector was transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of a soluble IL-21/TF/IL-15:IL-7/IL-15RaSu protein complex (referred to as 21tl5-7s; Figures 45 and Figure 46). The 21tl5-7s protein was purified from CHO-K1 cell culture supernatant using anti-TF antibody affinity chromatography and size exclusion chromatography resulting in soluble (non-aggregated) protein complexes consisting of IL-7/IL-15RaSu and IL-21/TF/IL-15 fusion proteins.

In some cases, the leader (signal sequence) peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

Example 32: Purification of 21tl5-7s by immunoaffinity chromatography

An anti-TF antibody affinity column was connected to a GE Healthcare™ AKTA Avant protein purification system. The flow rate was 4 mL/min for all steps except the elution step, which was 2 mL/min.

Cell culture harvest of 21tl5-7s was adjusted to pH 7.4 with IM Tris base and loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes PBS, followed by elution with 6 column volumes 0. IM acetic acid, pH 2.9. Absorbance at 280 nm was collected and then the sample was neutralized to pH 7.5-8.0 by adding IM Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon® centrifugal filters with a 30 KDa molecular weight cutoff. The buffer-exchanged protein sample was stored at 2-8°C for further biochemical analysis and biological activity testing.

After each elution, the anti-TF antibody affinity column was then stripped using 6 column volumes 0. IM glycine, pH 2.5. The column was then neutralized using 10 column volumes PBS, 0.05% sodium azide and stored at 2-8 °C.

Example 33: Size exclusion chromatography

A GE Healthcare Superdex® 200 Increase 10/300 GL gel filtration column was connected to a GE Healthcare AKTA™ Avant protein purification system. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min. A capillary loop was used to inject 200pL of 1 mg/mL of 7tl5-21scomplex onto the column. The injection was chased with 1.25 column volumes of PBS.

Example 34: SDS-PAGE of 21tl5-7s and 21tl5-TGFRs

To determine the purity and protein molecular weight, the purified 21tl5-7s or 21tl5-TGFRs protein sample were analyzed using 4-12% NuPage Bis-Tris protein gel SDS-PAGE. The gel will be stained with InstantBlue™ for about 30 min, followed by destaining overnight in purified water.

Example 35: Glycosylation of 21tl5-7s and 21tl5-TGFRs in CHO-K1 cells

Glycosylation of 21tl5-7s in CHO-K1 cells or 21tl5-TGFRs in CHO-K1 cells were confirmed using the Protein Deglycosylation Mix II kit (New England Biolabs), according to the manufacturer’s instructions.

Example 36: Recombinant protein quantitation of 21tl5-7s and 21tl5-TGFRs complexes

The 21tl5-7s complex or the 21tl5-TGFRs complex were detected and quantified using standard sandwich ELISA methods. Anti-human tissue factor antibody (IgGl) served as the capture antibody and biotinylated anti-human IL-21, IL-15, or IL-7 antibody (21tl5-7s) or biotinylated anti-human IL-21, IL-15, or TGF-3RII antibody (21tl5-TGFRs) served as the detection antibody. Tissue factor in purified 21tl5-7s or 21tl5-TGFRs protein complexes was detected using an anti-human tissue factor capture antibody, and anti-human tissue factor antibody (IgGl) detection antibody. The anti-TF antibody ELISA will be compared to purified tissue factor at similar concentrations.

Example 37: Expansion capacity of primary natural killer (NK) cells by 21tl5-7s complex + anti-TF IgGl antibody or 21tl5-TGFRs complex + anti-TF IgGl antibody

To assess the 21tl5-7s complex’s ability to expand primary natural killer (NK) cells, 21tl5-7s complex and 21tl5-7s complex + anti-TF IgGl antibody was added to NK cells obtained from samples of fresh human leukocytes. Cells were stimulated with 50nM of 21tl5-7s complex with or without 25 nM of anti-TF IgGl or anti-TF IgG4 antibody at 37°C and 5% CO2. Cells were maintained at concentration at 0.5 x 106/mL not exceeding 2.0 x 106/mL by counting every 48-72 hours and media was replenished with fresh stimulator. Cells stimulated with 21tl5-7s complex or anti-TF IgGl antibody or anti-TF IgG4 antibody, or anti-TF IgG4 + 21tl5-7s complex were maintained up to day 5. Figure 47 shows expansion of primary NK cells upon incubation with 21tl5-7s complex + anti-TF IgGl antibody. Figure 54 also shows a schematic of the results.

Example 38: Activation of expanded NK cells by the 21tl5-7s complex + anti-TF IgGl antibody or the 21tl5-TGFRs complex + anti-TF IgGl antibody

Primary NK cells can be induced ex vivo following overnight stimulation of purified NK cells with 21tl5-7s complex + anti-TF IgGl antibody. Fresh human leukocytes were obtained from a blood bank and CD56+ NK cells were isolated with the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >80% and confirmed by staining with CD56-BV421 and CD16-BV510 specific antibodies (BioLegend). Cells were counted and resuspended in 1 x 106/mL in a 24 well flat bottom plate in 1 mL of complete media (RPMI 1640 (Gibco), supplemented with 4 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), non-essential amino acid (Thermo Life Technologies), sodium pyruvate (Thermo Life Technologies), and 10% FBS (Hyclone)). Cells were stimulated with 50 nM of 21tl5-7s with or without 25 nM of anti-TF IgGl antibody at 37°C and 5% CO2. Cells were counted every 48-72 hours and maintained at a concentration of 0.5 x 106/mL to 2.0 x 106/mL until dayl4. Media was periodically replenished with fresh stimulator. Cells were harvested and surface stained at day 3 with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies (Biolegend and analyzed by Flow Cytometry-Celeste-BD Bioscience). Figure 48 shows the activation markers CD25 MFI and CD69 MFI. The activation marker CD25 MFI increased with 21tl5-7s complex + anti-TF IgGl antibody stimulation, but not 21tl5-7s complex stimulation. The activation marker CD69 MFI increased with both 21tl5-7s complex + anti-TF IgGl antibody and with 21tl5-7s complex, alone.

Example 39: Cytotoxicity of NK cells against human tumor cells

Fresh human blood buffy coat was obtained from a blood bank. NK cells were isolated via negative selection using the RosetteSep/human NK cell reagent (StemCell Technologies). The NK cells were cultured in complete RPMI-1640 medium with 21tl57s 100 nM and 50 nM of anti-TF IgGl antibody for up to 11 days at 37°C and 5% CO2. The activated NK cells were mixed with Celltrace violet-labeled K562 cells at E:T ratio equal to 2:1 and incubated at 37°C for 4 hours. The mixture was harvested and the percentage of dead K562 cells were determined by propidium iodide staining and flow cytometry. Figure 49 shows increased specific lysis of K562 cells when incubated with expanded NK cells.

Example 40: Creation of an IL-21/IL-15RaSu DNA construct

In a non-limiting example, an IL-21/IL-15RaSu DNA construct was created. The human IL-21 sequence and human IL-15RaSu sequence were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. A DNA construct was made linking the IL-21 sequence to the IL-15RaSu sequence. The final IL-21/IL15RaSu DNA construct sequence was synthesized by Genewiz. See Figure 50.

Example 41: Creation of an IL-7/TF/IL-15 DNA construct

In a non-limiting example, an IL-7/TF/IL-15 construct was made by linking the IL-7 sequence to the N-terminus coding region of tissue factor 219, and further linking the IL-7/TF construct with the N-terminus coding region of IL-15. See Figure 51.

Example 42: Creation of an IL-21/IL-15Ra Sushi DNA construct

In a non-limiting example, a second chimeric polypeptide of IL-21/IL-15RaSu was generated. The human IL-21 and human IL-15Ra sushi sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. A DNA construct was made linking the IL-21 sequence to the IL-15Ra sushi sequence.

The final IL-21/IL-15RaSu DNA construct sequence was synthesized by Genewiz.

The nucleic acid sequence encoding the second chimeric polypeptide of IL-21/IL15RaSu domain (including leader sequence), synthesized by Genewiz, is as follows (SEQ ID NO: 111):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC C (Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGA GCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAG

GAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAAC GTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The second chimeric polypeptide of IL-21/IL-15Ra sushi domain (including leader sequence) is as follows (SEQ ID NO: 110):

(Signal Sequence)

MKWVTFISLLFLF S SAYS (Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDS (Human IL-15Ra sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAH WTTPSLKCIR

Example 43: Creation of an IL-7/TF/IL-15 DNA construct

In a non-limiting example, an exemplary first chimeric polypeptide of IL7/TF/IL-15 was made by linking the IL-7 sequence to the N-terminus coding region of tissue factor 219, and further linking the IL-7/TF construct with the N-terminus coding region of IL-15. The nucleic acid sequence encoding the first chimeric polypeptide of IL-7/TF/IL-15 (including leader sequence), synthesized by Genewiz, is as follows (SEQ ID NO: 107):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC C (Human IL-7 fragment)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG

AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATC AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCA ACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACAC CGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACC ACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGA CCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGG TTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCG AGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAA GGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACAC CTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATT ACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGA GTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCT GTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGT GCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGT CCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGT AAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGA GAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCC AATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGT GCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCA CATTGTCCAGATGTTCATCAATACCTCC

The first chimeric polypeptide of IL-7/TF/IL-15 (including leader sequence), is as follows (SEQ ID NO: 106):

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL-7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (Human Tissue Factor 219) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTD TECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQ VGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTL YYWKS S S SG KKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFR E (Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS

Example 44: Secretion of IL-21/IL-15RaSu and IL-7/TF/IL-15 fusion proteins

The IL-21/IL-15RaSu and IL-7/TF/IL-15 DNA constructs were cloned into a pMSGV-1 modified retrovirus expression vector (as described by Hughes, Hum Gene Ther 16:457-72, 2005, hereby incorporated by reference), and the expression vector was transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of a soluble IL-7/TF/IL-15:IL-21/IL-15RaSu protein complex (referred to as 7tl5-21s). The 7tl5-21s protein was purified from CHO-K1 cell culture supernatant using anti-TF antibody (IgGl) affinity chromatography and size exclusion chromatography resulting in soluble (non-aggregated) protein complexes consisting of IL-21/IL-15RaSu and IL-7/TF/IL-15 fusion proteins. See Figure 52 and Figure 53.

Example 45: Analytical size exclusion chromatography (SEC) analysis of IL-21/IL15RaSu and IL-7/TF/IL-15 fusion proteins

To determine if anti-tissue factor monoclonal antibody and 7tl5-21s can form an antibody-fusion-molecule complex, analytical size exclusion chromatography (SEC) was performed. A Superdex 200 Increase 10/300 GL gel filtration column (from GE Healthcare) was connected to an AKTA Avant system (from GE Healthcare). The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min. Samples of the anti-TF mAb (1 mg/mL), 7tl5-21s (Img/mL), and a mixture of combined at a 1:1 ratio, so the final concentration of each protein is 0.5mg/mL) were in PBS. Each sample was injected into the Superdex 200 column using a capillary loop, and analyzed by SEC. The SEC chromatograph of each sample was shown in Figure 55. The SEC results indicated that there are two protein peaks for 7tl5-21s, likely representing a dimer (with an apparent molecular weight of 199.2kDa) and a higher oligomer of 7tl5-21s, and there is one peak (with an apparent molecular weight of 206.8kDa) for the anti-TF mAb. However, as expected, a new protein peak with a higher molecular weight (with an apparent molecular weight of 576.9 kDa) was formed in the mixture sample containing the anti-TF mAb and 7tl5-21s, indicating that the anti-TF mAb and 7tl5-21s form an antibody-antigen complex through the binding of anti-TF mAb to TF in the fusion protein complex.

Example 46: Expansion capacity of primary natural killer (NK) cells by 7tl5-21s complex + anti-TF IgGl antibody

To assess the 7tl5-21s complex’s ability to expand primary natural killer (NK) cells, 7tl5-21s complex and 7tl5-21s complex + anti-TF IgGl antibody are added to NK cells obtained from samples of fresh human leukocytes. Cells are stimulated with 50nM of 7tl5-21s complex with or without 25 nM of anti-TF IgGl or anti-TF IgG4 antibody at 37°C and 5% CO2. Cells are maintained at concentration at 0.5 x 106/mL not exceeding 2.0 x 106/mL by counting every 48-72 hours and media is replenished with fresh stimulator. Cells stimulated with 7tl5-21s complex or anti-TF IgGl antibody or anti-TF IgG4 antibody or anti-TF IgG4 + 7tl5-21s complex are maintained up to day 5.

Expansion of primary NK cells upon incubation with 21tl5-7s complex + anti-TF IgGl antibody is observed.

Example 47: Activation of expanded NK cells by the 7tl5-21s complex + anti-TF IgGl antibody

Primary NK cells are induced ex vivo following overnight stimulation of purified NK cells with 7tl5-21s complex + anti-TF IgGl antibody. Fresh human leukocytes are obtained from a blood bank and CD56+ NK cells are isolated with the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells is >80% and is confirmed by staining with CD56-BV421 and CD16-BV510 specific antibodies (BioLegend). Cells are counted and resuspended in 1 x 106/mL in a 24 well flat bottom plate in 1 mL of complete media (RPMI 1640 (Gibco), supplemented with 4 mM Lglutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), non-essential amino acid (Thermo Life Technologies), sodium pyruvate (Thermo Life Technologies), and 10% FBS (Hyclone)). Cells are stimulated with 50 nM of 7tl5-21s with or without 25 nM of anti-TF IgGl antibody at 37°C and 5% CO2. Cells are counted every 48-72 hours and maintained at a concentration of 0.5 x 106/mL to 2.0 x 106/mL until day 14. Media is periodically replenished with fresh stimulator. Cells are harvested and surface stained at day 3 with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies (Biolegend) and analyzed by Flow Cytometry-Celeste-BD Bioscience). The activation marker CD25 MFI are observed to increase with 7tl5-21s complex + anti-TF IgGl antibody stimulation, but not 7tl5-21s complex stimulation. The activation marker CD69 MFI is observed to increase with both 7tl5-21s complex + anti-TF IgGl antibody and with 7tl5-21s complex, alone.

Example 48: Increase in Glucose Metabolism in NK Cells Using 18tl5-12s

A set of experiments was performed to determine the effect of the construct of 18tl5-12s (Figure 6) on oxygen consumption rate and extracellular acidification rate (ECAR) on NK cells purified from human blood.

In these experiments, fresh human leukocytes were obtained from the blood bank from two different human donors and NK cells were isolated via negative selection using the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >80% and confirmed by staining with CD56-BV421 and CD16-BV510 specific Abs (BioLegend). The cells were counted and resuspended in 2 x 106/mL in 24-well, flatbottom plates in 1 mL of complete media (RPMI 1640 (Gibco) supplemented with 4 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), non-essential amino acid (Thermo Life Technologies), sodium pyruvate (Thermo Life Technologies) and 10% FBS (Hyclone)). The cells were stimulated with either (1) media alone, (2) 100 nM 18tl5-12s, or (3) mixture of single cytokines recombinant human IL-12 (0.25 pg), recombinant human IL15 (1.25 pg), and recombinant human IL-18 (1.25 pg) overnight at 37 °C and 5% CO2. On the next day, the cells were harvested and extracellular flux assays on expanded NK cells were performed using a XFp Analyzer (Seahorse Bioscience). The harvested cells washed and plated 2.0 x 105 cells/well in at least duplicate for extracellular flux analysis of OCR (Oxygen Consumption Rate) and ECAR (Extracellular Acidification Rate). The glycolysis stress tests were performed in Seahorse Media contain 2 mM of glutamine. The following were used during the assay: 10 mM glucose; 100 nM oligomycin; and 100 mM 2-deoxy-D-glycose (2DG).

The data show that the 18tl5-12s results in significantly increased oxygen consumption rate (Figure 56) and extracellular acidification rate (ECAR) as compared to the same cells activated with a combination of recombinant human IL-12, recombinant human IL-15, and recombinant human IL-18 (Figure 57).

Example 49: 7tl5-16s21 fusion protein generation and characterization

A fusion protein complex was generated comprising of anti-CD16scFv/IL15RaSu/IL-21 and IL-7/TF/IL-15 fusion proteins. The human IL-7 and IL-21 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made linking the IL-7 sequence to the Nterminus coding region of tissue factor 219 followed by the N-terminus coding region of IL-15.

The nucleic acid and protein sequences of a construct comprising IL-7 linked to the N-terminus of tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the IL-7/TF/IL-15 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human IL-7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCT GATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAAC TGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACA AGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAA GATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGC ACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTG CTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGA AGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGG AGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC

AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of IL-7/TF/IL-15 fusion protein (including the leader sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL-7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN KEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAAL GEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

Constructs were also made by linking the anti-CD16scFv sequence to the Nterminus coding region of IL-15RaSu chain followed by the N-terminus coding region of

IL-21 which was synthesized by Genewiz. The nucleic acid and protein sequences of a construct comprising the anti-CD16scFv linked to the N-terminus of IL-15RaSu chain followed by the N-terminus coding region of IL-21 are shown below.

The nucleic acid sequence of the anti-CD16SscFv/IL-15 RaSu/IL-21 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC ((Anti-human CD16scFv)

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACC GTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGT ACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAA CAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCG CCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTG CAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAG CTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGA GGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCT GGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTA CGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCC GGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCA GGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAA CTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCC CTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT

GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

The amino acid sequence of the anti-CD16scFv/IL-15RaSu/IL-21 construct (including signal peptide sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Anti-human CD16scFv)

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKL TVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGM SWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLR AEDTAVYYCARGRSLLFDYWGQGTLVTVSR (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR (Human IL-21)

Q GQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLP APED VETNCEWSAF SCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

The anti-CD16scFv/IL-15RaSu/IL-21 and IL-7/TF/IL-15 constructs were cloned into a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of the soluble IL-7/TF/IL-15:anti-CD16scFv/IL-15RaSu/IL-21 protein complex (referred to as 7tl5-16s21; Figure 58 and Figure 59), which can be purified by anti-TF IgGl-based affinity and other chromatography methods.

Binding of 7tl5-16s21 to CHO cells expressing human CD 16b

CHO cells were transfected with human CD 16b in a pMC plasmid and selected with 10 pg/mL of blasticidin for 10 days. The CHO cells stably expressing CD 16b were stained with 1.2 pg/mL of 7tl5-16s21, containing anti-human CD16 scFv or 18tl5-12s, which does not contain anti-human CD 16 scFv, as a negative control, and then stained with biotinylated anti-human tissue factor Ab and PE conjugated streptavidin. Only antihuman CD16scFv containing 7tl5-16s21 stained the cells as shown in Figure 60A. 18tl5-12s did not stain the CHO cells expressing human CD16b as showed in Figure 60B.

Detection of IL-15, IL-21, and IL-7 in 7tl5-16s21 using ELISA

A 96-well plate was coated with 100 pL (8 pg/mL) of anti-TF IgGl in R5 (coating buffer) and incubated at room temperature (RT) for 2 hrs. The plates were washed 3 times and blocked with 100 pL of 1% BSA in PBS. Serial dilution of 7tl516s21 (at a 1:3 ratio) were added to the wells, and incubated at RT for 60 min. Following 3 washes, 50 ng/mL of biotinylated-anti-IL15 antibody (BAM247, R&D Systems), 500 ng/mL of biotinylated-anti-IL-21 antibody (13 81, R&D Systems), or 500 ng/mL of biotinylated-anti-IL-7 antibody (506602, R&D Systems) was added to the wells and incubated at RT for 60 min. The plate was washed 3 times, and incubated with 0.25 pg/mL of HRP-SA (Jackson ImmunoResearch) at 100 pL per well for 30 min at RT, followed by 4 washes and incubation with 100 pl of ABTS for 2 mins at RT. Absorbance was read at 405 nm. As shown in Figures 61A-61C, the IL-15, IL-21, and IL-7 domains in 7tl5-16s21 were detected by the individual antibodies.

The IL-15 in 7tl5-16s21 promotes IL-2Rf and common γ chain containing 32Όβ cell proliferation

To analyze the activity of IL-15 in 7tl5-16s21, the IL-15 activity of 7tl5-16s21 was compared to recombinant IL-15 using 32ϋβ cells that express ΙΕ2Κβ and common γ chain, and evaluating their effects on promoting cell proliferation. IL-15 dependent 32ϋβ cells were washed 5 times with IMDM-10% FBS and seeded in the wells at 2 x 104 cells/well. Serially-diluted 7tl5-16s21 or IL-15 were added to the cells (Figure 62). Cells were incubated in a CO2 incubator at 37°C for 3 days. Cell proliferation was detected by adding 10 pl of WST1 to each well on day 3 and incubating for an additional 3 hours in a CO2 incubator at 37°C. The absorbance at 450 nm was measured by analyzing the amount of formazan dye produced. As shown in Figure 62, 7tl5-16s21 and IL-15 promoted 320β cell proliferation, with the EC50 of 7t15-16821 and H.-4 5 being 172.2 pM and 16.63 pM, respectively.

Purification elution chromatograph of 7tl5-16s21 from anti-TF antibody affinity column 7tl5-16s21 harvested from cell culture was loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. The column was then washed with 5 column volumes of PBS, followed by elution with 6 column volumes of 0. IM acetic acid (pH 2.9). A280 elution peak was collected and neutralized to pH 7.5-8.0 with IM Tris base. The neutralized sample was buffer exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular weight cutoff. Figure 63 is a line graph showing the chromatographic profile of 7tl5-16s21 protein containing cell culture supernatant following binding and elution on anti-TF antibody resin. As shown in Figure 63, the anti-TF antibody affinity column bound 7tl5-16s21 which contains TF. The buffer-exchanged protein sample was stored at 2-8 °C for further biochemical analyses and biological activity tests. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0. IM glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except for the elution step, which was 2 mL/min.

Analytical size exclusion chromatography (SEC) analysis of 7tl5-16s21

To perform size exclusion chromatography (SEC) analysis for 7tl5-16s21, a Superdex 200 Increase 10/300 GL gel filtration column (GE Healthcare) connected to an AKTA Avant system (GE Healthcare) was used. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min. A sample containing 7tl5-16s21 in PBS was injected into the Superdex 200 column using a capillary loop, and analyzed by SEC. As shown in Figure 64, the SEC results showed two protein peaks for 7tl516s21.

Example 50: TGFRtl5-16s21 fusion protein generation and characterization

A fusion protein complex was generated comprising anti-human CD16scFv/IL15RaSu/IL21 and TGF3 Receptor n/TF/IL-15 fusion proteins (Figure 65 and 66). The human TGF3 Receptor II (Ile24-Aspl59), tissue factor 219, and IL-15 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made linking two TGF3 Receptor II sequences with a G4S(3) linker to generate a single chain version of TGFp Receptor II and then directly linking to the N-terminus coding region of tissue factor 219 followed by the Nterminus coding region of IL-15.

The nucleic acid and protein sequences of a construct comprising two TGF3 Receptor II linked to the N-terminus of tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the two TGF3 Receptor II/TF/IL-15 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Two Human TGFf Receptor IIfragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT

GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT

TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGF3 Receptor II/TF/IL-15 fusion protein (including the leader sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human TGFp Receptor II)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

Constructs were also made by attaching anti-human CD16scFv directly linking to the N-terminus coding region of IL-15RaSu chain followed by the N-terminus coding region of IL-21 which was synthesized by Genewiz. The nucleic acid and protein sequences of a construct comprising the anti-human CD16scFv linked to the N-terminus of IL-15RaSu followed by the N-terminus coding region of IL-21 are shown below.

The nucleic acid sequence of the anti-CD16scFv/IL-15 RaSu/IL-21 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Anti-human CD16scFv)

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACC GTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGT ACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAA CAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCG CCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTG CAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAG CTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGA GGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCT GGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTA CGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCC GGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCA GGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAA CTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCC CTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC

AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

The amino acid sequence of the anti-CD16scFv/IL-15RaSu/IL-21 construct (including signal peptide sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Anti-human CD16scFv)

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKL TVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGM SWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLR AEDTAVYYCARGRSLLFDYWGQGTLVTVSR (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR (Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

The anti-CD16scFv/IL-15RaSu/IL-21 and TGFR/TF/IL-15 constructs were cloned into a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of the soluble TGFR/TF/IL-15:CD16scFv/IL15RaSu/IL-21 protein complex (referred to as TGFRtl5-16s21), which can be purified by anti-TF IgGl-based affinity and other chromatography methods.

Interaction between TGFRtl5-16s21 and CHO cells expressing human CD 16b

CHO cells were transfected with human CD 16b in a pMC plasmid and selected with 10 pg/mL of blasticidin for 10 days. Cells stably expressing CD 16b were stained with 1.2 pg/mL of TGFRtl5-16s21, containing anti-human CD 16 scFv, or 7tl5-21s, not containing anti-human CD 16 scFv, as a negative control, and with biotinylated antihuman tissue factor antibody and PE conjugated streptavidin. As shown in Figures 67A and 67B, TGFRtl5-16s21, which contains anti-human CD16scFv, showed positive binding, while 7tl5-21s did not show binding.

Effect of TGFRtl5-16s21 on TGFfl activity in HEK-Blue TGFf cells

To evaluate the activity of TGFpRII in TGFRtl5-16s21, the effect of TGFRtl516s21 on the activity of TGFpi in HEK-Blue TGF3 cells was analyzed. HEK-Blue TGF3 cells (Invivogen) were washed twice with pre-warmed PBS and resuspended in the testing medium (DMEM, 10% heat-inactivated FCS, lx glutamine, lx anti-anti, and 2x glutamine) at 5 x 105 cells/mL. In a flat-bottom 96-well plate, 50 pl cells were added to each well (2.5 x 104 cells/well) and followed with 50 pL O.lnM TGFpi (R&D systems). TGFRtl5-16s21 or TGFR-Fc (R&D Systems) prepared at a 1:3 serial dilution was then added to the plate to reach a total volume of 200 pL. After 24 hrs of incubation at 37°C, 40 pL of induced HEK-Blue TGF3 cell supernatant was added to 160 pL pre-warmed QUANTI-Blue (Invivogen) in a flat-bottom 96-well plate, and incubated at 37°C for 1-3 hrs. The OD values were then determined using a plate reader (Multiscan Sky) at 620-655 nM. The IC50 of each protein sample was calculated with GraphPad Prism 7.04. The IC50 of TGFRtl5-16s21 and TGFR-Fc were 9127 pM and 460.6 pM respectively. These results showed that the TGFpRII domain in TGFRtl5-16s21 was able to block the activity of TGF31־ in HEK-Blue TGF3 cells.

The IL-15 in TGFRtl5-16s21 promotes EL-2Rf and common γ chain containing 32Όβ cell proliferation

To analyze the activity of IL-15 in TGFRtl5-16s21, the IL-15 activity of TGFRtl5-16s21 was compared to recombinant IL-15 using 32ϋβ cells that express IL2Rβ and common γ chain, and evaluating their effects on promoting cell proliferation. IL-15 dependent 32ϋβ cells were washed 5 times with IMDM-10% FBS and seeded in the wells at 2 x 104 cells/well. Serially-diluted TGFRtl5-16s21 or IL-15 were added to the cells (Figure 68). Cells were incubated in a CO2 incubator at 37°C for 3 days. Cell proliferation was detected by adding 10 pL of WST1 to each well on day 3 and incubating for an additional 3 hours in a CO2 incubator at 37°C. The absorbance at 450 nm was measured by analyzing the amount of formazan dye produced. As shown in Figure 68, TGFRt! 5-16821 and IL-15 promoted 32Ι)β cell proliferation, with the ECso of TGFRtl5-16s21 and IL-15 being 51298 pM and 10.63 pM, respectively.

Detection of IL-15, IL-21, and TGFfRII in TGFRtl5-16s21 using ELISA

A 96-well plate was coated with 100 pL (8 pg/mL) of anti-TF IgGl in R5 (coating buffer) and incubated at room temperature (RT) for 2 hrs. The plates were washed 3 times and blocked with 100 pL of 1% BSA in PBS. TGFRtl5-16s21 serially diluted at a 1:3 ratio was added and incubated at RT for 60 min. Following three washes, 50 ng/mL of biotinylated-anti-IL-15 antibody (BAM247, R&D Systems), 500 ng/mL of biotinylated-anti-IL-21 antibody (13 81, R&D Systems), or 200 ng/mL of biotinylated-anti-TGFβRII antibody (BAF241, R&D Systems) was applied per well, and incubated at RT for 60 min. Following three washes, incubation with 0.25 pg/mL of HRP-SA (Jackson ImmunoResearch at 100 pL per well for 30 min at RT was carried out, followed by 4 washes and incubation with 100 pL of ABTS for 2 mins at RT. Absorbance was read at 405 nm. The data are shown in Figure 69. As shown in Figures 70A-70C, the IL-15, IL-21, and ΤΟΡβΚΙΙ domains in TGFRtl5-16s21 were detected by the respective antibodies.

Purification elution chromatograph ofTGFRt!5-16s21 using anti-TF antibody affinity column

TGFRtl5-16s21 harvested from cell culture was loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS, followed by elution with 6 column volumes of 0.1M acetic acid (pH 2.9). A280 elution peak was collected and then neutralized to pH 7.5-8.0 with IM Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular weight cutoff. As shown in Figure 71, the anti-TF antibody affinity column bound to TGFRtl516s21, which contains tissue factor as a fusion partner. The buffer-exchanged protein sample was stored at 2-8 °C for further biochemical analyses and biological activity tests. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except for the elution step, which was 2 mL/min.

Reduced SDS-PAGE ofTGFRt!5-16s21

To determine the purity and molecular weight of the TGFRtl5-16s21 protein, protein sample purified with anti-TF antibody affinity column was analyzed by sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDSPAGE) under reduced condition. After electrophoresis, the gel was stained with InstantBlue for about 30 min, followed by destaining overnight in purified water.

To verify that the TGFRtl5-16s21 protein undergoes glycosylation after translation in CHO cells, a deglycosylation experiment was conducted using the Protein Deglycosylation Mix II kit from New England Biolabs according to the manufacturer’s instructions. Figure 72 shows results from the reduced SDS-PAGE analysis of the sample in non-deglycosylated (lane 1 in red outline) and deglycosylated (lane 2 in yellow outline) state. The results showed that the TGFRtl5-16s21 protein is glycosylated when expressed in CHO cells. After deglycosylation, the purified sample showed expected molecular weights (69 kDa and 48 kDa) in the reduced SDS gel. Lane M was loaded with 10pL of SeeBlue Plus2 Prestained Standard.

Example 51: 7tl5-7s fusion protein generation and characterization

A fusion protein complex was generated comprising IL-7/TF/IL-15 and IL-7/IL15RaSu fusion proteins (Figure 73 and Figure 74). The human IL-7, tissue factor 219, and IL-15 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made linking the IL-7 sequence to the N-terminus coding region of tissue factor 219 followed by the Nterminus coding region of IL-15.

The nucleic acid and protein sequences of a construct comprising IL-7 linked to the N-terminus of tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of 7tl5 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human IL7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCT GATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAAC TGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACA AGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAA GATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGC ACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTG CTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGA AGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGG AGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT

TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of 7tl5 fusion protein (including the leader sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN KEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAAL GEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL

ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

Constructs were also made by linking the IL-7 sequence to the N-terminus coding region of IL-15RaSu chain which was synthesized by Genewiz. The nucleic acid and protein sequences of a construct comprising the IL-7 linked to the N-terminus of IL15RaSu chain are shown below.

The nucleic acid sequence of 7s construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGG CC (Human IL7)

GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTA ATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATT GCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAG GAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAAT GAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAA CAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCT GGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGA ACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATA AAAACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACAC (Human IL-15R a sushi domain)

ATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTC AAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAA

GCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACG AATGTCGCCCACTGGACAACCCCCAGTCTCAAATGCATTAGA

The amino acid sequence of 7s fusion protein (including the leader sequence) is as follows:

(Signal peptide)

MGVKVLFALICIAVAEA (Human IL7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN KEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAAL GEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR

The IL-7/TF/IL-15 and IL-7/IL-15RaSu constructs were cloned into a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of the soluble IL-7/TF/IL-15:IL-7/IL-15RaSu protein complex referred to as 7tl5-7s, which can be purified by anti-TF IgGl affinity and other chromatography methods.

Purification elution chromatograph of 7tl5-7s using anti-TF antibody affinity column

7tl5-7s harvested from cell culture was loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS, followed by elution with 6 column volumes of 0. IM acetic acid (pH 2.9). A280 elution peak was collected and then neutralized to pH 7.5-8.0 with IM Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular weight cutoff. As shown in Figure 75, the anti-TF antibody affinity column bound to 7tl5-7s which contains tissue factor (TF) as a fusion partner. The buffer-exchanged protein sample was stored at 2-8 °C for further biochemical analyses and biological activity tests. After each elution, the antiTF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except the elution step, which was 2 mL/min.

Immunostimulation of 7tl5-7s in C57BL/6 mice

7tl5-7s is a multi-chain polypeptide (a type A multi-chain polypeptide described herein) that includes the first polypeptide that is a soluble fusion of human IL-7, human tissue factor 219 fragment and human IL-15 (7tl5), and the second polypeptide that is a soluble fusion of human IL-7 and sushi domain of human IL-15 receptor alpha chain (7s).

CHO cells were co-transfected with the IL7-TF-IL15 (7tl5) and IL7-IL15Ra sushi domain (7s) vectors. The 7tl5-7s complex was purified from the transfected CHO cell culture supernatant. The IL-7, IL-15 and tissue factor (TF) components were demonstrated in the complex by ELISA as shown in Figure 76. A humanized anti-TF monoclonal antibody (anti-TF IgGl) was used as the capture antibody to determine TF in 7tl5-7s, and biotinylated anti-human IL-15 antibody (R&D systems) and biotinylated anti-human IL-7 antibody (R&D Systems) were used as the detection antibodies to respectively detect IL-15 and IL-7 in 7tl5-7s, followed by peroxidase conjugated streptavidin (Jackson ImmunoResearch Lab) and ABTS substrate (Surmodics IVD, Inc.).

Example 52: TGFRtl5-TGFRs fusion protein generation and characterization

A fusion protein complex was generated comprising of TGF3 Receptor II/IL15RaSu and TGF3 Receptor II/TF/IL-15 fusion proteins (Figure 77 and Figure 78). The human TGF3 Receptor II (Ile24-Aspl59), tissue factor 219, and IL-15 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made linking two TGF3 Receptor II sequences with a G4S(3) linker to generate a single chain version of TGF3 Receptor II and then directly linking to the N-terminus coding region of tissue factor 219 followed by the Nterminus coding region of IL-15.

The nucleic acid and protein sequences of a construct comprising two ΤΘΡβ Receptor II linked to the N-terminus of tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the two ΤΘΡβ Receptor II/TF/IL-15 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Two Human ΤΟΡβ Receptor IIfragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT

TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGF3 Receptor II/TF/IL-15 fusion protein (including the leader sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human TGFf Receptor II) IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

Constructs were also made by attaching two TGF3 Receptor II directly to the IL15RaSu chain which was synthesized by Genewiz. The nucleic acid and protein sequences of a construct comprising the TGF3 Receptor II linked to the N-terminus of IL-15RaSu are shown below.

The nucleic acid sequence of the TGFp Receptor II/IL-15 RaSu construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Two human TGFf Receptor IIfragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCC

GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The amino acid sequence of the two TGF3 Receptor II/IL-15RaSu construct (including signal peptide sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Two human ΤΟΡβ Receptor II extra-cellular domains)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

The TGFβR/IL-15RaSu and TGF3R/TF/IL-15 constructs were cloned into a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHOKI cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of the soluble TGFβR/TF/IL-15:TGFβR/IL-15RaSu protein complex (referred to as TGFRtl5-TGFRs), which can be purified by anti-TF IgGl affinity and other chromatography methods.

Effect of TGFRt 15-TGFRs on TGFpi activity in HEK-Blue TGFp cells

To evaluate the activity of TGFpRII in TGFRtl 5-TGFRs, the effect of TGFRtl 5TGFRs on the activity of TGFpi in HEK-Blue TGF3 cells was analyzed. HEK-Blue TGF3 cells (Invivogen) were washed twice with pre-warmed PBS and resuspended in the testing medium (DMEM, 10% heat-inactivated FCS, lx glutamine, lx anti-anti, and 2x glutamine) at 5 x 105 cells/mL. In a flat-bottom 96-well plate, 50 pL cells were added to each well (2.5 x 104 cells/well) and followed with 50 pL O.lnM TGFpi (R&D systems). TGFRtl5-TGFRsor TGFR-Fc (R&D Systems) prepared at a 1:3 serial dilution was then added to the plate to reach a total volume of 200 pL. After 24hrs of incubation at 37°C, 40 pL of induced HEK-Blue TGF3 cell supernatant was added to 160 pL pre-warmed QUANTI-Blue (Invivogen) in a flat-bottom 96-well plate, and incubated at 37°C for 1-3 hrs. The OD values were then determined using a plate reader (Multiscan Sky) at 620-655 nM. The IC50 of each protein sample was calculated with GraphPad Prism 7.04. The IC50 of TGFRtl 5-TGFRs and TGFR-Fc were 216.9 pM and 460.6 pM respectively. These results showed that the TGFpRII domain in TGFRtl 5-TGFRs was able to block the activity of TGFpi in HEK-Blue TGF3 cells.

The IL-15 in TGFRtl5-TGFRs promotes IL-2Rf> and common γ chain containing 321ff cell proliferation

To evaluate the activity of IL-15 in TGFRtl 5-TGFRs, the IL-15 activity of TGFRtl 5-TGFRs was compared to recombinant IL-15 using 32ϋβ cells that express

IL2R3 and common γ chain, and evaluating their effects on promoting cell proliferation. IL-15 dependent 32ϋβ cells were washed 5 times with IMDM-10% FBS and seeded in the wells at 2 x 104 cells/well. Serially-diluted TGFRtl5-TGFRs or IL-15 were added to the cells (Figure 79). Cells were incubated in a CO2 incubator at 37°C for 3 days. Cell proliferation was detected by adding 10 pL of WST1 to each well on day 3 and incubating for an additional 3 hours in a CO2 incubator at 37°C. The absorbance at 450 nm was measured by analyzing the amount of formazan dye produced. As shown in Figure 79, TGFRtl5-TGFRs and IL-15 promoted 320β cell proliferation, with the EC50 of TGFRtl5-TGFRsand IL-15 being 1901 pM and 10.63 pXL respectively.

Detection ofIL-15 and TGFfRII domains in TGFRtl5-TGFRs with corresponding antibodies using ELISA

A 96-well plate was coated with 100 pL (8 pg/mL) of anti-TF IgGl in R5 (coating buffer) and incubated at room temperature (RT) for 2 hrs. The plates were washed 3 times and blocked with 100 pL of 1% BSA in PBS. TGFRtl5-TGFRs was added at a 1:3 serial dilution, and incubated at RT for 60 min. After 3 washes, 50 ng/mL of biotinylated-anti-IL-15 antibody (BAM247, R&D Systems), or 200 ng/mL of biotinylated-anti-TGFβRII antibody (BAF241, R&D Systems) was added to the wells and incubated at RT for 60 min. Next the plates were washed 3 times, and 0.25 pg/mL of HRP-SA (Jackson ImmunoResearch) at 100 pL per well was added and incubated for 30 min at RT, followed by 4 washes and incubation with 100 pL of ABTS for 2 mins at RT. Absorbance at 405 nm was read. As shown in Figure 81A and 8 IB, the IL-15 and ΤΟΡβΙΗΙ domains in TGFRtl5-TGFRs were detected by the individual antibodies.

Purification elution chromatograph of TGFRtl5-TGFRs from anti-TF antibody affinity column

TGFRtl5-TGFRs harvested from cell culture was loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS, followed by elution with 6 column volumes of 0. IM acetic acid (pH 2.9). A280 elution peak was collected and then neutralized to pH 7.5-8.0 with IM Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular weight cutoff. As shown in Figure 82, the anti-TF antibody affinity column bound to TGFRtl5-TGFRs which contains TF as a fusion partner. The buffer-exchanged protein sample was stored at 2-8 °C for further biochemical analyses and biological activity tests. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except for the elution step, which was 2 mL/min.

Analytical size exclusion chromatography (SEC) analysis of TGFRtl 5-TGFRs

A Superdex 200 Increase 10/300 GL gel filtration column (from GE Healthcare) was connected to an AKTA Avant system (from GE Healthcare). The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min. A sample containing TGFRtl5-TGFRs in PBS was injected into the Superdex 200 column using a capillary loop, and analyzed by SEC. The SEC chromatograph of the sample is shown in Figure 83. The SEC results showed four protein peaks for TGFRtl 5-TGFRs.

Reduced SDS-PAGE analysis of TGFRtl5-TGFRs

To determine the purity and molecular weight of the TGFRtl 5-TGFRs protein, protein sample purified with anti-TF antibody affinity column was analyzed by sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDSPAGE) method under reduced condition. After electrophoresis, the gel was stained with InstantBlue for about 30 min, followed by destaining overnight in purified water.

To verify that the TGFRtl 5-TGFRs protein undergoes glycosylation after translation in CHO cells, a deglycosylation experiment was conducted using the Protein Deglycosylation Mix II kit from New England Biolabs and the manufacturer’s instructions. Figure 84 shows the reduced SDS-PAGE analysis of the sample in nondeglycosylated (lane 1 in red outline) and deglycosylated (lane 2 in yellow outline) state. The results showed that the TGFRtl 5-TGFRs protein is glycosylated when expressed in CHO cells. After deglycosylation, the purified sample showed expected molecular weights (69 kDa and 39 kDa) in the reduced SDS gel. Lane M was loaded with 10 ul of SeeBlue Plus2 Prestained Standard.

Immunostimulatory activity ofTGFRt!5-TGFRs in C57BL/6 mice

TGFRtl5-TGFRs is a multi-chain polypeptide (a type A multi-chain polypeptide described herein) that includes a first polypeptide that is a soluble fusion of two TGFpRII domains, human tissue factor 219 fragment and human IL-15, and the second polypeptide that is a soluble fusion of two TGFpRII domains and sushi domain of human IL-15 receptor alpha chain.

Wild type C57BL/6 mice were treated subcutaneously with either control solution or with TGFRtl5-TGFRs at a dosage of 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 10 mg/kg. Four days after treatment, spleen weight and the percentages of various immune cell types present in the spleen were evaluated. As shown in Figure 85A, the spleen weight in mice treated with TGFRtl5-TGFRs increased with increasing dosage of TGFRtl5-TGFRs. Moreover, the spleen weight in mice treated with 1 mg/kg, 3 mg/kg, and 10 mg/kg of TGFRtl5-TGFRs were higher as compared to mice treated with the control solution, respectively. In addition, the percentages of CD4+ T cells, CD8+ T cells, NK cells, and CD19+ B cells present in the spleen of control-treated and TGFRtl5-TGFRs-treated mice were evaluated. As shown in Figure 85B, in the spleens of mice treated with TGFRtl5TGFRs, the percentages of CD8+ T cells and NK cells both increased with increasing dosage of TGFRtl5-TGFRs. Specifically, the percentages of CD8+ T cells were higher in mice treated with 0.3 mg/kg, 3 mg/kg, and 10 mg/kg of TGFRtl5-TGFRs compared to control-treated mice, and the percentages of NK cells were higher in mice treated with 0.3 mg/kg, 1 mg/kg, 3 mg/kg, and 10 mg/kg of TGFRtl5-TGFRs compared to controltreated mice. These results demonstrate that TGFRtl5-TGFRs is able to stimulate immune cells in the spleen, in particular CD8+ T cells and NK cells.

The pharmacokinetics of TGFRtl5-TGFRs molecules were evaluated in wild type C57BL/6 mice. The mice were treated subcutaneously with TGFRtl5-TGFRs at a dosage of 3 mg/kg. The mouse blood was drained from tail vein at various time points and the serum was prepared. The TGFRtl5-TGFRs concentrations in mouse serum was determined with ELISA (capture: anti-human tissue factor antibody; detection:

biotinylated anti-human ΤΘΡβ receptor antibody and followed by peroxidase conjugated streptavidin and ABTS substrate). The results showed that the half-life of TGFRtl5TGFRs was 12.66 hours in C57BL/6 mice.

The mouse splenocytes were prepared in order to evaluate the immunostimulatory activity of TGFRt 15-TGFRs over time in mice. As shown in Figure 86A, the spleen weight in mice treated with TGFRtl5-TGFRs increased 48 hours posttreatment and continued to increase over time. In addition, the percentages of CD4+ T cells, CD8+ T cells, NK cells, and CD19+ B cells present in the spleen of control-treated and TGFRtl5TGFRs-treated mice were evaluated. As shown in Figure 86B, in the spleens of mice treated with TGFRtl5-TGFRs, the percentages of CD8+ T cells and NK cells both increased at 48 hours after treatment and were higher and higher overtime after the single dose treatment. These results further demonstrate that TGFRtl5-TGFRs is able to stimulate immune cells in the spleen, in particular CD8+ T cells and NK cells.

Furthermore, the dynamic proliferation of immune cells based on Ki67 expression of splenocytes and cytotoxicity potential based on granzyme B expression were evaluated in splenocytes isolated from mice following a single dose (3 mg/kg) of TGFRt 15-TGFRs. As shown in Figure 87A and 87B, in the spleens of mice treated with TGFRt 15-TGFRs, the expression of Ki67 and granzyme B by NK cells increased at 24 hours after treatment and its expression of CD8+ T cells and NK cells both increased at 48 hours and later time points after the single dose treatment. These results demonstrate that TGFRtl5-TGFRs not only increases the numbers of CD8+ T cells and NK cells but also enhance the cytotoxicity of these cells. The single dose treatment of TGFRtl5-TGFRs led CD8+ T cells and NK cells to proliferate for at least 4 days.

The cytotoxicity of the splenocytes from TGFRt 15-TGFRs-treated mice against tumor cells was also evaluated. Mouse Moloney leukemia cells (Yac-1) were labeled with CellTrace Violet and were used as tumor target cells. Splenocytes were prepared from TGFRtl5-TGFRs (3 mg/kg)-treated mouse spleens at various time points post treatment and were used as effector cells. The target cells were mixed with effector cells at an E:T ratio =10:1 and incubated at 37°C for 20 hours. Target cell viability was assessed by analysis of propidium iodide positive, violet-labeled Yac-1 cells using flow cytometry. Percentage of Yac-1 tumor inhibition was calculated using the formula, (1398

[viable Yac-1 cell number in experimental sample]/[viable Yac-1 cell number in the sample without splenocytes]) x 100. As shown in Figure 88, splenocytes from TGFRtl5TGFRs-treated mice had stronger cytotoxicity against Yac-1 cells than the control mouse splenocytes.

Tumor size analysis in response to chemotherapy and/or TGFRtl 5-TGFRs

Pancreatic cancer cells (SW1990, ATCC® CRL-2172) were subcutaneously (s.c.) injected into C57BL/6 scid mice (The Jackson Laboratory, 001913, 2xl06 cells/mouse, in 100pL HBSS) to establish the pancreatic cancer mouse model. Two weeks after tumor cell injection, chemotherapy was initiated in these mice intraperitoneally with a combination of Abraxane (Celgene, 68817-134, 5 mg/kg, i.p.) and Gemcitabine (Sigma Aldrich, G6423, 40 mg/kg, i.p.), followed by immunotherapy with TGFRtl5-TGFRs (3 mg/kg, s.c.) in 2 days. The procedure above was considered one treatment cycle and was repeated for another 3 cycles (1 cycle/week). Control groups were set up as the SW1990injected mice that received PBS, chemotherapy (Gemcitabine and Abraxane), or TGFRtl 5-TGFRs alone. Along with the treatment cycles, tumor size of each animal was measured and recorded every other day, until the termination of the experiment 2 months after the SW1990 cells were injected. Measurement of the tumor volumes were analyzed by group and the results indicated that the animals receiving a combination of chemotherapy and TGFRtl 5-TGFRs had significantly smaller tumors comparing to the PBS group, whereas neither chemotherapy nor TGFRtl5-TGFRs therapy alone work as sufficiently as the combination (Figure 89).

In vitro senescent Bl 6F10 melanoma model

Next, in vitro killing of senescent Bl6F10 melanoma cells by activated mouse NK cells was evaluated. B16F10 senescence cells (B16F10-SNC) cells were labelled with CellTrace violet and incubated for 16 hrs with different E:T ratio of in vitro 2t2activated mouse NK cells (isolated from spleen of C57BL/6 mice injected with TGFRtl 5-TGFRs 10 mg/kg for 4 days). The cells were trypsinized, washed and resuspended in complete media containing propidium iodide (PI) solution. The cytotoxicity was assessed by flow cytometry (Figure 90).

Example 53: 7tl5-21sl37L (long version) fusion protein creation and characterization

A fusion protein complex was generated comprising of IL-21/IL15RaSu/CD137L and IL-7/TF/IL-15 fusion proteins (Figure 91 and Figure 92). Specifically, a construct was made linking the IL-7 sequence to the N-terminus coding region of tissue factor 219 followed by the N-terminus coding region of IL-15. The nucleic acid and protein sequences of a construct comprising IL-7 linked to the Nterminus of tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the 7tl 5 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human IL7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCT GATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAAC TGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACA AGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAA GATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGC ACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTG CTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGA AGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGG AGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC

ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of 7tl5 fusion protein (including the leader sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN KEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAAL GEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

The nucleic acid and protein sequences of the 21sl37L are shown below. The nucleic acid sequence of the 21sl37L construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG ((G4S)3 linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human CD137L)

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGAC CTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCG ATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGAC GGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGC

TGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCG AGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCT GCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGA GGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCG GCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTG GCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAA ATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA

The amino acid sequence of 21sl37L fusion protein (including the leader sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR ((G4S)3 linker)

GGGGSGGGGSGGGGS (Human CD137L)

REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVS LTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSA AGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAW QLTQGATVLGLFRVTPEIPAGLPSPRSE

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

The IL-21/IL-15RaSu/CD137L and IL-7/TF/IL-15 constructs were cloned into a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY,

Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHOKI cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of the soluble IL-7/TF/IL-15: IL-21/IL-15RaSu/CD137L protein complex (referred to as 7tl5-21sl37L), which can be purified by anti-TF IgGl affinity and other chromatography methods.

Purification elution chromatograph of 7tl5-21sl37L usinganti-TFantibody affinity column

7tl5-21sl37L harvested from cell culture was loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS, followed by elution with 6 column volumes of 0.1M acetic acid (pH 2.9). A280 elution peak was collected and then neutralized to pH 7.5-8.0 with IM Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular weight cutoff. As shown in Figure 93, the anti-TF antibody affinity column bound to 7tl521sl37L which contains TF as a fusion partner. The buffer-exchanged protein sample was stored at 2-8 °C for further biochemical analyses and biological activity tests. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0. IM glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except for the elution step, which was 2 mL/min. Figure 94 shows the analytical SEC profile of 7tl5-21sl37L.

Example 54: 7tl5-21sl37L (short version) fusion protein generation and characterization

A fusion protein complex was generated comprising of IL-21/IL15RaSu/CD137L and IL-7/TF/IL-15 fusion proteins. Specifically, a construct was made linking the IL-7 sequence to the N-terminus coding region of tissue factor 219 followed by the N-terminus coding region of IL-15. The nucleic acid and protein sequences of a construct comprising IL-7 linked to the N-terminus of tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of 7tl5 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human IL7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCT GATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAAC TGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACA AGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAA GATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGC ACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTG CTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGA AGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGG AGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC

AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of 7tl5 fusion protein (including the leader sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN KEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAAL GEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

The nucleic acid and protein sequences of the 21sl37L (short version) are shown below. The nucleic acid sequence of 21sl37L (short version) construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG ((G4S)3 linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human CD 137 Ligand short version)

GATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTG GTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACC CAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACA CGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTA GAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGC TGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACC GTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCA GGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCAC

ACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCT TGGGACTCTTCCGGGTGACCCCCGAAATC

The amino acid sequence of the 21sl37L (short version) construct (including signal peptide sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR ((G4S)3 linker)

GGGGSGGGGSGGGGS (Human CD 137 Ligand short version)

DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKE DTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALAL TVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEI

The IL-21/IL-15RaSu/CD137L (short version) and IL-7/TF/IL-15 constructs were cloned into a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of the soluble IL-7/TF/IL-15: IL-21/IL15RaSu/CD137L protein complex (referred to as 7tl5-21sl37L (short version)), which can be purified by anti-TF IgGl affinity and other chromatography methods.

Binding of 7tl5-21sl37L (short version) to CD 137 (4.IBB)

On day 1, a 96-well plate was coated with 100 pL (2.5 pg/mL) of GAH IgG Fc (G C, R&D Systems) in R5 (coating buffer) or R5 only and incubated at 4°C, overnight. On day 2, the plates were washed three times and blocked with 300 pL of 1% BSA in PBS at 37°C for 2 hrs. 10 ng/mL of 4.1BB/Fc (838-4B, R&D Systems) was added at 100 pL/well and incubated for 2 hrs at RT. After three washes, the 7tl521sl37L or 7tl5-21s serially diluted at a 1/3 ratio (starting at 10 nM), and incubated at 4°C overnight. On day 3, following 3 washes, 300 ng/mL of biotinylated-anti-hTF antibody (BAF2339, R&D Systems) was added at 100 pL per well and incubated at RT for 2 hrs. The plate was then washed three times and incubated with 0.25 pg/mL of HRPSA (Jackson ImmuneResearch) at 100 pL per well for 30 min, followed by 3 washes and incubation with 100 pL of ABTS for 2 mins at RT. Absorbance was read at 405 nm. As shown in Figure 95, 7tl5-21sl37L (short version) showed significant interaction with 4.1BB/Fc (blue line) as compared to 7tl5-21s.

Detection of IL-15, IL-21, and IL-7 in 7tl5-21sl37L (short version) with ELISA

A 96-well plate was coated with 100 pL (8 pg/mL) of anti-TF IgGl in R5 (coating buffer) and incubated at RT for 2 hrs. The plates were washed 3 times and blocked with 100 pL of 1% BSA in PBS. 7tl5-21sl37L (short version), serially diluted at a 1:3 ratio was added, and incubated at RT for 60 min. After three washes, 50 ng/mL of biotinylated-anti-IL15 antibody (BAM247, R&D Systems), 500 ng/mL of biotinylated-anti-IL21 antibody (13 81, R&D Systems), or 500 ng/mL of biotinylated-anti-IL7 antibody (506602, R&D Systems) was added to the wells and incubated at RT for 60 min. After three washes and incubation with 0.25 pg/mL of HRPSA (Jackson ImmunoResearch) at 100 pL per well was carried out for 30 min at RT, followed by four washes and incubation with 100 pL of ABTS for 2 mins at RT. Absorbance was read at 405 nm. As shown in Figures 96A-96C, the IL-15, IL-21, and IL-7 domains in 7tl5-21sl37L (short version) were detected by the respective antibodies.

The IL-15 in 7tl5-lsl37L (short version) promotes IL2Rafy containing CTLL2 cell proliferation

To evaluate the IL-15 activity of 7tl5-21sl37L (short version), 7tl5-21sl37L (short version) was compared with recombinant IL15 in promoting proliferation of IL2R0^y expressing CTLL2 cells. IL dependent CTLL2 cells were washed 5 times with IMDM-10% FBS and seeded to the wells at 2 x 104 cells/well. Serially diluted 7tl521sl37L (short version) or IL-15 were added to the cells (Figure 97). Cells were incubated in a CO2 incubator at 37°C for 3 days. Cell proliferation was detected by adding 10 pL of WST1 to each well on day 3 and incubated for an additional 3 hours in a CO2 incubator at 37°C. The amount of formazan dye produced was analyzed by measuring the absorbance at 450 nm. As shown in Figure 97, 7tl5-21sl37L (short version) and IL-15 promoted CTLL2 cell proliferation The ECso of 7tl5-21sl37L (short version) and IL-15 was 55.91 pM and 6.22 pM. respectively.

The IL-21 in 7tl5-lsl37L (short version)promotes IL21R containingB9 cell proliferation

To evaluate the IL-21 activity of 7tl5-21sl37L (short version), 7tl5-21sl37L (short version) was compared with recombinant IL-21 in promoting proliferation of IL21R expressing B9 cells. IL-21R containing B9 cells were washed 5 times with RPMI10% FBS and seeded to the wells at 1 x 104 cells/well. Serially diluted 7tl5-21sl37L (short version) or IL-21 were added to the cells (Figure 98). Cells were incubated in a CO2 incubator at 37°C for 5 days. Cell proliferation was detected by adding 10 pL of WST1 to each well on day 5 and incubated for an additional 4 hours in a CO2 incubator at 37°C. The amount of formazan dye produced w7as analyzed by measuring the absorbance at 450 nm. As shown in Figure 98, 7tl5-21sl37L (short version) and IL-21 promoted B9 cell proliferation. The ECso of 7tl5-21sl37L (short version) and IL-21 was 104.1 nM and 72.55 nM. respectively.

Example 55: 7tl5-TGFRs fusion protein generation and characterization

A fusion protein complex was generated comprising of TGF3 Receptor II/IL15RaSu and IL-7/TF/IL-15 fusion proteins (Figure 99 and Figure 100). The human TGF3

Receptor II (Ile24-Aspl59), tissue factor 219, IL-15, and IL-7 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made linking the IL-7 sequence to the N-terminus coding region of tissue factor 219 followed by the N-terminus coding region of IL-15. The nucleic acid and protein sequences of a construct comprising IL-7 linked to the Nterminus of tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the 7tl 5 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human IL7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCT GATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAAC TGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACA AGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAA GATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGC ACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTG CTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGA AGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGG AGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT

GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of 7tl5 fusion protein (including the leader sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDAN KEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAAL GEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

Constructs were also made by attaching two TGF3 Receptor II directly to the IL15RaSu chain which was synthesized by Genewiz. The nucleic acid and protein sequences of a construct comprising the TGF3 Receptor II linked to the N-terminus of IL-15RaSu are shown below.

The nucleic acid sequence of the TGFRs construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human TGFf Receptor IIfragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA

AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The amino acid sequence of TGFRs fusion protein (including the leader sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human TGFf Receptor II)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR

Effect of 7tl5-TGFRs on TGFfl activity in HEK-Blue TGFf cells

To evaluate the activity of TGF3R in 7tl5-TGFRs, the effect of 7tl5-TGFRs on the activity of TGFpi in HEK-Blue TGF3 cells was analyzed. HEK-Blue TGF3 cells (Invivogen) were washed twice with pre-warmed PBS and resuspended in the testing medium (DMEM, 10% heat-inactivated FCS, lx glutamine, lx anti-anti, and 2x glutamine) at 5 x 105 cells/mL. In a flat-bottom 96-well plate, 50 pL cells were added to each well (2.5 x 104 cells/well) and followed with 50 pL O.lnM TGFpi (R&D systems). 7tl5-TGFRs or TGFR-Fc (R&D Systems) prepared at al :3 serial dilution was then added to the plate to reach a total volume of 200 pL. After 24hrs of incubation at 37°C, 40 pL of induced HEK-Blue TGF3 cell supernatant was added to 160 pL pre-warmed QUANTI-Blue (Invivogen) in a flat-bottom 96-well plate, and incubated at 37°C for 1-3 hrs. The OD values were then determined using a plate reader (Multiscan Sky) at 620-655 nM. The IC50 of each protein sample was calculated with GraphPad Prism 7.04. The

IC50 of 7tl5-TGFRs and TGFR-Fc were 1142 pM and 558.6 pM respectively. These results showed that the TGF3R in 7tl 5-TGFRs was able to block the activity of TGFpi in HEK-Blue TGFp cells.

Detection of IL-15, TGFfRII, and IL-7 in 7tl5-TGFRs with ELISA

A 96-well plate was coated with 100 pL (8 pg/mL) of anti-TF IgGl in R5 (coating buffer) and incubated at room temperature (RT) for 2 hrs. The plates were washed three times and blocked with 100 pL of 1% BSA in PBS. Serial dilution of 7tl5TGFRs (1:3 ratio) was added, and incubated at RT for 60 mins. After 3 washes, 50 ng/mL of biotinylated-anti-IL-15 antibody (BAM247, R&D Systems), 200 ng/mL of biotinylated-anti-TGFbRII antibody (BAF241, R&D Systems), or 500 ng/mL of biotinylated-anti-IL-7 antibody (506602, R&D Systems) was added and incubated at RT for 60 min. Following three washes, incubation with 0.25 pg/mL of HRP-SA (Jackson ImmunoResearch) at 100 pL per well was carried out for 30 min at RT, followed by 4 washes and incubation with 100 pL of ABTS for 2 mins at RT. Absorbance was read at 405 nm. The data are shown in Figure 101. As shown in Figures 102A-102C, the IL-15, TGFR, and IL-7 in 7tl 5-TGFRs were detected by the respective antibodies.

The IL-15 in 7tl5-TGFRspromotes IL-2Rf and common γ chain containing 32Df cell proliferation

To evaluate the activity of IL-15 in 7tl 5-TGFRs, 7tl 5-TGFRs was compared to recombinant IL-15 using 32ϋβ cells that express IL2Rβ and common γ chain, and evaluating their effects on promoting cell proliferation. IL-15 dependent 32ϋβ cells were washed 5 times with IMDM-10% FBS and seeded in the wells at 2 x 104 cells/well. Serially-diluted 7tl 5-TGFRs or IL-15 were added to the cells (Figure 103). Cells were incubated in a CO2 incubator at 37°C for 3 days. Cell proliferation was detected by adding 10 pL of WST1 to each well on day 3 and incubating for an additional 3 hours in a CO2 incubator at 37°C. The amount of formazan dye produced was analyzed by measuring the absorbance at 450 nm. As shown in Figure 103, 7tl5-TGFRs and IL-15 promoted 32Ι)β cell proliferation, with the EC50 of 7tl5-TGFRs and IL-15 being 126 nM and 16.63 pM, respectively.

Purification elution chromatograph of 7tl5-TGFRs using anti-TF antibody affinity column

7tl5-TGFRs harvested from cell culture was loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS, followed by elution with 6 column volumes of 0.1M acetic acid (pH 2.9). A280 elution peak was collected and then neutralized to pH 7.5-8.0 with IM Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular weight cutoff. As shown in Figure 104, the anti-TF antibody affinity column can bind 7tl5TGFRs which contains TF as a fusion partner of 7tl5-TGFRs. The buffer-exchanged protein sample was stored at 2-8 °C for further biochemical analyses and biological activity tests. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except for the elution step, which was 2 mL/min.

Reduced SDS-PAGE analysis of 7tl5-TGFRs

To determine the purity and molecular weight of the protein, 7tl5-TGFRs protein sample purified with anti-TF antibody affinity column was analyzed by sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS-PAGE) method under reduced condition. After electrophoresis, the gel was stained with InstantBlue for about 30 min, followed by destaining overnight in purified water.

To verify that the 7tl5-TGFRs protein undergoes glycosylation after translation in CHO cells, a deglycosylation experiment was conducted using the Protein Deglycosylation Mix II kit from New England Biolabs and the manufacturer’s instructions. Figure 105 shows reduced SDS-PAGE analysis of the sample in nondeglycosylated (lane 1 in red outline) and deglycosylated (lane 2 in yellow outline) state. These results showed that the protein is glycosylated when it is expressed in CHO cells. After deglycosylation, the purified sample showed expected molecular weights (55 kDa and 39 kDa) in reduced SDS gel. Lane M was loaded with 10 ul of SeeBlue Plus2 Prestained Standard.

Characterization of 7tl5-TGFRs

7tl5-TGFRs is a multi-chain polypeptide (a type A multi-chain polypeptide described herein) that includes the first polypeptide that is a soluble fusion of human IL7, human tissue factor 219 fragment and human IL-15 (7tl5), and the second polypeptide that is a soluble fusion of single chain two TGFpRII domains and sushi domain of human IL-15 receptor alpha chain (TGFRs).

CHO cells were co-transfected with 7tl 5 and TGFRs vectors. The 7tl5-TGFRs complex was purified from the transfected CHO cell culture supernatant. The IL-7, IL15, TGF3 receptor and tissue factor (TF) components were demonstrated in the complex by ELISA as shown in Figure 106. A humanized anti-TF monoclonal antibody (anti-TF IgGl) was used as the capture antibody to determine TF in 7tl5-TGFRs, and biotinylated antibodies against human IL-15 antibody (R&D systems), human IL-7 (Biolegend), antiTGFp receptor (R&D Systems) were used as the detection antibodies to respectively determine IL-7, IL-15 and TGF3 receptor in 7tl5-TGFRs. Peroxidase conjugated streptavidin (Jackson ImmunoResearch Lab) and ABTS substrate (Surmodics IVD, Inc.) were then used to detect the bound biotinylated antibodies. The results were analyzed by ELISA (Figure 106).

In vivo characterization of 7tl5-TGFRs in C57BL/6 mice

To determine the immunostimulatory activity of 7tl5-TGFRs in vivo, C57BL/6 mice were subcutaneously treated with control solution (PBS) or 7tl5-TGFRs at 0.3, 1, 3 and 10 mg/kg. The treated mice were euthanized. The mouse spleens were collected and weighed day 4 post treatment. Single splenocyte suspensions were prepared and stained with fluorochrome-conjugated anti-CD4, anti-CD8, and anti-NKl.l antibodies and the percentage of CD4+ T cells, CD8+ T cells, and NK cells was analyzed by flow cytometry. The results showed that 7tl5-TGFRs was effective at expanding splenocytes based on spleen weight (Figure 107A), especially at 1-10 mg/kg. The percentages of CD8+ T cells and NK cells were higher compared to control-treated mice (Figure 107B) at all doses tested.

CD44 Expression of CD4+ and CD8+ T cells

It has been known that IL-15 induces CD44 expression on T cells and development of memory T cells. CD44 expression of CD4+ and CD8+ T cells in the 7tl5-TGFRs treated mice were assessed. C57BL/6 mice were subcutaneously treated with 7tl5-TGFRs. The splenocytes were stained with fluorochrome-conjugated antiCD4, anti-CD8 and anti-CD44 monoclonal antibodies for immunocyte subsets. The percentages of CD4^CD44hlgh T cells of total CD4+ T cells and CD8~CD44hlgh T cells of total CD8+ T cells were analyzed by flow cytometry. As shown in Figures 108A and 108B, 7tl5-TGFRs significantly activated CD4+ and CD8+ T cells to differentiate into memory T cells.

Furthermore, the dynamic proliferation of immune cells based on Ki67 expression of splenocytes and cytotoxicity potential based on granzyme B expression of the splenocytes induced by 7tl5-TGFRs after the single dose treatment of mouse were also evaluated. C57BL/6 mice were subcutaneously treated with 7tl5-TGFRs at 3 mg/kg. The treated mice were euthanized and the splenocytes were prepared. The prepared splenocytes were stained with fluorochrome-conjugated anti-CD4, anti-CD8, and antiNK1.1 (NK) antibodies for immunocyte subsets and then intracellularly stained with antiKi67 antibody for cell proliferation and anti-granzyme B antibody for cytotoxic marker. The mean fluorescent intensity (MFI) of Ki67 and granzyme B of corresponding immunocyte subsets was analyzed by flow cytometry. As shown in Figures 109A and 109B, in the spleens of mice treated with 7tl5-TGFRs, the expression of Ki67 and granzyme B by CD8+ T cells and NK cells increased compared with PBS control treatment. These results demonstrate that 7tl5-TGFRs is not only to increase numbers of CD8+ T cells and NK cells but also enhance potential cytotoxicity of these cells.

Additionally, cytotoxicity of the mouse splenocytes against tumor cells was also evaluated. Mouse Yac-1 cells were labeled with CellTrace Violet and used as tumor target cells. The splenocytes were prepared from 7tl5-TGFRs-treated mice and used as effector cells. The target cells were mixed with effector cells at E:T ratio = 10:1 in

RPMI-10 medium with or without 7tl5-TGFRs at 100 nM and incubated at 37°C for 20 hours. Target Yac-1 cell inhibition was assessed by analysis of viable violet-labeled Yac-1 cells using flow cytometry. Percentage of Yac-1 inhibition was calculated using a formula, (1-viable Yac-1 cell number in experimental sample/viable Yac-1 cell number in the sample without splenocytes) x 100. As shown in Figure 110, 7tl5-TGFRs-treated mouse splenocytes had stronger cytotoxicity against Yac-1 cells than the control mouse splenocytes and addition of 7tl5-TGFRs during cytotoxic assay further enhanced cytotoxicity of splenocytes against Yac-1 target cells.

Example 56: TGFRtl5-21sl37L fusion protein generation and characterization

A fusion protein complex was generated comprising IL-21/IL-15RaSu/CD137L and TGF3 Receptor II/TF/IL-15 fusion proteins (Figure 111 and Figure 112). The human TGF3 Receptor II (Ile24-Aspl59), tissue factor 219, and IL-15 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made linking two TGF3 Receptor II sequences with a G4S(3) linker to generate a single chain version of TGFp Receptor II and then directly linking to the N-terminus coding region of tissue factor 219 followed by the N-terminus coding region of IL-15.

The nucleic acid sequence of the TGFRtl5 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human TGFf Receptor IIfragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT

AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG

GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGFRtl5 fusion protein (including the leader sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human TGFf Receptor II)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

The nucleic acid and protein sequences of the 21sl37L are shown below. The nucleic acid sequence of the 21sl37L construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACAT CGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCT GCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTC AGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCA ACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGA AGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGAT CCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG ((G4S)3 linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human CD137L)

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGAC CTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCG ATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGAC GGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGC TGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCG AGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCT GCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGA GGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCG GCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTG GCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAA ATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA

The amino acid sequence of 21sl37L fusion protein (including the leader sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR ((G4S)3 linker)

GGGGSGGGGSGGGGS (Human CD137L)

REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVS LTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSA AGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAW QLTQGATVLGLFRVTPEIPAGLPSPRSE

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

The IL-21/IL-15RaSu/CD137L and TGFR/TF/IL-15 constructs were cloned into a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHOKI cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of the soluble TGFR/TF/IL-15: IL-21/IL-15RaSu/CD137L protein complex (referred to as TGFRtl5-21sl37L), which can be purified by anti-TF IgGl affinity and other chromatography methods.

Purification elution chromatograph ofTGFRt!5-21sl37L using anti-TFantibody affinity column

TGFRtl5-21sl37L harvest from cell culture was loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS, followed by elution with 6 column volumes of 0.1M acetic acid (pH 2.9). A280 elution peak was collected and then neutralized to pH 7.5-8.0 with IM Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular weight cutoff. As shown in Figure 113, the anti-TF antibody affinity column bound to TGFRtl521sl37L which contains TF as a fusion partner of TGFRtl5-21sl37L. The bufferexchanged protein sample was stored at 2-8 °C for further biochemical analyses and biological activity tests. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0. IM glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except for the elution step, which was 2 mL/min.

Example 57: TGFRtl5-TGFRs21 fusion protein generation and characterization

A fusion protein complex was generated comprising of TGF3 Receptor II/IL15RaSu/IL-21 and TGF3 Receptor II/TF/IL-15 fusion proteins (Figure 114 and Figure 115). The human TGF3 Receptor II (Ile24-Aspl59), tissue factor 219, IL-21, and IL-15 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made linking two TGF3 Receptor II sequences with a G4S(3) linker to generate a single chain version of TGF3 Receptor II and then directly linking to the N-terminus coding region of tissue factor 219 followed by the N-terminus coding region of IL-15.

The nucleic acid and protein sequences of a construct comprising two TGF3 Receptor II linked to the N-terminus of tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the TGFRtl5 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human TGFf Receptor IIfragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC

ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGFRtl5 fusion protein (including the leader sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human TGFf Receptor II)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW

KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

Constructs were also made by attaching two TGF3 Receptor II directly to the IL15RaSu chain, followed by the N-terminus coding region of IL-21, which was synthesized by Genewiz. The nucleic acid and protein sequences of a construct comprising the TGF3 Receptor II linked to the N-terminus of IL-15RaSu following with the N-terminus of IL-21 are shown below.

The nucleic acid sequence of the TGFRs21 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human ΤΟΡβ Receptor IIfragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC

TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (Human IL-21)

CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATT GTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGC TCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGA AGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATG TATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAA GACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACC ACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCAT CAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC

The amino acid sequence of TGFRs21 fusion protein (including the leader sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human TGFp Receptor II)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR (Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCF QKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

The TGFR/IL-15RaSu/IL-21 and TGFR/TF/IL-15 constructs were cloned into a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHOKI cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of the soluble TGFR/TF/IL-15:TGFR/IL-15RaSu/IL-21 protein complex (referred to as TGFRtl5-TGFRs21), which can be purified by anti-TF antibody IgGl affinity and other chromatography methods.

Purification elution chromatograph of TGFRtl 5-TGFRs21 using anti-TF antibody affinity column

TGFRtl 5-TGFRs21 harvested from cell culture was loaded onto the anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS, followed by elution with 6 column volumes of 0. IM acetic acid (pH 2.9). A280 elution peak was collected and then neutralized to pH 7.5-8.0 with IM Tris base. The neutralized sample was then buffer exchanged into PBS using Amicon centrifugal filters with a 30 KDa molecular weight cutoff. As shown in Figure 116, the anti-TF antibody affinity column bound to TGFRtl 5-TGFRs21 which contains TF as a fusion partner. The buffer-exchanged protein sample was stored at 2-8 °C for further biochemical analyses and biological activity tests. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS, and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except for the elution step, which was 2 mL/min.

Reduced SDS-PAGE analysis of TGFRtl5-TGFRs21

To determine the purity and molecular weight of the protein, TGFRtl 5-TGFRs21 protein sample purified with anti-TF antibody affinity column was analyzed by sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDSPAGE) method under reduced condition. After electrophoresis, the gel was stained with InstantBlue for about 30 min, followed by destaining overnight in purified water.

To verify that the TGFRtl 5-TGFRs21 protein undergoes glycosylation after translation in CHO cells, a deglycosylation experiment was conducted using the Protein Deglycosylation Mix II kit from New England Biolabs and the manufacturer’s instructions. Figure 117 shows the reduced SDS-PAGE analysis of the sample in nondeglycosylated (lane 1 in red outline) and deglycosylated (lane 2 in yellow outline) state. It is clear that the protein is glycosylated when it is expressed in CHO cells. After deglycosylation, the purified sample showed expected molecular weights (69 kDa and 55 kDa) in reduced SDS gel. Lane M was loaded with 10 ul of SeeBlue Plus2 Prestained Standard.

Immunostimulation of TGFRtl5-TGFRs21 in C57BL/6 mice

TGFRtl 5-TGFRs21 is a multi-chain polypeptide (a type A multi-chain polypeptide described herein) that includes the first polypeptide that is a soluble fusion of single chain two TGFpRII domains, human tissue factor 219 fragment and human IL-15 (TGFRtl 5), and the second polypeptide that is a soluble fusion of single chain two TGFpRII domains, sushi domain of human IL-15 receptor alpha chain and human IL-21 (TGFRs21).

CHO cells were co-transfected with TGFRtl 5 and TGFRs21 vectors. The TGFRtl 5-TGFRs21 complex was purified from the transfected CHO cell culture supernatant. The TGF3 receptor, IL-15, IL-21 and tissue factor (TF) components were demonstrated in the complex by ELISA as shown in Figure 118A-B. A humanized antiTF monoclonal antibody (anti-TF IgGl) was used as the capture antibody to determine TF in TGFRtl5-TGFRs21, biotinylated anti-human IL-15 antibody (R&D systems), biotinylated anti-human TGF3 receptor antibody (R&D systems, and biotinylated antihuman IL-21 antibody (R&D Systems) were used as the detection antibodies to respectively determine IL-15, TGF3 receptor, and IL-21 in TGFRtl5-TGFRs21. For detection, peroxidase conjugated streptavidin (Jackson ImmunoResearch Lab) and ABTS were used.

Wild type C57BL/6 mice were treated subcutaneously with either control solution (PBS) or with TGFRtl5-TGFRs21 at 3 mg/kg. Four days after treatment, spleen weight and the percentages of various immune cell types present in the spleen were evaluated. As shown in Figure 119A, the percentages of CD4+ T cells, CD8+ T cells, and NK cells present in the spleen of control-treated and TGFRtl 5-TGFRs21-treated mice were evaluated. The dynamic proliferation of immune cells based on Ki67 expression after TGFRtl 5-TGFRs21 treatment was also evaluated. The splenocytes were stained with fluorochrome-conjugated anti-CD4, anti-CD8, and anti-NKl.l (NK) antibodies and then intracellularly stained with anti-Ki67 antibody. The percentage of CD4+ T cells, CD8+ T cells, and NK cells and the mean fluorescent intensity (MFI) of Ki67 of corresponding immunocyte subsets were analyzed by flow cytometry (Figures 119Aand 119B). Furthermore, cytotoxicity potential based on granzyme B expression of the splenocytes induced by TGFRtl 5-TGFRs21 after the single dose treatment of mouse was also evaluated. As shown in Figure 120, in the spleens of mice treated with TGFRtl 5TGFRs21, the expression of granzyme B by NK cells increased after treatment. The splenocytes from TGFRtl 5-TGFRs21-treated mice were stained with fluorochromeconjugated anti-CD4, anti-CD8, and anti-NKl.l (NK) antibodies and then intracellularly stained with anti-granzyme B antibody. The mean fluorescent intensity (MFI) of granzyme B of corresponding immunocyte subsets was analyzed by flow cytometry (Figure 120).

As shown in Figure 119A, in the spleens of mice treated with TGFRtl 5TGFRs21, the percentages of CD8+ T cells and NK cells both increased on day 4 after a single TGFRtl 5-TGFRs21 treatment. These results demonstrate that TGFRtl 5431

TGFRs21 is able to induce immune cells to proliferate in mouse spleen, in particular CD8+ T cells and NK cells.

Additionally, cytotoxicity of the mouse splenocytes against tumor cells was also evaluated. Mouse Yac-1 cells were labeled with CellTrace Violet and used as tumor target cells. The splenocytes were prepared from TGFRtl5-TGFRs21-treated mice and used as effector cells. The target cells were mixed with effector cells at E:T ratio =10:1 in RPMI-10 medium with or without TGFRtl5-TGFRs21 at 100 nM and incubated at 37°C for 24 hours. Target Yac-1 cell inhibition was assessed by analysis of viable violetlabeled Yac-1 cells using flow cytometry. Percentage of Yac-1 inhibition was calculated using a formula, (!-[viable Yac-1 cell number in experimental sample]/[viable Yac-1 cell number in the sample without splenocytes]) x 100. As shown in Figure 121, TGFRtl5TGFRs21-treated mouse splenocytes had stronger cytotoxicity against Yac-1 cells than the control mouse cells in the presence of TGFRtl5-TGFRs21 during cytotoxic assay (Figure 121).

Example 58: TGFRtl5-TGFRsl6 fusion protein generation

A fusion protein complex was generated comprising of TGF3 Receptor II/IL15RaSu/ anti-CD16scFv and TGF3 Receptor II/TF/IL-15 fusion proteins (Figure 122 and Figure 123). The human TGF3 Receptor II (Ile24-Aspl59), tissue factor 219, and IL-15 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made linking two TGF3 Receptor II sequences with a G4S(3) linker to generate a single chain version of TGF3 Receptor II and then directly linking to the N-terminus coding region of tissue factor 219 followed by the N-terminus coding region of IL-15.

The nucleic acid and protein sequences of a construct comprising two TGF3 Receptor II linked to the N-terminus of tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the TGFRtl5 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human TGFf Receptor IIfragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA

AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGFRtl5 fusion protein (including the leader sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human TGFf Receptor II)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

Constructs were also made by attaching two TGF3 Receptor II directly to the IL15RaSu chain, followed by the anti-CD16scFv sequence, which was synthesized by Genewiz. The nucleic acid and protein sequences of a construct comprising the TGF3 Receptor II linked to the N-terminus of IL-15RaSu following with the anti-CD16scFv sequence are shown below.

The nucleic acid sequence of the TGFRsl6 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human TGFβReceptor IIfragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA

GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (Anti-human CD16scFv)

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACC GTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGT ACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAA CAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCG CCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTG CAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAG CTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGA GGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCT GGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTA CGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCC GGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCA GGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAA CTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCC CTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG

The amino acid sequence of TGFRsl6 fusion protein (including the leader sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human TGFβReceptor II)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN

NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR (Anti-human CD16scFv)

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKL TVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGM SWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLR AEDTAVYYCARGRSLLFDYWGQGTLVTVSR

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

The TGFR/IL-15RaSu/anti-CD16scFv and TGFR/TF/IL-15 constructs were cloned into a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of the soluble TGFR/TF/IL-15:TGFR/IL15RaSu/anti-CD16scFv protein complex (referred to as TGFRtl5-TGFRsl6), which can be purified by anti-TF IgGl affinity and other chromatography methods.

Example 59: The TGFRtl5-TGFRsl37L fusion protein generation

A fusion protein complex was generated comprising of TGF3 Receptor II/IL15RaSu/ CD137L and TGF3 Receptor n/TF/IL-15 fusion proteins (Figure 124 and Figure 125). The human TGF3 Receptor II (Ile24-Aspl59), tissue factor 219, CD137L, and IL-15 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made linking two

TGF3 Receptor II sequences with a G4S(3) linker to generate a single chain version of TGF3 Receptor II and then directly linking to the N-terminus coding region of tissue factor 219 followed by the N-terminus coding region of IL-15.

The nucleic acid and protein sequences of a construct comprising two TGF3 Receptor II linked to the N-terminus of tissue factor 219 following with the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the TGFRtl5 construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human TGFβReceptor IIfragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAG CACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTT TACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAA ACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGC ACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTA CCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGC ACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAAC AACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACT GTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACA AACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGC AAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGT TGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATT CAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTC TTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTT TAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTT AGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAG GAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTG TGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGFRtl5 fusion protein (including the leader sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human TGFβReceptor II)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV

WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKC FYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYL ETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYW KSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

Constructs were also made by attaching two TGF3 Receptor II directly to the IL15RaSu chain, followed by a (G4S)3 linker and the CD137L sequence, which was synthesized by Genewiz. The nucleic acid and protein sequences of a construct comprising the TGF3 Receptor II linked to the N-terminus of IL-15RaSu following with a (G4S)3 linker and the CD137L sequence are shown below.

The nucleic acid sequence of the TGFRsl37L construct (including signal peptide sequence) is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC (Human TGFβReceptor IIfragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACC GACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCA GGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCAC CTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATC ACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAA GAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGT

AACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAG GTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCA CGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCC GTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGA CAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAG CCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCC TGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGA AGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGA GACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCT TTAGCGAGGAATACAATACCAGCAACCCCGAC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTG AAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCA AGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTA CCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG ((G4S)3 linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human CD137L)

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGAC CTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCG ATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGAC GGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGC TGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCG AGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCT GCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGA GGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCG GCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTG GCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAA ATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA

The amino acid sequence of TGFRsl37L fusion protein (including the leader sequence) is as follows:

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human TGFβReceptor II)

IPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCD VRF STCDNQKSCMSNC SI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVN NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE CNDNIIFSEEYNTSNPD (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR ((G4S)3 linker)

GGGGSGGGGSGGGGS (Human CD137L)

REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVS LTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSA AGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAW QLTQGATVLGLFRVTPEIPAGLPSPRSE

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form that may be soluble or secreted.

The TGFR/IL-15RaSu/CD137L and TGFR/TF/IL-15 constructs were cloned into a modified retrovirus expression vectors as described previously (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for formation and secretion of the soluble TGFR/TF/IL-15:TGFR/IL-15RaSu/CD137L protein complex (referred to as TGFRtl5-TGFRsl37L), which can be purified by anti-TF IgGl affinity and other chromatography methods.

Example 60: Stimulation of NK cells in vitro by multi-chain chimeric polypeptide constructs

A set of experiments was performed to assess changes in the surface phenotype of lymphocyte populations after stimulation with 18tl5-12s, 18tl5-12sl6, and 7tl5-21s. In these experiments, fresh human leukocytes were obtained from the blood bank. Peripheral blood lymphocytes were isolated with the Ficoll-PAQUE Plus (GE Healthcare) density gradient media. The cells were counted and resuspended at 0.2 x 106/mL in a 96-well flat-bottom plate in 0.2 mL of complete media (RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). The cells were stimulated with: 18tl5-12s (100 nM); 18tl5-12sl6 (100 nM), a mixture of single cytokines rhIL15 (50 ng/mL) (Miltenyi), rhIL18 (50 ng/mL) (Invivogen), and rhIL-12 (10 ng/mL) (Peprotech); 7tl5-21s (100 nM) + anti-TF antibody (50 nM); 7tl521s (100 nM); or anti-TF antibody (50 nM) at 37 °C and 5% CO2 for 16 hours. The next day, the cells were harvested and surface stained for 30 minutes with antibodies specific for CD4 or CD8, CD62L, and CD69. After surface staining, cells were washed (1500 RPM for 5 minutes at room temperature) in FACS buffer (IX PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)). After two washes, the cells were analyzed by flow cytometry (Celesta-BD Bioscience). Figure 126 shows that overnight incubation of purified lymphocyte populations (CD4 and CD8 T cells) with 18tl5-12s, 18tl5-12sl6, or 7tl5-21s + anti-TF antibody resulted in an increase in the percentage of CD8 and CD4 T cells expressing CD69. Additionally, incubation with 7tl5-21s + anti-TF antibody resulted in an increase in the percentage of CD8 and CD4 T cells expressing CD62L (Figure 126).

A set of experiments was performed to determine the increase in phospho-ST AT4 and phospho-STAT5 levels in NK cells after stimulation with 18tl5-12s. In these experiments, fresh human leukocytes were obtained from the blood bank and CD56+ NK cells were isolated with the RosetteSep/human NK cell reagent (StemCell Technologies).

The purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16BV510, CD25-PE, and CD69-APCFire750 specific antibodies (BioLegend). The cells were counted and resuspended in 0.05 x 106/mL in a 96-well flat-bottom plate in 0.1 mL of complete media (RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). The cells were stimulated with hIL-12 (10 ng/mL) (Biolegend) or hIL-15 (50 ng/mL) (NCI) (Single cytokines), or 18tl5-12s (100 nM) at 37 °C and 5% CO2 for 90 minutes. Unstimulated NK cells (US) were used as a control. The cells were harvested and fixed in paraformaldehyde (Sigma) to a final concentration of 1.6%. Plates were incubated in the dark at room temperature for 10 minutes. FACS buffer (IX PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) (100 pL) was added and cells were transferred to 96-well “V” bottom plate. The cells were washed for 1500 RPM for 5 minutes at room temperature. The cell pellet was mixed with 100 pL chilled methanol by gently pipetting up and down, and cells were incubated for 30 minutes at 4 °C. The cells were mixed with 100 mL of FACS buffer and washed for 1500 RPM for 5 minutes at room temperature. The cell pellets were mixed with 50 mL of FACS buffer containing 4 mL of pSTAT4 (BD Bioscience) and pSTAT5 antibodies (BD Bioscience) followed by incubation for 30 minutes at room temperature in the dark. The cells were mixed with 100 mL of FACS buffer and washed for 1500 RPM for 5 minutes at room temperature. The cell pellets were mixed with 50 mL of FACS buffer and cells were analyzed by flow cytometry (Celesta-BD Bioscience). Figure 127 shows that incubation of NK cells with 18tl5-12s induced an increase in pSTAT4 and pSTAT5 (plotted data, normalized fold-change).

Example 61: Stimulation of NK cells in vivo by TGFRtl5-TGFRs

A set of experiments was performed to determine the effect of the TGFRtl 5TGFRs construct on immune stimulation in ApoE_/ mice fed with a Western diet. In these experiments, 6-week old female B6.129P2-ApoEtmlUnc/J mice (Jackson Laboratory) were fed with aWestern diet containing 21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein, and 15% starch (TD88137, Envigo Laboratories). After 8-weeks of the Western diet, the mice were injected subcutaneously with TGFRtl5-TGFRs at 3 mg/kg.

Three days post treatment, mice were fasted for 16 hours and then blood samples were collected through retro-orbital venous plexus puncture. The blood was mixed with 10 pL 0.5 M EDTA, and 20 pL blood was taken for lymphocyte subsets analysis. The red blood cells were lysed with ACK (0.15 MNH4C1, 1.0 mMKHC03, 0.1 mMNa2EDTA, pH 7.4) and the lymphocytes were stained with anti-mouse CD8a and anti-mouse NK1.1 antibodies for 30 minutes at 4 °C in FACS staining buffer (1% BSA in PBS). The cells were washed once and analyzed with a BD FACS Celesta. For Treg staining, ACK treated blood lymphocytes were stained with anti-mouse CD4 and anti-mouse CD25 antibodies for 30 minutes at 4 °C in FACS staining buffer. The cells were washed once and resuspended in fixation/permeabilization working solution and incubated at room temperature for 60 minutes. The cells were washed once and resuspended in permeabilization buffer. The samples were centrifuged at 300-400 x g for 5 minutes at room temperature and the supernatant was then discarded. The cell pellet was resuspended in residual volume and the volume adjusted to about 100 pL with 1 x permeabilization buffer. Anti-F0xp3 antibody was added to the cells, and the cells were incubated for 30 minutes at room temperature. Permeabilization buffer (200 pL) was added to the cells, and the cells were centrifuged at 300-400 x g for 5 minutes at room temperature. The cells were resuspended in flow cytometry staining buffer and analyzed on a flow cytometer. Figures 128A-128C show that treatment with TGFRtl5-TGFRs increased the percentage of NK cells and CD8+ T cells in ApoE_/ mice fed with Western diet.

Example 62: Induction of proliferation of immune cells in vivo

A set of experiments was performed to determine the effect of the TGFRtl5TGFRs construct on immune stimulation in C57BL/6 mice. In these experiments, C57BL/6 mice were subcutaneously treated with control solution (PBS) or TGFRtl5TGFRs at 0.1, 0.3, 1, 3, and 10 mg/kg. The treated mice were euthanized 4 days posttreatment. Spleen weight was measured and splenocyte suspensions were prepared. The splenocyte suspensions were stained with conjugated anti-CD4, anti-CD8, and antiNK1.1 (NK) antibodies. The cells were additionally stained for proliferation marker Ki67. Figure 129A shows that spleen weight in mice treated with TGFRtl5-TGFRs increased with increasing dosage of TGFRtl5-TGFRs. Additionally, spleen weight in mice treated with 1 mg/kg, 3 mg/kg, and 10 mg/kg of TGFRtl5-TGFRs was higher as compared to mice treated with just the control solution. The percentages of CD8+ T cells and NK cells both increased with increasing dosage of TGFRtl5-TGFRs (Figure 129B). Finally, TGFRtl5-TGFRs significantly upregulated expression of cell proliferation marker Ki67 in both CD8+ T cells and NK cells at all doses of TGFRtl5-TGFRs tested (Figure 129C). These results demonstrate that TGFRtl5-TGFRs treatment induced proliferation of both CD8+ T cells and NK cells in C57BL/6 mice.

A set of experiments was performed to determine the effect of the TGFRtl5TGFRs construct on immune stimulation in ApoE_/ mice fed with a Western diet. In these experiments, 6-week old female B6.129P2-ApoEtmlUnc/J mice (Jackson Laboratory) were fed with aWestern diet containing 21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein, and 15% starch (TD88137, Envigo Laboratories). After 8-week of the Western diet, the mice were injected subcutaneously with TGFRtl5-TGFRs at 3 mg/kg. Three days post-treatment, the mice were fasted for 16 hours and then blood samples were collected through retro-orbital venous plexus puncture. The blood was mixed with 10 pL 0.5 M EDTA and 20 pL blood was taken for lymphocyte subsets analysis. The red blood cells were lysed with ACK (0.15 MNH4C1, 1.0 mMKHC03, 0.1 mMNa2EDTA, pH 7.4) and the lymphocytes were stained with anti-mouse CD8a and anti-mouse NK1.1 antibodies for 30 minutes at 4 °C in FACS staining buffer (1% BSA in PBS). The cells were washed once and resuspended in Fixation Buffer (BioLegend Cat# 420801) for 20 minutes at room temperature. The cells were centrifuged at 350 x g for 5 minutes, the fixed cells were resuspended in Intracellular Staining Permeabilization Wash Buffer (BioLegend Cat# 421002) and then centrifuged at 350 x g for 5 minutes. The cells were then stained with anti-Ki67 antibody for 20 minutes at RT. The cells were washed twice with Intracellular Staining Permeabilization Wash Buffer and centrifuged at 350 x g for 5 minutes. The cells were then resuspended in FACS staining buffer. Lymphocyte subsets were analyzed with a BD FACS Celesta. As described in Figure 130A and 130B, treatment of ApoE_/ mice with TGFRtl5-TGFRs induced proliferation (Ki67-positive staining) in NK and CD8+ T cells.

Example 63: NK-mediated cytotoxicity following treatment with multi-chain construct

A set of experiments was performed to determine if treatment of NK cells with TGFRtl5-TGFRs enhanced cytotoxicity of NK cells. In these experiments, Human Daudi B lymphoma cells were labeled with CellTrace Violet (CTV) and used as tumor target cells. Mouse NK effector cells were isolated with NK1.1-positive selection using a magnetic cell sorting method (Miltenyi Biotec) of C57BL/6 female mouse spleens 4 days post TGFRtl5-TGFRs subcutaneous treatment at 3 mg/kg. Human NK effector cells were isolated from peripheral blood mononuclear cells derived from human blood buffy coats with the RosetteSep/human NK cell reagent (Stemcell Technologies). The target cells (Human Daudi B lymphoma cells) were mixed with effector cells (either mouse NK effector cells or human NK effector cells) in the presence of 50 nM TGFRtl5-TGFRs or in the absence of TGFRtl5-TGFRs (control) and incubated at 37 °C for 44 hours for mouse NK cells and for 20 hours for human NK cells. Target cell (Daudi) viability was assessed by analysis of propidium iodide-positive, CTV-labeled cells using flow cytometry. The percentage of Daudi inhibition was calculated using the formula (1viable tumor cell number in experimental sample/viable tumor cell number in the sample without NK cells) x 100. Figure 131 shows that mouse (Figure 131 A) and human (Figure 13 IB) NK cells had significantly stronger cytotoxicity against Daudi B cells following NK cell activation with TGFRtl5-TGFRs than in the absence of TGFRtl5-TGFRs activation.

A set of experiments was performed to determine antibody-dependent cellular cytotoxicity (ADCC) of mouse and human NK cells following treatment with TGFRtl5TGFRs. In these experiments, human Daudi B lymphoma cells were labeled with CellTrace Violet (CTV) and used as tumor target cells. Mouse NK effector cells were isolated with NK 1.1-positive selection using a magnetic cell sorting method (Miltenyi Biotec) of C57BL/6 female mouse spleens 4 days post-TGFRtl5-TGFRs subcutaneous treatment at 3 mg/kg. Human NK effector cells were isolated from peripheral blood mononuclear cells derived from human blood buffy coats with the RosetteSep/human NK cell reagent (Stemcell Technologies). The target cells (Daudi B cells) were mixed with effector cells (either mouse NK effector cells or human NK effector cells) in the presence of anti-CD20 antibody (10 nM Rituximab, Genentech) and in the presence of 50 nM TGFRtl5-TGFRs, or in the absence of TGFRtl5-TGFRs (control) and incubated at 37 °C for 44 hours for mouse NK cells and for 20 hours for human NK cells. The Daudi B cells express the CD20 targets for the anti-CD20 antibody. Target cell viability was assessed after incubation by analysis of propidium iodide-positive, CTV-labeled target cells using flow cytometry. The percentage of Daudi inhibition was calculated using the formula (1viable tumor cell number in experimental sample/viable tumor cell number in the sample without NK cells) x 100. Figure 132 shows that mouse NK cells (Figure 132A) and human NK cells (Figure 132B) had stronger ADCC activity against Daudi B cells following NK cell activation with TGFRtl 5-TGFRs than in the absence of TGFRtl 5TGFRs activation.

Example 64: Treatment of Cancer

A set of experiments was performed to assess antitumor activity of TGFRtl 5TGFRs plus anti-TRPl antibody (TA99) in combination with chemotherapy in a melanoma mouse model. In these experiments, C57BL/6 mice were subcutaneously injected with 0.5 x 106 B16F10 melanoma cells. The mice were treated with three doses of chemotherapy docetaxel (10 mg/kg) (DTX) on day 1, day 4, and day 7, followed by treatment with single dose of combination immunotherapy TGFRtl 5-TGFRs (3 mg/kg) + anti-TRPl antibody TA99 (200 pg) on day 9. Figure 133A shows a schematic of the treatement regimen. Tumor growth was monitored by caliper measurement, and tumor volume was calculated using the formula V = (L * W2)/2, where L is the largest tumor diameter and W is the perpendicular tumor diameter. Figure 133B shows that treatment with DTX + TGFRtl 5-TGFRs + TA99 significantly reduced tumor growth compared to saline control and DTX treatment groups (N=10, ****p <0.001, Multiple t test analyses).

To assess immune cell subsets in the B16F10 tumor model, peripheral blood analysis was performed. In these experiments, C57BL/6 mice were injected with Bl6F10 cells and treated with DTX, DTX + TGFRtl 5-TGFRs + TA99, or saline. Blood was drawn from the submandibular vein of Bl 6F10 tumor-bearing mice on days 2, 5, and 8 post-immunotherapy for the DTX + TGFRtl 5-TGFRs + TA99 group and day 11 posttumor injection for the DTX and saline groups. RBCs were lysed in ACK lysis buffer and the lymphocytes were washed and stained with anti-NKl.l, anti-CD8, and anti-CD4 antibodies. The cells were analyzed by flow cytometry (Celesta-BD Bioscience). Figures 133C-133E show that DTX + TGFRtl5-TGFRs + TA99 treatment induced an increase in the percentage of NK cells and CD8+ T cells in the tumors compared to the saline and DTX treatment groups.

On day 17, total RNA was extracted from tumors of mice treated with saline, DTX or DTX r TGFRtl 5-TGFRs ;־ TA99 using Trizol Total RNA (1 pg) was used for cDNA synthesis using the QuantiTect Reverse Transcription Kit (Qiagen). Real-time PCR was carried out with CFX96 Detection System (Bio-Rad) using FAM-labeled predesigned primers for senescence cell markers, (F) p21 (G) DPP4 and (H) IL6. The housekeeping gene 18S ribosomal RNA was used as an internal control to normalize the variability in expression levels. The expression of each target mRNA relative to 18S rRNA was calculated based on Ct as 2־Δ(Δα), in which ACt = Cttarget- Ct18s. The data is presented as fold-change as compared to saline control. Figure 133F-133H show that DTX treatment induced an increase in senescent tumor cells that were subsequently reduced following treatment with TGFRtl 5-TGFRs + TA99 immunotherapy.

A set of experiments was performed to investigate amelioration of Western dietinduced hyperglycemia in ApoE_/ mice by TGFRtl 5-TGFRs. In these experiments, 6week old female B6.129P2-ApoEtmlUnc/J mice (Jackson Laboratory) were fed with a Western diet containing 21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein, and 15% starch (TD88137, Envigo Laboratories). After 8-weeks of the Western diet, the mice were injected subcutaneously with TGFRtl5-TGFRs at 3 mg/kg. Three days posttreatment, the mice were fasted for 16 hours and then blood samples were collected through retro-orbital venous plexus puncture. Blood glucose was detected with a glucose meter (OneTouch UltraMini) and GenUltimated test strips using a drop of fresh blood. As shown in Figure 134A, TGFRtl 5-TGFRs treatment reduced hyperglycemia induced by the Western diet. The plasma insulin and resistin levels were analyzed with Mouse Rat Metabolic Array by Eve Technologies. HOMA-IR was calculated using the following formula: homeostatic model assessment-insulin resistance = Glucose (mg/dL) * Insulin (mU/mL)/405. As shown in Figure 134B, TGFRtl 5-TGFRs treatment reduced insulin resistance compared to the untreated group. TGFRtl 5-TGFRs (p<0.05) reduced resistin levels significantly compared to the untreated group as shown in Figure 142C, which may relate to the reduced insulin resistance induced by TGFRtl5-TGFRs (Figure 134B).

Example 65: Induction of differentiation of NK cells into cytokine-induced memory like NK cells

A set of experiments was performed to assess the differentiation of NK cells into cytokine-induced memory like NK Cells (CIMK-NK Cells) after stimulation with 18tl512s. In these experiments, fresh human leukocytes were obtained from the blood bank and CD56+ NK cells were isolated with the RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >90% and confirmed by staining with CD56BV421, CD16-BV510, CD25-PE, and CD69-APCFire750 antibodies (BioLegend). The cells were counted and resuspended in 2 x 106/mL in a 24-well flat-bottom plate in 2 mL of complete media (RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). The cells were unstimulated (“No Spike”) or stimulated with 18tl5-12s (100 nM) or a mixture of single cytokines including rhIL15 (50 ng/mL) (Miltenyi), rhIL18 (50 ng/mL) (Invivogen), and rhIL-12 (10 ng/mL) (Peprotech) (“single cytokines”) at 37 °C and 5% CO2 for 16 hrs. The next day, the cells were harvested, and washed two times with warm complete media at 1000 RPM for 10 minutes at room temperature. The cells were resuspended at 2 x 106/mL in a 24-well flatbottom plate in 2 mL of complete media with rhIL15 (1 ng/mL). After every 2 days, half of the medium was replaced with fresh complete media containing rhIL15.

To assess the change in memory phenotype of NK cells at day 7, the cells were stained with antibodies to cell-surface CD56, CD16, CD27, CD62L, NKp30, and NKp44 (BioLegend). After surface staining, the cells were washed (1500 RPM for 5 minutes at room temperature) in FACS buffer (IX PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)). After two washes, the cells were analyzed by flow cytometry (Celesta-BD Bioscience). Figure 135 shows that incubation of NK cells with 18tl5-12s resulted in an increase in the percentage of CD16+CD56+ NK cells expressing

CD27, CD62L, and NKp44, and an increase in the levels (MFI) of NKp30 in CD 16+CD56+NK cells.

Example 66: Upregulation of CD44 memory T cells

A set of experiments was performed to assess upregulation of CD44 memory T cells upon treatment with TGFRtl 5-TGFRs. In these experiments, C57BL/6 mice were subcutaneously treated with TGFRtl 5-TGFRs. The treated mice were euthanized and the single splenocyte suspensions were prepared 4 days (TGFRtl 5-TGFRs) following the treatment. The prepared splenocytes were stained with fluorochrome-conjugated antiCD4, anti-CD8 and anti-CD44 antibodies and the percentages of CD44hlgh T cells in CD4+ T cells or CD8+ T cells were analyzed by flow cytometry. The results show that TGFRtl 5-TGFRs upregulated expression of the memory marker CD44 on CD4+ and CD8+ T cells (Figures 136). These findings indicate that TGFRtl5-TGFRs was able to induce mouse T cells to differentiate into memory T cells.

Example 67: Tissue factor coagulation assays following treatment with single-chain or multi-chain chimeric polypeptides

A set of experiments was performed to assess blood coagulation following treatment with single-chain or multi-chain chimeric polypeptides. To initiate the blood coagulation cascade pathway, tissue factor (TF) binds to Factor Vila (FVIIa) to form a TF/FVIIa complex. The TF/FVIIa complex then binds Factor X (FX) and converts FX to FXa.

Factor Vila (FVIIa) activity Assay

One assay to measure blood coagulation involves measuring Factor Vila (FVIIa) activity. This type of assay requires the presence of tissue factor and calcium. The TF/FVIIa complex activity can be measured by a small substrate or by a natural protein substrate, for example, Factor X (FX). When FX is used as a substrate, phospholipids are also required for TF/FVIIa activity. In this assay, FVIIa activity is determined with FVIIa-specific chromogenic substrate S-2288 (Diapharma, West Chester, OH). The color change of the S-2288 substrate can be measured spectrophotometrically and is proportional to the proteolytic activity of FVIIa (e.g., the TF/FVIIa complex).

In these experiments, the FVIIa activity of the following groups were compared: the 219-amino acid extracellular domain of tissue factor domain (TF219), a multi-chain chimeric polypeptide with a wild-type tissue factor domain, and a multi-chain chimeric polypeptide with a mutant tissue factor domain. The chimeric polypeptides containing mutant tissue factor molecules were constructed with mutations to the TF domain at amino acid sites: Lys20, Ile22, Asp58, Argl35, and Phel40.

In order to assess activity of FVIIa, FVIIa, and TF219 or a TF219 -containing multichain chimeric polypeptide were mixed at an equal molar concentration (10 nM) in all wells of a 96-well ELISA plate in a total volume of 70 pL. After incubation for 10 minutes at 37 °C, 10 pL of 8 mM S-2288 substrate was added to start the reaction. The incubation was then kept at 37 °C for 20 minutes. Finally, color change was monitored by reading absorbance at 405 nm. The OD values of different TF/VIIa complexes are shown in Table 1 and Table 2. Table 1 shows a comparison of TF219,21tl5-21s wild-type (WT) and 21tl5-21s mutant (Mut). Table 2 shows a comparison of TF219,21tl5-TGFRs wild-type (WT), and 2Itl 5-TGFRs mutant (Mut). These data show that TF219-containing multi-chain chimeric polypeptides (e.g., 21tl5-21s-WT, 21tl5-21s-Mut, 2Itl 5-TGFRSWT, and 21tl5-TGFRS-Mut) have lower FVIIa activity than TF219 when the chromogenic S-2288 was used as a substrate. Notably, the multi-chain chimeric polypeptides containing TF219 mutations showed much lower FVIIa activity when compared to multi-chain chimeric polypeptides containing wild type TF219.

Table 1. FVIIa activity

<td> Molecule</td><td> OD value at 405 nm</td>

<td> TF219</td><td> 0.307</td>

<td> 21tl5/21S-WT</td><td> 0.136</td>

<td> 21tl5/21S-Mut</td><td> 0.095</td>

WT: wild type of TF219,Mut: TF219 containing mutations.

Table 2. FVIIa activity

<td> Molecule</td><td> OD value at 405 nm</td>

<td> TF219</td><td> 0.345</td>

<td> 21tl5/TGFRS-WT</td><td> 0.227</td>

<td> 21tl5/TGFRS-Mut</td><td> 0.100</td>

WT: wild type of TF219,Mut: TF219 containing mutations.

Factor X (FX) Activation Assay

An additional assay to measure blood coagulation involves measuring activation of Factor X (FX). Briefly, TF/VIIa activates blood coagulation Factor X (FX) to Factor Xa (FXa) in the presence of calcium and phospholipids. TF243, which contains the transmembrane domain of TF, has much higher activity in activating FX to FXa than TF219, which does not contain the transmembrane domain. TF/VIIa dependent activation of FX is determined by measuring FXa activity using an FXa-specific chromogenic substrate S-2765 (Diapharma, West Chester, OH). The color change of S-2765 can be monitored spectrophotometrically and is proportional to the proteolytic activity of FXa.

In these experiments, FX activation with a multi-chain chimeric polypeptide (18tl5-12s, mouse (m)21tl5, 21tl5-TGFRs, and 21tl5-7s) was compared with a positive control (Innovin) or TF219. TF219 (or TF219-containing multi-chain chimeric polypeptides)/FVIIa complexes were mixed at an equal molar concentration (0.1 nM each) in a volume of 50 pL in round bottom wells of a 96-well ELISA plate, after which 10 pL of 180 nM FX was added. After 15 minutes of incubation at 37 °C, during which time FX was converted to FXa, 8 pL of 0.5 M EDTA (which chelates calcium and thus terminates FX activation by TF/VIIa) was added to each well to stop FX activation. Next, 10 pL of 3.2 mM S-2765 substrate was added to the reaction mixture. Immediately, the plate absorbance was measured at 405 nm and was recorded as the absorbance at time 0. The plate was then incubated for 10-20 minutes at 37 °C. The color change was monitored by reading absorbance at 405 nm following the incubation. Results of FX activation as measured by FXa activity using chromogenic substrate S2765 are shown in Figure 137. In this experiment, Innovin, which is a commercial prothrombin reagent containing lipidated recombinant human TF243, was used as a positive control for FX activation. Innovin was reconstituted with purified water to about 10 nM of TF243. Next, 0.1 nM TF/VIIa complex was made by mixing an equal volume of 0.2nM of FVIIa with 0.2 nM of Innovin. Innovin demonstrated very potent FX activation activity, while TF219 and TF219-containing multi-chain chimeric polypeptides had very low FX activation activity, confirming that TF219 is not active in a TF/FVIIa complex for activating natural substrate FX in vivo.

Prothrombin Time Test

A third assay to measure blood coagulation is the prothrombin time (PT) test, which measures blood clotting activity. Here, the PT test was performed using commercially available normal human plasma (Ci-Trol Coagulation Control, Level I). For a standard PT test, clot reactions were initiated by addition of Innovin, a lipidated recombinant human TF243, in the presence of calcium. Clotting time was monitored and reported by STart PT analyzer (Diagnostica Stago, Parsippany, N.J.). PT assays were started by injecting 0.2 mL of various dilutions of Innovin diluted in PT assay buffer (50 mM Tris-HCl, pH 7.5, 14.6 mM CaC12, 0.1% BSA) into cuvettes containing 0.1 mL of normal human plasma prewarmed at 37 °C. In the PT assay, shorter PT time (clotting time) indicates a higher TF-dependent clotting activity while longer PT (clotting time) means lower TF-dependent clotting activity.

As seen in Figure 138, addition of different amounts of Innovin (e.g., Innovin reconstituted with purified water equivalent to 10 nM of lipidated recombinant human TF243 was considered to be 100% Innovin) to the PT assay demonstrated a dose-response relationship, where lower concentrations of TF243 resulted in a longer PT time (lower clotting activity). For example, 0.001% Innovin had a PT time greater than 110 seconds, which was almost the same as buffer alone.

In another experiment, the PT test was conducted on TF219 and multi-chain chimeric polypeptides including: 18tl5-12s, 7tl5-21s, 21tl5-TGFRs-WT, and 21tl5TGFRs-Mut. Figure 139 show that TF219 and TF219-containing multi-chain chimeric polypeptides (at a concentration of 100 nM) had prolonged PT times indicating extremely low or no clotting activity.

Studies were also conducted to evaluate whether incubating the multi-chain chimeric polypeptides in the presence of other cells carrying receptors for the cytokine components of the multi-chain chimeric polypeptide (32&amp;#971;β or human PBMCs) would affect the clotting time in the PT assay. To examine whether cells that express IL-15 receptor (32&amp;#971;β cells) or IL-15 and IL-21 receptors (PBMCs) would bind IL-15 containing multi-chain chimeric polypeptides to mimic natural TF as a cellular FVIIa receptor, TF219-containing multi-chain chimeric polypeptides (at a concentration of 100 nM for each molecule) were diluted in the PT assay buffer and preincubated with 32&amp;#971;β cells (at 2 x 105 cells/mL) or PBMC (at 1 x 105 cells/mL) for 20-30 minutes at room temperature. The PT assay was then conducted as described above. Figures 140 and 141 shows that TF219 and TF219-containing multi-chain chimeric polypeptides mixed with 32&amp;#971;β cells (Figure 140) or PBMC (Figure 141) at a final concentration of 100 nM had prolonged PT times similar to 0.001-0.01% Innovin (equivalent to 0.1 pM to 1.0 pM of TF243). Expressed in percentage of relative TF243 activity, TF219-containing multi-chain chimeric polypeptides had 100,000 to 1,000,000 times lower TF dependent clotting activity when compared to Innovin. This demonstrated that TF219-containing multi-chain chimeric polypeptides had extremely low or no TF-dependent clotting activity, even while the molecules were bound to an intact cell membrane surface, such as 32&amp;#971;β or PBMCs.

Example 68: Characterization of 7tl5-21sl37L (long version)

The nucleic acid sequence of the 7tl 5 construct (including signal peptide sequence) is as follows (SEQ ID NO: 107):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC C (Human IL7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATG GTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCC TCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGA

GGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATG AACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCA CCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCT GGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGA GCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATC AAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (Human Tissue Factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCA ACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACAC CGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACC ACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGA CCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGG TTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCG AGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAA GGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACAC CTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATT ACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGA GTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCT GTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGT GCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGT CCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGT AAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGA GAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCC AATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGT GCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCA CATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of 7tl5 fusion protein (including the leader sequence) is as follows (SEQ ID NO: 106):

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGM FLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (Human Tissue Factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTD TECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQ VGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTL YYWKS S S SG KKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFR E (Human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG DASIH

DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

The nucleic acid sequence of the 21sl37L construct (including signal peptide sequence) is as follows (SEQ ID NO: 225):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTC C (Human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCG ACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCC CGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAG GCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTG AGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGG CAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCC

CCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCA GCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human IL-15R a sushi domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGA GCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAG GAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAAC GTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG ((G4S)3 linker)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human CD137L)

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGC GGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGG GCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGG GGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGA GTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGG CTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTG GGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCT CGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCC AGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCA GCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC CCAGCCGGACTCCCTTCACCGAGGTCGGAA

The amino acid sequence of 21sl37L fusion protein (including the leader sequence) is as follows (SEQ ID NO: 226):

(Signal peptide)

MKWVTFISLLFLF S SAYS (Human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDS (Human IL-15R a sushi domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAH WTTPS

LKCIR ((G4S)3 linker)

GGGGSGGGGSGGGGS (Human CD137L)

REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGL SYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQ GATVLGLFRVTPEIPAGLPSPRSE

The following experiment was conducted to evaluate whether the CD137L portion in 7tl5-21sl37L was intact to bind to CD137 (4. IBB). On day 1, a 96-well plate was coated with 100 pL (2.5 pg/mL) of GAH IgGFc (G C, R&amp;D Systems) in R5 (coating buffer), overnight. On day 2, the plates were washed three times and blocked with 300 pL of 1% BSA in PBS at 37°C for 2 hrs. 10 ng/ml of 4. IBB/Fc (838-4B, R&amp;D Systems) was added at 100 pl/well for 2 hrs at room temperature. Following three washes, 7tl5-21sl37L (long version) or 7tl5-21sl37Ls (short version) was added starting at 10 nM, or recombinant human 4.1BBL starting at 180ng/mL, with 1/3 dilution, followed by incubation at 4°C overnight. On day 3, the plates were washed three times, and 500 ng/mL of biotinylate-goat anti-human 4.1BBL (BAF2295, R&amp;D Systems) was applied at 100 pL per well, followed by incubation at RT for 2 hrs. The plates were washed three times, and incubated with 0.25 pg/mL of HRP-SA (Jackson ImmuneResearch) at 100 pL per well for 30 min. The plates were then washed three times, and incubated with 100 pL of ABTS for 2 mins at RT. The results were read at 405 nm. As shown in Figure 142, both 7tl5-21sl37L (long version) and 7tl5-21sl37L (short version) could interact with 4. IBB/Fc (dark diamond and gray square) compared to the recombinant human 4. IBB ligand (rhCD137L, light gray star). 7tl5-21sl37L (long version) (dark diamond) interacted better with 4.1BB/Fc as compared to 7tl5-21sl37L (short version) (gray square).

The following experiments were conducted to evaluate whether the components IL7, IL21, IL15, and 4.1BBL in 7tl5-21sl37L (long version) were intact to be detected by the individual antibody using ELISA. A 96-well plate was coated with 100 pL (4 pg/mL) of anti-TF (human IgGl) in R5 (coating buffer) and incubated at RT for 2 hrs. The plates were washed three times, and blocked with 100 pL of 1% BSA in PBS. Purified 7tl5-21sl37L (long version) was added starting at 10 nM, and at 1/3 dilution, followed by incubation at RT for 60 min. The plates were washed three times, and 500 ng/mL of biotinylate-anti-IL7 (506602, R&amp;D Systems), 500 ng/mL of biotinylate-antiIL21 (13 81, R&amp;D Systems), 50 ng/mL of biotinylate-anti-IL15 (BAM247, R&amp;D Systems), or 500 ng/ml of biotinylate-goat anti-human 4.1BBL (BAF2295, R&amp;D Systems) was added per well and incubated at room temperature for 60 min. The plates were washed three times and incubated with 0.25 pg/mL of HRP-SA (Jackson ImmunoResearch) at 100 pL per well for 30 min at RT. The plates were washed four times, and incubated with 100 pL of ABTS for 2 mins at room temperature. The absorbance results were read at 405 nm. As shown in Figure 143A-143D, the components including IL7, IL21, IL15, and 4.1BBL in 7tl5-21sl37L (long version) were detected by the individual antibodies.

The following experiment was conducted to evaluate the activity of IL 15 in 7tl521sl37L (long version) and 7tl5-21sl37L (short version). The ability of 7tl5-21sl37L (long version) and 7tl5-21sl37L (short version) to promote proliferation of IL2Ra3yexpressing CTLL2 cells was compared with that of recombinant IL15. IL15 dependent CTLL2 cells were washed five times with IMDM-10% FBS and seeded to the wells at 2 x 104 cells/well. Serially diluted 7tl5-21sl37L (long version), 7tl5-21sl37L (short version), or IL15 were added to the cells. Cells were incubated in a CO2 incubator at 37 °C for 3 days. Cell proliferation was detected by adding 20 pL of PrestoBlue (Al3261, ThermoFisher) to each well on day 3 and incubated for an additional 4 hours in a CO2 incubator at 37 °C. Raw absorbance at 570-610 nm was read in a micro-titer plate reader. As shown in Figure 144, 7tl5-21sl37L (long version), 7tl5-21sl37L (short version), and IL15 all promoted CTLL2 cell proliferation. The EC50 of 7tl5-21sl37L (long version), 7tl5-21sl37L (short version), and IL15 is 51.19 pM, 55.75 pM, and 4.947 pM, respectively.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Exemplary Embodiments

Embodiment Al. A multi-chain chimeric polypeptide comprising:

(a) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and (iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and (ii) a second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains.

Embodiment A2. The multi-chain chimeric polypeptide of embodiment Al, wherein the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide.

Embodiment A3. The multi-chain chimeric polypeptide of embodiment Al, wherein the first chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

Embodiment A4. The multi-chain chimeric polypeptide of any one of embodiments A1-A3, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide.

Embodiment A5. The multi-chain chimeric polypeptide of any one of embodiments A1-A3, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment A6. The multi-chain chimeric polypeptide of any one of embodiments A1-A5, wherein the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide.

Embodiment A7. The multi-chain chimeric polypeptide of any one of embodiments A1-A5, wherein second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

Embodiment A8. The multi-chain chimeric polypeptide of any one of embodiments A1-A7, wherein the first target-binding domain and the second targetbinding domain bind specifically to the same antigen.

Embodiment A9. The multi-chain chimeric polypeptide of embodiment A8, wherein the first target-binding domain and the second target-binding domain bind specifically to the same epitope.

Embodiment A10. The multi-chain chimeric polypeptide of embodiment A9, wherein the first target-binding domain and the second target-binding domain comprise the same amino acid sequence.

Embodiment All. The multi-chain chimeric polypeptide of any one of embodiments A1-A7, wherein the first target-binding domain and the second targetbinding domain bind specifically to different antigens.

Embodiment A12. The multi-chain chimeric polypeptide of any one of embodiments Al-Al 1, wherein one or both of the first target-binding domain and the second target-binding domain is an antigen-binding domain.

Embodiment A13. The multi-chain chimeric polypeptide of embodiment A12, wherein the first target-binding domain and the second target-binding domain are each antigen-binding domains.

Embodiment A14. The multi-chain chimeric polypeptide of embodiment A12 or A13, wherein antigen-binding domain comprises a scFv or a single domain antibody.

Embodiment Al 5. The multi-chain chimeric polypeptide of any one of embodiments A1-A14, wherein one or both of the first target-binding domain and the second target-binding domain bind specifically to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, Pcadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF- β receptor II (TGF- βΒΠ), a ligand of TGF- βΙΜΙΙ, a ligand of DNAM-1, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKP30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD28.

Embodiment Al 6. The multi-chain chimeric polypeptide of any one of embodiments A1-A14, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin or cytokine protein.

Embodiment Al 7. The multi-chain chimeric polypeptide of embodiment Al 6, wherein the soluble interleukin, cytokine, or ligand protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L.

Embodiment Al 8. The multi-chain chimeric polypeptide of any one of embodiments A1-A14, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin or cytokine receptor.

Embodiment Al 9. The multi-chain chimeric polypeptide of embodiment Al 8, wherein the soluble receptor is a soluble TGF-β receptor II (TGF-β RII), a soluble TGFβΚΙΙΙ, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-1, a scMHCI, a scMHCII, a scTCR, a soluble CD155, or a soluble CD28.

Embodiment A20. The multi-chain chimeric polypeptide of any one of embodiments Al-Al9, wherein the first chimeric polypeptide further comprises one or more additional target-binding domain(s), where at least one of the one or more additional antigen-binding domain(s) is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment A21. The multi-chain chimeric polypeptide of embodiment A20, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the at least one of the one or more additional antigenbinding domain(s), and/or a linker sequence between the at least one of the one or more additional antigen-binding domain(s) and the first domain of the pair of affinity domains.

Embodiment A22. The multi-chain chimeric polypeptide of any one of embodiments Al-Al9, wherein the first chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal and/or C-terminal end of the first chimeric polypeptide.

Embodiment A23. The multi-chain chimeric polypeptide of embodiment A22, wherein at least one of the one or more additional target-binding domains directly abuts the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment A24. The multi-chain chimeric polypeptide of embodiment A22, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first domain of the pair of affinity domains.

Embodiment A25. The multi-chain chimeric polypeptide of embodiment A22, wherein the at least one of the one or more additional target-binding domains directly abuts the first target-binding domain in the first chimeric polypeptide.

Embodiment A26. The multi-chain chimeric polypeptide of embodiment A22, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first target-binding domain.

Embodiment A27. The multi-chain chimeric polypeptide of embodiment A22, wherein at least one of the one or more additional target-binding domains is disposed at the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target-binding domains is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment A28. The multi-chain chimeric polypeptide of embodiment A27, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the N-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment A29. The multi-chain chimeric polypeptide of embodiment A27, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment A30. The multi-chain chimeric polypeptide of embodiment A27, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the C-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment A31. The multi-chain chimeric polypeptide of embodiment A27, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment A32. The multi-chain chimeric polypeptide of embodiment AT, wherein the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, directly abuts the soluble tissue factor domain and/or the first domain of the pair of affinity domains.

Embodiment A33. The multi-chain chimeric polypeptide of embodiment A27, wherein the first chimeric polypeptide further comprises a linker sequence disposed (i) between the soluble tissue factor domain and the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, and/or (ii) between the first domain of the pair of affinity domains and the at least one of the one or more additional targetbinding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment A34. The multi-chain chimeric polypeptide of any one of embodiments A1-A33, wherein the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

Embodiment A3 5. The multi-chain chimeric polypeptide of embodiment A34, wherein at least one of the one or more additional target-binding domains directly abuts the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment A36. The multi-chain chimeric polypeptide of embodiment A34, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment A37. The multi-chain chimeric polypeptide of embodiment A34, wherein at least one of the one or more additional target-binding domains directly abuts the second target-binding domain in the second chimeric polypeptide.

Embodiment A38. The multi-chain chimeric polypeptide of embodiment A34, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second targetbinding domain in the second chimeric polypeptide.

Embodiment A39. The multi-chain chimeric polypeptide of any one of embodiments A20-A38, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen.

Embodiment A40. The multi-chain chimeric polypeptide of embodiment A39, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope.

Embodiment A41. The multi-chain chimeric polypeptide of embodiment A40, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence.

Embodiment A42. The multi-chain chimeric polypeptide of embodiment A39, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same antigen.

Embodiment A43. The multi-chain chimeric polypeptide of embodiment A42, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each bind specifically to the same epitope.

Embodiment A44. The multi-chain chimeric polypeptide of embodiment A43, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains each comprise the same amino acid sequence.

Embodiment A45. The multi-chain chimeric polypeptide of any one of embodiments A20-A38, wherein the first target-binding domain, the second targetbinding domain, and the one or more additional target-binding domains bind specifically to different antigens.

Embodiment A46. The multi-chain chimeric polypeptide of any one of embodiments A20-A45, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains is an antigenbinding domain.

Embodiment A47. The multi-chain chimeric polypeptide of embodiment A46, wherein the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains are each an antigen-binding domain.

Embodiment A48. The multi-chain chimeric polypeptide of embodiment A47, wherein antigen-binding domain comprises a scFv.

Embodiment A49. The multi-chain chimeric polypeptide of any one of embodiments A20-A48, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more target-binding domains bind specifically to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-β receptor II (TGF-βRII), a ligand of TGF-βRIII, a ligand of DNAM-1, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKP30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD 155, a receptor for CD 122, and a receptor for CD3, and a receptor for CD28.

Embodiment A50. The multi-chain chimeric polypeptide of any one of embodiments A20-A48, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin or cytokine protein.

Embodiment A51. The multi-chain chimeric polypeptide of embodiment A50, wherein the soluble interleukin, cytokine, or ligand protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L.

Embodiment A52. The multi-chain chimeric polypeptide of any one of embodiments A20-A48, wherein one or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains is a soluble interleukin or cytokine receptor.

Embodiment A53. The multi-chain chimeric polypeptide of embodiment A52, wherein the soluble receptor is a soluble TGF-β receptor II (TGF-β RII), a soluble TGFβΚΙΙΙ, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-1, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a soluble CD3, or a soluble CD28.

Embodiment A54. The multi-chain chimeric polypeptide of any one of embodiments A1-A53, wherein the first chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the first chimeric polypeptide.

Embodiment A55. The multi-chain chimeric polypeptide of any one of embodiments A1-A53, wherein the second chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

Embodiment A56. The multi-chain chimeric polypeptide of any one of embodiments A1-A55, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment A57. The multi-chain chimeric polypeptide of embodiment A56, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1.

Embodiment A58. The multi-chain chimeric polypeptide of embodiment A57, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1.

Embodiment A59. The multi-chain chimeric polypeptide of embodiment A58, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1.

Embodiment A60. The multi-chain chimeric polypeptide of any one of embodiments A56-A59, wherein the soluble human tissue factor domain does not comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment A61. The multi-chain chimeric polypeptide of embodiment A60, wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment A62. The multi-chain chimeric polypeptide of any one of embodiments A1-A61, wherein the soluble tissue factor domain is not capable of binding to Factor Vila.

Embodiment A63. The multi-chain chimeric polypeptide of any one of embodiments A1-A62, wherein the soluble tissue factor domain does not convert inactive Factor X into Factor Xa.

Embodiment A64. The multi-chain chimeric polypeptide of any one of embodiments A1-A63, wherein the multi-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

Embodiment A65. The multi-chain chimeric polypeptide of any one of embodiments A1-A64, wherein the pair of affinity domains is a sushi domain from an alpha chain of human IL-15 receptor (IL15Ra) and a soluble IL-15.

Embodiment A66. The multi-chain chimeric polypeptide of embodiment A65, wherein the soluble IL15 has a D8N or D8A amino acid substitution.

Embodiment A67. The multi-chain chimeric polypeptide of embodiment A65 or A66, wherein the human IL15Ra is a mature full-length IL15Ra.

Embodiment A68. The multi-chain chimeric polypeptide of any one of embodiments A1-A64, wherein the pair of affinity domains is selected from the group consisting of: bamase and bamstar, a PKA and an AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25.

Embodiment A69. The multi-chain chimeric polypeptide of any one of embodiments A1-A68, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminal end.

Embodiment A70. A composition comprising any of the multi-chain chimeric polypeptides of embodiments A1-A69.

Embodiment A71. The composition of embodiment A70, wherein the composition is a pharmaceutical composition.

Embodiment A72. A kit comprising at least one dose of the composition of embodiment A70 or A71.

Embodiment A73. A method of stimulating an immune cell, the method comprising:

contacting an immune cell with an effective amount of any of the multi-chain chimeric polypeptides of embodiments A1-A69 or the composition of embodiment A70 or A71.

Embodiment A74. The method of embodiment A73, wherein the immune cell is contacted in vitro.

Embodiment A75. The method of embodiment A74, wherein the immune cell was previously obtained from a subject.

Embodiment A76. The method of embodiment A75, wherein the method further comprises obtaining the immune cell from the subject prior to the contacting step.

Embodiment A77. The method of embodiment A73, wherein the immune cell is contacted in vivo.

Embodiment A78. The method of any one of embodiments A73-A77, wherein the immune cell is selected from the group consisting of: an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, a Th 17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, and a natural killer cell.

Embodiment A79. The method of any one of embodiments A73-A78, wherein the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor.

Embodiment A80. The method of any one of embodiments A73-A78, wherein the method further comprises, after the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor.

Embodiment A81. The method of any one of embodiments A73-A80, wherein the method further comprises administering the immune cell to a subject in need thereof.

Embodiment A82. The method of embodiment A81, wherein the subject has been identified or diagnosed as having an age-related disease or condition.

Embodiment A83. The method of embodiment A82, wherein the age-related disease or condition is selected from the group consisting of Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment A84. The method of embodiment A81, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment A85. The method of embodiment A84, wherein the cancer is selected from the group consisting of solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment A86. The method of embodiment A81, wherein the subject has been diagnosed or identified as having an infectious disease.

Embodiment A87. The method of embodiment A86, wherein the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Embodiment A88. A method of inducing or increasing proliferation of an immune cell, the method comprising:

contacting an immune cell with an effective amount of any of the multi-chain chimeric polypeptides of embodiments A1-A69 or the composition of embodiment A70 or A71.

Embodiment A89. The method of embodiment A88, wherein the immune cell is contacted in vitro.

Embodiment A90. The method of embodiment A89, wherein the immune cell was previously obtained from a subject.

Embodiment A91. The method of embodiment A90, wherein the method further comprises obtaining the immune cell from the subject prior to the contacting step.

Embodiment A92. The method of embodiment A88, wherein the immune cell is contacted in vivo.

Embodiment A93. The method of any one of embodiments A88-A92, wherein the immune cell is selected from the group consisting of: an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, a Th 17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, and a natural killer cell.

Embodiment A94. The method of any one of embodiments A88-A93, wherein the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor.

Embodiment A95. The method of any one of embodiments A88-A93, wherein the method further comprises, after the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor.

Embodiment A96. The method of any one of embodiments A88-A95, wherein the method further comprises administering the immune cell to a subject in need thereof.

Embodiment A97. The method of embodiment A96, wherein the subject has been identified or diagnosed as having an age-related disease or condition.

Embodiment A98. The method of embodiment A97, wherein the age-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment A99. The method of embodiment A96, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment Al00. The method of embodiment A99, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment A101. The method of embodiment A96, wherein the subject has been diagnosed or identified as having an infectious disease.

Embodiment Al 02. The method of embodiment A96, wherein the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Embodiment Al 03. A method of inducing differentiation of an immune cell into a memory or memory-like immune cell, the method comprising:

contacting an immune cell with an effective amount of any of the multi-chain chimeric polypeptides of embodiments A1-A69 or the composition of embodiment A70 or A71.

Embodiment A104. The method of embodiment A103, wherein the immune cell is contacted in vitro.

Embodiment Al 05. The method of embodiment Al 04, wherein the immune cell was previously obtained from a subject.

Embodiment Al06. The method of embodiment Al05, wherein the method further comprises obtaining the immune cell from the subject prior to the contacting step.

Embodiment A107. The method of embodiment A103, wherein the immune cell is contacted in vivo.

Embodiment A108. The method of any one of embodiments A103-A107, wherein the immune cell is selected from the group consisting of: an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a Th 17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, and a natural killer cell.

Embodiment A109. The method of any one of embodiments A103-A108, wherein the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor.

Embodiment A110. The method of any one of embodiments A103-A108, wherein the method further comprises, after the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor.

Embodiment Alli. The method of any one of embodiments A103-A110, wherein the method further comprises administering the immune cell to a subject in need thereof.

Embodiment Al 12. The method of embodiment Al 11, wherein the subject has been identified or diagnosed as having an age-related disease or condition.

Embodiment Al 13. The method of embodiment Al 12, wherein the age-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment Al 14. The method of embodiment Al 11, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment Al 15. The method of embodiment Al 14, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment Al 16. The method of embodiment Alli, wherein the subject has been diagnosed or identified as having an infectious disease.

Embodiment Al 17. The method of embodiment Al 16, wherein the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Embodiment Al 18. A method of killing a cancer cell, an infected cell, or a senescent cell in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the multi-chain chimeric polypeptides of embodiments A1-A69 or the composition of embodiment A70 or A71.

Embodiment Al 19. The method of embodiment Al 18, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment A120. The method of embodiment Al 19, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment A121. The method of embodiment Al 18, wherein the subject has been identified or diagnosed as having an aging-related disease or condition.

Embodiment A122. The method of embodiment A121, wherein the aging-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment A123. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the multi-chain chimeric polypeptides of embodiments A1-A69 or the composition of embodiment A70 or A71.

Embodiment A124. The method of embodiment A123, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment A125. The method of embodiment A124, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment A126. The method of embodiment A123, wherein the subject has been identified or diagnosed as having an aging-related disease or condition.

Embodiment A127. The method of embodiment A126, wherein the aging-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment A128. The method of embodiment A123, wherein the subject has been diagnosed or identified as having an infectious disease.

Embodiment A129. The method of embodiment A128, wherein the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Embodiment A130. Nucleic acid encoding any of the multi-chain chimeric polypeptides of any one of embodiments A1-A69.

Embodiment A131. A vector comprising the nucleic acid of embodiment A130.

Embodiment A132. The vector of embodiment A131, wherein the vector is an expression vector.

Embodiment A133. A cell comprising the nucleic acid of embodiment A130 or 5 the vector of embodiment A131 or A132.

Embodiment Al 34. A method of producing a multi-chain chimeric polypeptide, the method comprising:

culturing the cell of embodiment A133 in a culture medium under conditions sufficient to result in the production of the multi-chain chimeric polypeptide; and recovering the multi-chain chimeric polypeptide from the cell and/or the culture medium.

Embodiment Al35. A multi-chain chimeric polypeptide produced by the method 15 of embodiment Al 34.

Embodiment A136. The multi-chain chimeric polypeptide of embodiment A56, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 3.

Embodiment A137. The multi-chain chimeric polypeptide of embodiment A136, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 3.

Embodiment A138. The multi-chain chimeric polypeptide of embodiment A137, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 3.

Embodiment A139. The multi-chain chimeric polypeptide of embodiment Al 3 8, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO: 3.

Embodiment A140. The multi-chain chimeric polypeptide of embodiment A56, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 4.

Embodiment A141. The multi-chain chimeric polypeptide of embodiment A140, 5 wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 4.

Embodiment A142. The multi-chain chimeric polypeptide of embodiment A141, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 4.

Embodiment A143. The multi-chain chimeric polypeptide of embodiment A142, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO: 4.

Embodiment A144. The multi-chain chimeric polypeptide of embodiment 56, wherein the human soluble tissue factor domain does not initiate coagulation.

Embodiment A145. The multi-chain chimeric polypeptide of any one of claims 1- , wherein the soluble tissue factor domain comprises or consists of a sequence from a wildtype soluble human tissue factor.

B. Exemplary Embodiments

Embodiment Bl. A multi-chain chimeric polypeptide comprising:

(a) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and (iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and (ii) a second target-binding domain, wherein:

the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains;

the first target-binding domain and the second targeting-binding domain each independently bind specifically to a receptor of IL-18 or a receptor of IL-12.

Embodiment B2. The multi-chain chimeric polypeptide of embodiment Bl, wherein the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide.

Embodiment B3. The multi-chain chimeric polypeptide of embodiment Bl, wherein the first chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

Embodiment B4. The multi-chain chimeric polypeptide of any one of embodiments B1-B3, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide.

Embodiment B5. The multi-chain chimeric polypeptide of any one of embodiments B1-B3, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment B6. The multi-chain chimeric polypeptide of any one of embodiments B1-B5, wherein the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide.

Embodiment B7. The multi-chain chimeric polypeptide of any one of embodiments B1-B5, wherein second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

Embodiment B8. The multi-chain chimeric polypeptide of any one of embodiments B1-B7, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment B9. The multi-chain chimeric polypeptide of embodiment B8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1.

Embodiment B10. The multi-chain chimeric polypeptide of embodiment B9, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1.

Embodiment Bl 1. The multi-chain chimeric polypeptide of embodiment B10, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1.

Embodiment B12. The multi-chain chimeric polypeptide of any one of embodiments B8-B11, wherein the soluble human tissue factor domain does not comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment B13. The multi-chain chimeric polypeptide of embodiment Bl2, wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment B14. The multi-chain chimeric polypeptide of any one of embodiments B1-B13, wherein the soluble tissue factor domain is not capable of binding to Factor Vila.

Embodiment Bl5. The multi-chain chimeric polypeptide of any one of embodiments Bl-Bl 4, wherein the soluble tissue factor domain does not convert inactive Factor X into Factor Xa.

Embodiment Bl6. The multi-chain chimeric polypeptide of any one of embodiments Bl-Bl 5, wherein the multi-chain chimeric polypeptide does not stimulate coagulation in a mammal.

Embodiment Bl7. The multi-chain chimeric polypeptide of any one of embodiments Bl-Bl 6, wherein the first chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the first chimeric polypeptide.

Embodiment Bl8. The multi-chain chimeric polypeptide of any one of embodiments Bl-Bl 7, wherein the second chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

Embodiment Bl9. The multi-chain chimeric polypeptide of any one of embodiments B 1-18, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminal end.

Embodiment B20. The multi-chain chimeric polypeptide of embodiment Bl9, wherein the signal sequence comprises SEQ ID NO: 31.

Embodiment B21. The multi-chain chimeric polypeptide of embodiment B20, wherein the signal sequence is SEQ ID NO: 31.

Embodiment B22. The multi-chain chimeric polypeptide of any one of embodiments B1-B21, wherein the pair of affinity domains is a sushi domain from an alpha chain of human IL-15 receptor (IL15Ra) and a soluble IL-15.

Embodiment B23. The multi-chain chimeric polypeptide of embodiment B22, wherein the soluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment B24. The multi-chain chimeric polypeptide of embodiment B22, wherein the soluble IL-15 comprises a sequence that is 80% identical to SEQ ID NO: 14.

Embodiment B25. The multi-chain chimeric polypeptide of embodiment B24, wherein the soluble IL-15 comprises a sequence that is 90% identical to SEQ ID NO: 14.

Embodiment B26. The multi-chain chimeric polypeptide of embodiment B25, wherein the soluble IL-15 comprises a sequence that is 95% identical to SEQ ID NO: 14.

Embodiment B27. The multi-chain chimeric polypeptide of embodiment B26, wherein the soluble IL-15 comprises SEQ ID NO: 14.

Embodiment B28. The multi-chain chimeric polypeptide of any one of embodiments B22-B27, wherein the sushi domain of IL15Ra comprises a sushi domain from human IL15Ra.

Embodiment B29. The multi-chain chimeric polypeptide of embodiment B28, wherein the sushi domain from human IL15Ra comprises a sequence that is 80% identical to SEQ ID NO: 28.

Embodiment B30. The multi-chain chimeric polypeptide of embodiment B29, wherein the sushi domain from human IL15Ra comprises a sequence that is 90% identical to SEQ ID NO: 28.

Embodiment B31. The multi-chain chimeric polypeptide of embodiment B30, wherein the sushi domain from human IL15Ra comprises a sequence that is 95% identical to SEQ ID NO: 28.

Embodiment B32. The multi-chain chimeric polypeptide of embodiment B31, wherein the sushi domain from human IL15Ra comprises SEQ ID NO: 28.

Embodiment B33. The multi-chain chimeric polypeptide of embodiment B28, wherein the sushi domain from human IL15Ra is a mature full-length IL15Ra.

Embodiment B34. The multi-chain chimeric polypeptide of any one of embodiments B1-B21, wherein the pair of affinity domains is selected from the group consisting of: bamase and bamstar, a PKA and an AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25.

Embodiment B35. The multi-chain chimeric polypeptide of any one of embodiments B1-B34, wherein one or both of the first target-binding domain and the second target-binding domain is an agonistic antigen-binding domain.

Embodiment B36. The multi-chain chimeric polypeptide of embodiment B35, wherein the first target-binding domain and the second target-binding domain are each agonistic antigen-binding domains.

Embodiment B37. The multi-chain chimeric polypeptide of embodiment B35 or B36, wherein antigen-binding domain comprises a scFv or single-domain antibody.

Embodiment B38. The multi-chain chimeric polypeptide of any one of embodiments B1-B34, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble IL-15 or a soluble IL-18.

Embodiment B39. The multi-chain chimeric polypeptide of embodiment B38, wherein the first target-binding domain and the second target-binding domain are each independently a soluble IL-15 or a soluble IL-18.

Embodiment B40. The multi-chain chimeric polypeptide of any one of embodiments B1-B39, wherein the first target-binding domain and the second targetbinding domain both bind specifically to a receptor of IL-18 or a receptor of IL-12.

Embodiment B41. The multi-chain chimeric polypeptide of embodiment B40, wherein the first target-binding domain and the second target-binding domain bind specifically to the same epitope.

Embodiment B42. The multi-chain chimeric polypeptide of embodiment B41, wherein the first target-binding domain and the second target-binding domain comprise the same amino acid sequence.

Embodiment B43. The multi-chain chimeric polypeptide of any one of embodiments B1-B39, wherein the first target-binding domain binds specifically to a receptor for IL-12, and the second target-binding domain binds specifically to a receptor for IL-18.

Embodiment B44. The multi-chain chimeric polypeptide of any one of embodiments B1-B39, wherein the first target-binding domain binds specifically to a receptor for IL-18, and the second target-binding domain bind specifically to a receptor for IL-12.

Embodiment B45. The multi-chain chimeric polypeptide of embodiment B44, wherein the first target-binding domain comprises a soluble IL-18.

Embodiment B46. The multi-chain chimeric polypeptide of embodiment B45, wherein the soluble IL-18 is a soluble human IL-18.

Embodiment B47. The multi-chain chimeric polypeptide of embodiment B46, wherein the soluble human IL-18 comprises a sequence at least 80% identical to SEQ ID NO: 16.

Embodiment B48. The multi-chain chimeric polypeptide of embodiment B47, wherein the soluble human IL-18 comprises a sequence at least 90% identical to SEQ ID NO: 16.

Embodiment B49. The multi-chain chimeric polypeptide of embodiment B48, wherein the soluble human IL-18 comprises a sequence at least 95% identical to SEQ ID NO: 16.

Embodiment B50. The multi-chain chimeric polypeptide of embodiment B49, wherein the soluble human IL-18 comprises a sequence of SEQ ID NO: 16.

Embodiment B51. The multi-chain chimeric polypeptide of any one of embodiments B44-B50, wherein the second target-binding domain comprises a soluble IL-12.

Embodiment B52. The multi-chain chimeric polypeptide of embodiment B51, wherein the soluble IL-18 is a soluble human IL-12.

Embodiment B53. The multi-chain chimeric polypeptide of embodiment B52, wherein the soluble human IL-15 comprises a sequence of soluble human ΙΕ-12β (p40) and a sequence of soluble human IL-12a (p35).

Embodiment B54. The multi-chain chimeric polypeptide of embodiment B53, wherein the soluble human IL-15 further comprises a linker sequence between the sequence of soluble ΙΕ-12β (p40) and the sequence of soluble human IL-12a (p35).

Embodiment B55. The multi-chain chimeric polypeptide of embodiment B54, wherein the linker sequence comprises SEQ ID NO: 7.

Embodiment B56. The multi-chain chimeric polypeptide of any one of embodiments B53-B55, wherein the sequence of soluble human ΙΕ-12β (p40) comprises a sequence that is at least 80% identical to SEQ ID NO: 66.

Embodiment B57. The multi-chain chimeric polypeptide of embodiment B56, wherein the sequence of soluble human ΙΕ-12β (p40) comprises a sequence that is at least 90% identical to SEQ ID NO: 66.

Embodiment B58. The multi-chain chimeric polypeptide of embodiment B57, wherein the sequence of soluble human ΙΕ-12β (p40) comprises a sequence that is at least 95% identical to SEQ ID NO: 66.

Embodiment B59. The multi-chain chimeric polypeptide of embodiment B58, wherein the sequence of soluble human ΙΕ-12β (p40) comprises SEQ ID NO: 66.

Embodiment B60. The multi-chain chimeric polypeptide of any one of embodiments B53-B59, wherein the sequence of soluble human IL-12a (p35) comprises a sequence that is at least 80% identical to SEQ ID NO: 68.

Embodiment B61. The multi-chain chimeric polypeptide of embodiment B60, wherein the sequence of soluble human IL-12a (p35) comprises a sequence that is at least 90% identical to SEQ ID NO: 68.

Embodiment B62. The mule-chain chimeric polypeptide of embodiment B61, wherein the sequence of soluble human IL-12a (p35) comprises a sequence that is at least 95% identical to SEQ ID NO: 68.

Embodiment B63. The multi-chain chimeric polypeptide of embodiment B62, wherein the sequence of soluble human IL-12a (p35) comprises SEQ ID NO: 68.

Embodiment B64. The multi-chain chimeric polypeptide of embodiment Bl, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 70.

Embodiment B65. The multi-chain chimeric polypeptide of embodiment B64, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 70.

Embodiment B66. The multi-chain chimeric polypeptide of embodiment B65, wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 70.

Embodiment B67. The multi-chain chimeric polypeptide of embodiment B66, wherein the first chimeric polypeptide comprises SEQ ID NO: 70.

Embodiment B68. The multi-chain chimeric polypeptide of embodiment B67, wherein the first chimeric polypeptide comprises SEQ ID NO: 72.

Embodiment B69. The multi-chain chimeric polypeptide of any one of embodiments Bl and B64-B68, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 74.

Embodiment B70. The multi-chain chimeric polypeptide of embodiment B69, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 74.

Embodiment B71. The multi-chain chimeric polypeptide of embodiment B70, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 74.

Embodiment B72. The multi-chain chimeric polypeptide of embodiment B71, wherein the second chimeric polypeptide comprises SEQ ID NO: 74.

Embodiment B73. The multi-chain chimeric polypeptide of embodiment B72, wherein the second chimeric polypeptide comprises SEQ ID NO: 74.

Embodiment B74. The multi-chain chimeric polypeptide of any one of embodiments B1-B63, wherein the first chimeric polypeptide further comprises one or more additional target-binding domain(s), where at least one of the one or more additional antigen-binding domain(s) is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment B75. The multi-chain chimeric polypeptide of embodiment B74, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the at least one of the one or more additional antigenbinding domain(s), and/or a linker sequence between the at least one of the one or more additional antigen-binding domain(s)and the first domain of the pair of affinity domains.

Embodiment B76. The multi-chain chimeric polypeptide of any one of embodiments B1-B63, wherein the first chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal and/or C-terminal end of the first chimeric polypeptide.

Embodiment B77. The multi-chain chimeric polypeptide of embodiment B76, wherein at least one of the one or more additional target-binding domains directly abuts the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment B78. The multi-chain chimeric polypeptide of embodiment B76, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first domain of the pair of affinity domains.

Embodiment B79. The multi-chain chimeric polypeptide of embodiment B76, wherein the at least one of the one or more additional target-binding domains directly abuts the first target-binding domain in the first chimeric polypeptide.

Embodiment B80. The multi-chain chimeric polypeptide of embodiment B76, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first target-binding domain.

Embodiment B81. The multi-chain chimeric polypeptide of embodiment B76, wherein at least one of the one or more additional target-binding domains is disposed at the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target-binding domains is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment B82. The multi-chain chimeric polypeptide of embodiment B81, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the N-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment B83. The multi-chain chimeric polypeptide of embodiment B81, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment B84. The multi-chain chimeric polypeptide of embodiment B81, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the C-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment B85. The multi-chain chimeric polypeptide of embodiment B81, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment B86. The multi-chain chimeric polypeptide of embodiment B81, wherein the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, directly abuts the soluble tissue factor domain and/or the first domain of the pair of affinity domains.

Embodiment B87. The multi-chain chimeric polypeptide of embodiment B81, wherein the first chimeric polypeptide further comprises a linker sequence disposed (i) between the soluble tissue factor domain and the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, and/or (ii) between the first domain of the pair of affinity domains and the at least one of the one or more additional targetbinding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment B88. The multi-chain chimeric polypeptide of any one of embodiments B1-B63 and B74-B87, wherein the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the Cterminal end of the second chimeric polypeptide.

Embodiment B89. The multi-chain chimeric polypeptide of embodiment B88, wherein at least one of the one or more additional target-binding domains directly abuts the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment B90. The multi-chain chimeric polypeptide of embodiment B88, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment B91. The multi-chain chimeric polypeptide of embodiment B88, wherein at least one of the one or more additional target-binding domains directly abuts the second target-binding domain in the second chimeric polypeptide.

Embodiment B92. The multi-chain chimeric polypeptide of embodiment B88, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second targetbinding domain in the second chimeric polypeptide.

Embodiment B93. The multi-chain chimeric polypeptide of any one of embodiments B74-B92, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen.

Embodiment B94. The multi-chain chimeric polypeptide of embodiment B93, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope.

Embodiment B95. The multi-chain chimeric polypeptide of embodiment B94, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence.

Embodiment B96. The multi-chain chimeric polypeptide of any one of embodiments B74-B92, wherein the first target-binding domain, the second targetbinding domain, and the one or more additional target-binding domains bind specifically to different antigens.

Embodiment B97. The multi-chain chimeric polypeptide of any one of embodiments B74-B96, wherein the one or more additional antigen-binding domains bind specifically to a target selected from the group consisting of: CD 16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-β receptor II (TGF^RII), a ligand of TGF^RIII, a ligand of DNAM-1, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKP30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor for

IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD 155, and a receptor for CD28.

Embodiment B98. The multi-chain chimeric polypeptide of any one of embodiments B74-B96, wherein the one or more additional target-binding domains is a soluble interleukin or cytokine protein.

Embodiment B99. The multi-chain chimeric polypeptide of embodiment B98, wherein the soluble interleukin, cytokine, or ligand protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L.

Embodiment Bl00. The multi-chain chimeric polypeptide of any one of embodiments B74-B96, wherein the one or more additional target-binding domains is a soluble interleukin or cytokine receptor.

Embodiment BIOL The multi-chain chimeric polypeptide of embodiment Bl00, wherein the soluble receptor is a soluble TGF-β receptor II (TGF-β RII), a soluble TGFβΚΙΙΙ, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-1, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, or a soluble CD28.

Embodiment Bl 02. A composition comprising any of the multi-chain chimeric polypeptides of embodiments Bl-B 101.

Embodiment Bl03. The composition of embodiment Bl02, wherein the composition is a pharmaceutical composition.

Embodiment Bl 04. A kit comprising at least one dose of the composition of embodiment B102 or B103.

Embodiment Bl05. A method of stimulating an immune cell, the method comprising:

contacting an immune cell with an effective amount of any of the multi-chain chimeric polypeptides of embodiments B1-B101 or the composition of embodiment Bl 02 orB103.

Embodiment Bl06. The method of embodiment Bl05, wherein the immune cell is contacted in vitro.

Embodiment Bl07. The method of embodiment Bl06, wherein the immune cell was previously obtained from a subject.

Embodiment Bl08. The method of embodiment Bl07, wherein the method further comprises obtaining the immune cell from the subject prior to the contacting step.

Embodiment Bl09. The method of embodiment Bl05, wherein the immune cell is contacted in vivo.

Embodiment Bl 10. The method of any one of embodiments Bl 05-B109, wherein the immune cell is selected from the group consisting of: an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, a Th 17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, and a natural killer cell.

Embodiment Bl 11. The method of any one of embodiments Bl 05-B110, wherein the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor.

Embodiment Bl 12. The method of any one of embodiments Bl05-B110, wherein the method further comprises, after the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor.

Embodiment Bl 13. The method of any one of embodiments B105-B112, wherein the method further comprises administering the immune cell to a subject in need thereof.

Embodiment Bl 14. The method of embodiment Bl 13, wherein the subject has been identified or diagnosed as having age-related disease or condition.

Embodiment Bl 15. The method of embodiment Bl 14, wherein the age-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in panceatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment Bl 16. The method of embodiment Bl 13, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment Bl 17. The method of embodiment Bl 16, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment Bl 18. The method of embodiment Bl 13, wherein the subject has been diagnosed or identified as having an infectious disease.

Embodiment Bl 19. The method of embodiment Bl 18, wherein the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Embodiment B120. A method of inducing or increasing proliferation of an immune cell, the method comprising:

contacting an immune cell with an effective amount of any of the multi-chain chimeric polypeptides of embodiments B1-B101 or the composition of embodiment Bl 02 orB103.

Embodiment B121. The method of embodiment Bl20, wherein the immune cell is contacted in vitro.

Embodiment B122. The method of embodiment B121, wherein the immune cell was previously obtained from a subject.

Embodiment Bl23. The method of embodiment Bl22, wherein the method further comprises obtaining the immune cell from the subject prior to the contacting step.

Embodiment B124. The method of embodiment Bl20, wherein the immune cell is contacted in vivo.

Embodiment Bl 25. The method of any one of embodiments Bl20-B 124, wherein the immune cell is selected from the group consisting of: an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, a Th 17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, and a natural killer cell.

Embodiment B126. The method of any one of embodiments B120-B125, wherein the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor.

Embodiment B127. The method of any one of embodiments B120-B125, wherein the method further comprises, after the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor.

Embodiment Bl 28. The method of any one of embodiments Bl 20-B 127, wherein the method further comprises administering the immune cell to a subject in need thereof.

Embodiment B129. The method of embodiment Bl28, wherein the subject has been identified or diagnosed as having an age-related disease or condition.

Embodiment B130. The method of embodiment Bl29, wherein the age-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in panceatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment B131. The method of embodiment Bl28, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment Bl32. The method of embodiment B131, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment Bl33. The method of embodiment Bl28, wherein the subject has been diagnosed or identified as having an infectious disease.

Embodiment Bl34. The method of embodiment Bl28, wherein the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Embodiment B135. A method of inducing differentiation of an immune cell into a memory or memory-like immune cell, the method comprising:

contacting an immune cell with an effective amount of any of the multi-chain chimeric polypeptides of embodiments B1-B101 or the composition of embodiment Bl 02 orB103.

Embodiment B136. The method of embodiment B135, wherein the immune cell is contacted in vitro.

Embodiment B137. The method of embodiment Bl36, wherein the immune cell was previously obtained from a subject.

Embodiment B138. The method of embodiment Bl37, wherein the method further comprises obtaining the immune cell from the subject prior to the contacting step.

Embodiment B139. The method of embodiment B135, wherein the immune cell is contacted in vivo.

Embodiment B140. The method of any one of embodiments B135-B139, wherein the immune cell is selected from the group consisting of: an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a Th 17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, and a natural killer cell.

Embodiment B141. The method of any one of embodiments B135-B140, wherein the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor.

Embodiment B142. The method of any one of embodiments B135-B140, wherein the method further comprises, after the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor.

Embodiment Bl43. The method of any one of embodiments B135-B142, wherein the method further comprises administering the immune cell to a subject in need thereof.

Embodiment B144. The method of embodiment Bl43, wherein the subject has been identified or diagnosed as having an age-related disease or condition.

Embodiment Bl45. The method of embodiment Bl44, wherein the age-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in panceatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment B146. The method of embodiment Bl43, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment B147. The method of embodiment Bl46, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment Bl48. The method of embodiment Bl43, wherein the subject has been diagnosed or identified as having an infectious disease.

Embodiment B149. The method of embodiment Bl48, wherein the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Embodiment Bl50. A method of killing a cancer cell, an infected cell, or a senescent cell in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the multi-chain chimeric polypeptides of embodiments B1-B101 or the composition of embodiment Bl02 or Bl03.

Embodiment B151. The method of embodiment Bl50, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment Bl52. The method of embodiment B151, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment Bl53. The method of embodiment Bl50, wherein the subject has been identified or diagnosed as having an aging-related disease or condition.

Embodiment Bl54. The method of embodiment Bl53, wherein the aging-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in panceatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction

Embodiment Bl 55. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the multi-chain chimeric polypeptides of embodiments Bl-B 101 or the composition of embodiment B102 or B103.

Embodiment Bl56. The method of embodiment Bl55, wherein the subject has been identified or diagnosed as having a cancer or infectious disease.

Embodiment Bl57. The method of embodiment Bl56, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment Bl58. The method of embodiment Bl55, wherein the subject has been identified or diagnosed as having age-related disease or condition.

Embodiment Bl59. The method of embodiment Bl58, wherein the age-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment Bl60. The method of embodiment Bl55, wherein the subject has been diagnosed or identified as having an infectious disease.

Embodiment B161. The method of embodiment Bl60, wherein the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Embodiment Bl 62. Nucleic acid encoding any of the multi-chain chimeric polypeptides of any one of embodiments B1-B101.

Embodiment Bl 63. A vector comprising the nucleic acid of embodiment Bl 62.

Embodiment Bl64. The vector of embodiment Bl63, wherein the vector is an expression vector.

Embodiment Bl65. A cell comprising the nucleic acid of embodiment Bl62 or the vector of embodiment Bl 63 or Bl 64.

Embodiment Bl66. A method of producing a multi-chain chimeric polypeptide, the method comprising:

culturing the cell of embodiment Bl65 in a culture medium under conditions sufficient to result in the production of the multi-chain chimeric polypeptide; and recovering the multi-chain chimeric polypeptide from the cell and/or the culture medium.

Embodiment Bl67. A multi-chain chimeric polypeptide produced by the method of embodiment Bl66.

Embodiment Bl68. The multi-chain chimeric polypeptide of embodiment B8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 3.

Embodiment Bl69. The multi-chain chimeric polypeptide of embodiment Bl68, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 3.

Embodiment Bl70. The multi-chain chimeric polypeptide of embodiment Bl69, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 3.

Embodiment B171. The multi-chain chimeric polypeptide of embodiment Bl70, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO: 3.

Embodiment Bl72. The multi-chain chimeric polypeptide of embodiment B8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 4.

Embodiment Bl73. The multi-chain chimeric polypeptide of embodiment Bl72, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 4.

Embodiment Bl74. The multi-chain chimeric polypeptide of embodiment Bl73, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 4.

Embodiment Bl75. The multi-chain chimeric polypeptide of embodiment Bl74, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO: 4.

C. Exemplary Embodiments

Embodiment CL A multi-chain chimeric polypeptide comprising:

(a) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and (iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and (ii) a second target-binding domain, wherein:

the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and the first target-binding domain and the second targeting-binding domain each independently bind specifically to a receptor of IL-21 or a ligand of tumor growth factor receptor β II (ΤΘΕβΚΙΙ).

Embodiment C2. The multi-chain chimeric polypeptide of embodiment Cl, wherein the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide.

Embodiment C3. The multi-chain chimeric polypeptide of embodiments Cl, wherein the first chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

Embodiment C4. The multi-chain chimeric polypeptide of any one of embodiments C1-C3, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide.

Embodiment C5. The multi-chain chimeric polypeptide of any one of embodiments C1-C3, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment C6. The multi-chain chimeric polypeptide of any one of embodiments C1-C5, wherein the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide.

Embodiment C7. The multi-chain chimeric polypeptide of any one of embodiments C1-C5, wherein second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

Embodiment C8. The multi-chain chimeric polypeptide of any one of embodiments C1-C7, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment C9. The multi-chain chimeric polypeptide of embodiment C8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1.

Embodiment CIO. The multi-chain chimeric polypeptide of embodiment C9, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1.

Embodiment Cl 1. The multi-chain chimeric polypeptide of embodiment CIO, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1.

Embodiment C12. The multi-chain chimeric polypeptide of any one of embodiments C8-C11, wherein the soluble human tissue factor domain does not comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment C13. The multi-chain chimeric polypeptide of embodiment Cl2, wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment C14. The multi-chain chimeric polypeptide of any one of embodiments C1-C13, wherein the soluble tissue factor domain is not capable of binding to Factor Vila.

Embodiment Cl 5. The multi-chain chimeric polypeptide of any one of embodiments Cl-Cl4, wherein the soluble tissue factor domain does not convert inactive Factor X into Factor Xa.

Embodiment Cl6. The multi-chain chimeric polypeptide of any one of embodiments Cl-Cl5, wherein the multi-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

Embodiment Cl7. The multi-chain chimeric polypeptide of any one of embodiments Cl-Cl6, wherein the first chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the first chimeric polypeptide.

Embodiment Cl 8. The multi-chain chimeric polypeptide of any one of embodiments Cl-Cl7, wherein the second chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

Embodiment Cl9. The multi-chain chimeric polypeptide of any one of embodiments Cl-Cl8, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminal end.

Embodiment C20. The multi-chain chimeric polypeptide of embodiment Cl9, wherein the signal sequence comprises SEQ ID NO: 31.

Embodiment C21. The multi-chain chimeric polypeptide of embodiment C20, wherein the signal sequence is SEQ ID NO: 31.

Embodiment C22. The multi-chain chimeric polypeptide of any one of embodiments C1-C21, wherein the pair of affinity domains is a sushi domain from an alpha chain of human IL-15 receptor (IL15Ra and a soluble IL-15.

Embodiment C23. The multi-chain chimeric polypeptide of embodiment C22, wherein the soluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment C24. The multi-chain chimeric polypeptide of embodiment C22, wherein the soluble IL-15 comprises a sequence that is 80% identical to SEQ ID NO: 14.

Embodiment C25. The multi-chain chimeric polypeptide of embodiment C24, wherein the soluble IL-15 comprises a sequence that is 90% identical to SEQ ID NO: 14.

Embodiment C26. The multi-chain chimeric polypeptide of embodiment C25, wherein the soluble IL-15 comprises a sequence that is 95% identical to SEQ ID NO: 14.

Embodiment C27. The multi-chain chimeric polypeptide of embodiment C26, wherein the soluble IL-15 comprises SEQ ID NO: 14.

Embodiment C28. The multi-chain chimeric polypeptide of any one of embodiments C22-C27, wherein the sushi domain of IL15Ra comprises a sushi domain from human IL15Ra.

Embodiment C29. The multi-chain chimeric polypeptide of embodiment C28, wherein the sushi domain from human IL15Ra comprises a sequence that is 80% identical to SEQ ID NO: 28.

Embodiment C30. The multi-chain chimeric polypeptide of embodiment C29, wherein the sushi domain from human IL15Ra comprises a sequence that is 90% identical to SEQ ID NO: 28.

Embodiment C31. The multi-chain chimeric polypeptide of embodiment C30, wherein the sushi domain from human IL15Ra comprises a sequence that is 95% identical to SEQ ID NO: 28.

Embodiment C32. The multi-chain chimeric polypeptide of embodiment C31, wherein the sushi domain from human IL15Ra comprises SEQ ID NO: 28.

Embodiment C33. The multi-chain chimeric polypeptide of embodiment C28, wherein the sushi domain from human IL15Ra is a mature full-length IL15Ra.

Embodiment C34. The multi-chain chimeric polypeptide of any one of embodiments C1-C21, wherein the pair of affinity domains is selected from the group consisting of: bamase and bamstar, a PKA and an AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25.

Embodiment C35. The multi-chain chimeric polypeptide of any one of embodiments C1-C34, wherein one or both of the first target-binding domain and the second target-binding domain is an antigen-binding domain.

Embodiment C36. The multi-chain chimeric polypeptide of embodiment C35, wherein the first target-binding domain and the second target-binding domain are antigen-binding domains.

Embodiment C37. The multi-chain chimeric polypeptide of embodiment C35 or C36, wherein antigen-binding domain comprises a scFv or single-domain antibody.

Embodiment C38. The multi-chain chimeric polypeptide of any one of embodiments C1-C34, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble IL-21 or a soluble TGFpRII.

Embodiment C39. The multi-chain chimeric polypeptide of any one of embodiments C1-C38, wherein the first target-binding domain and the second targetbinding domain both bind specifically to a receptor of IL-21 or a ligand of TGFpRII.

Embodiment C40. The multi-chain chimeric polypeptide of embodiment C39, wherein the first target-binding domain and the second target-binding domain bind specifically to the same epitope.

Embodiment C41. The multi-chain chimeric polypeptide of embodiment C40, wherein the first target-binding domain and the second target-binding domain comprise the same amino acid sequence.

Embodiment C42. The multi-chain chimeric polypeptide of any one of embodiments C1-C38, wherein the first target-binding domain binds specifically to a ligand of TGFpRII, and the second target-binding domain binds specifically to a receptor for IL-21.

Embodiment C43. The multi-chain chimeric polypeptide of any one of embodiments C1-C38, wherein the first target-binding domain binds specifically to a receptor for IL-21, and the second target-binding domain bind specifically to a ligand of TGFpRII.

Embodiment C44. The multi-chain chimeric polypeptide of embodiment C43, wherein the first target-binding domain comprises a soluble IL-21.

Embodiment C45. The multi-chain chimeric polypeptide of embodiment C44, wherein the soluble IL-21 is a soluble human IL-21.

Embodiment C46. The multi-chain chimeric polypeptide of embodiment C45, wherein the soluble human IL-21 comprises a sequence at least 80% identical to SEQ ID NO: 78.

Embodiment C47. The multi-chain chimeric polypeptide of embodiment C46, wherein the soluble human IL-21 comprises a sequence at least 90% identical to SEQ ID NO: 78.

Embodiment C48. The multi-chain chimeric polypeptide of embodiment C47, wherein the soluble human IL-21 comprises a sequence at least 95% identical to SEQ ID NO: 78.

Embodiment C49. The multi-chain chimeric polypeptide of embodiment C48, wherein the soluble human IL-21 comprises a sequence of SEQ ID NO: 78.

Embodiment C50. The multi-chain chimeric polypeptide of any one of embodiments C43-C49, wherein the second target-binding domain comprises a soluble TGFpRII.

Embodiment C51. The multi-chain chimeric polypeptide of embodiment C50, wherein the soluble TGFpRII is a soluble human TGFpRII.

Embodiment C52. The multi-chain chimeric polypeptide of embodiment C51, wherein the soluble human TGFpRII comprises a first sequence of soluble human TGFpRII and a second sequence of soluble human TGFpRII.

Embodiment C53. The multi-chain chimeric polypeptide of embodiment C52, wherein the soluble human TGFpRII further comprises a linker sequence between the first sequence of soluble human TGFpRII and the second sequence of soluble human TGFpRII.

Embodiment C54. The multi-chain chimeric polypeptide of embodiment C53, wherein the linker sequence comprises SEQ ID NO: 7.

Embodiment C55. The multi-chain chimeric polypeptide of any one of embodiments C52-C54, wherein the first sequence of soluble human TGFpRII comprises a sequence that is at least 80% identical to SEQ ID NO: 80.

Embodiment C56. The multi-chain chimeric polypeptide of embodiment C55, wherein the first sequence of soluble human TGFpRII comprises a sequence that is at least 90% identical to SEQ ID NO: 80.

Embodiment C57. The multi-chain chimeric polypeptide of embodiment C56, wherein the first sequence of soluble human TGFpRII comprises a sequence that is at least 95% identical to SEQ ID NO: 80.

Embodiment C58. The multi-chain chimeric polypeptide of embodiment C57, wherein the first sequence of soluble human TGFpRII comprises SEQ ID NO: 80.

Embodiment C59. The multi-chain chimeric polypeptide of any one of embodiments C52-C58, wherein the second sequence of soluble human TGFpRII comprises a sequence that is at least 80% identical to SEQ ID NO: 81.

Embodiment C60. The multi-chain chimeric polypeptide of embodiment C59, wherein the second sequence of soluble human TGFpRII comprises a sequence that is at least 90% identical to SEQ ID NO: 81.

Embodiment C61. The mule-chain chimeric polypeptide of embodiment C60, wherein the second sequence of soluble human TGFpRII comprises a sequence that is at least 95% identical to SEQ ID NO: 81.

Embodiment C62. The multi-chain chimeric polypeptide of embodiment C61, wherein the second sequence of soluble human TGFpRII comprises SEQ ID NO: 81.

Embodiment C63. The multi-chain chimeric polypeptide of embodiment Cl, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 86.

Embodiment C64. The multi-chain chimeric polypeptide of embodiment C63, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 86.

Embodiment C65. The multi-chain chimeric polypeptide of embodiment C64, wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 86.

Embodiment C66. The multi-chain chimeric polypeptide of embodiment C65, wherein the first chimeric polypeptide comprises SEQ ID NO: 86.

Embodiment C67. The multi-chain chimeric polypeptide of embodiment C66, wherein the first chimeric polypeptide comprises SEQ ID NO: 88.

Embodiment C68. The multi-chain chimeric polypeptide of any one of embodiments Cl and C63-C67, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 90.

Embodiment C69. The multi-chain chimeric polypeptide of embodiment C68, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 90.

Embodiment C70. The multi-chain chimeric polypeptide of embodiment C69, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 90.

Embodiment C71. The multi-chain chimeric polypeptide of embodiment C70, wherein the second chimeric polypeptide comprises SEQ ID NO: 90.

Embodiment C72. The multi-chain chimeric polypeptide of embodiment C71, wherein the second chimeric polypeptide comprises SEQ ID NO: 92.

Embodiment C73. The multi-chain chimeric polypeptide of any one of embodiments C1-C62, wherein the first chimeric polypeptide further comprises one or more additional target-binding domain(s), where at least one of the one or more additional antigen-binding domain(s) is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment C74. The multi-chain chimeric polypeptide of embodiment C73, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the at least one of the one or more additional antigenbinding domain(s), and/or a linker sequence between the at least one of the one or more additional antigen-binding domain(s) and the first domain of the pair of affinity domains.

Embodiment C75. The multi-chain chimeric polypeptide of any one of embodiments C1-C62, wherein the first chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal and/or C-terminal end of the first chimeric polypeptide.

Embodiment C76. The multi-chain chimeric polypeptide of embodiment C75, wherein at least one of the one or more additional target-binding domains directly abuts the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment C77. The multi-chain chimeric polypeptide of embodiment C75, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first domain of the pair of affinity domains.

Embodiment C78. The multi-chain chimeric polypeptide of embodiment C75, wherein the at least one of the one or more additional target-binding domains directly abuts the first target-binding domain in the first chimeric polypeptide.

Embodiment C79. The multi-chain chimeric polypeptide of embodiment C75, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first target-binding domain.

Embodiment C80. The multi-chain chimeric polypeptide of embodiment C75, wherein at least one of the one or more additional target-binding domains is disposed at the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target-binding domains is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment C81. The multi-chain chimeric polypeptide of embodiment C80, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the N-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment C82. The multi-chain chimeric polypeptide of embodiment C80, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment C83. The multi-chain chimeric polypeptide of embodiment C80, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the C-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment C84. The multi-chain chimeric polypeptide of embodiment C80, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment C85. The multi-chain chimeric polypeptide of embodiment C80, wherein the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, directly abuts the soluble tissue factor domain and/or the first domain of the pair of affinity domains.

Embodiment C86. The multi-chain chimeric polypeptide of embodiment C80, wherein the first chimeric polypeptide further comprises a linker sequence disposed (i) between the soluble tissue factor domain and the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, and/or (ii) between the first domain of the pair of affinity domains and the at least one of the one or more additional target binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment C87. The multi-chain chimeric polypeptide of any one of embodiments C1-C62 and C73-C86, wherein the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the Cterminal end of the second chimeric polypeptide.

Embodiment C88. The multi-chain chimeric polypeptide of embodiment C87, wherein at least one of the one or more additional target-binding domains directly abuts the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment C89. The multi-chain chimeric polypeptide of embodiment C87, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment C90. The multi-chain chimeric polypeptide of embodiment C87, wherein at least one of the one or more additional target-binding domains directly abuts the second target-binding domain in the second chimeric polypeptide.

Embodiment C91. The multi-chain chimeric polypeptide of embodiment C87, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second targetbinding domain in the second chimeric polypeptide.

Embodiment C92. The multi-chain chimeric polypeptide of any one of embodiments C73-C91, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen.

Embodiment C93. The multi-chain chimeric polypeptide of embodiment C92, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope.

Embodiment C94. The multi-chain chimeric polypeptide of embodiment C93, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence.

Embodiment C95. The multi-chain chimeric polypeptide of any one of embodiments C73-C91, wherein the first target-binding domain, the second targetbinding domain, and the one or more additional target-binding domains bind specifically to different antigens.

Embodiment C96. The multi-chain chimeric polypeptide of any one of embodiments C73-C95, wherein the one or more additional antigen-binding domains bind specifically to a target selected from the group consisting of: CD 16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-D, a ligand of TGF-β receptor II (TGF-RII), a ligand of TGF-βRIII, a ligand of DNAM-1, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD 155, and a receptor for CD28.

Embodiment C97. The multi-chain chimeric polypeptide of any one of embodiments C73-C95, wherein the one or more additional target-binding domains is a soluble interleukin or cytokine protein.

Embodiment C98. The multi-chain chimeric polypeptide of embodiment C97, wherein the soluble interleukin, cytokine, or ligand protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L.

Embodiment C99. The multi-chain chimeric polypeptide of any one of embodiments C73-C95, wherein the one or more additional target-binding domains is a soluble interleukin or cytokine receptor.

Embodiment Cl00. The multi-chain chimeric polypeptide of embodiment C99, wherein the soluble receptor is a soluble TGF-β receptor II (TGF-β RII), a soluble TGFβΚΙΙΙ, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-1, a scMHCI, a scMHCII, a scTCR, a soluble CD155,, or a soluble CD28.

Embodiment C101. A composition comprising any of the multi-chain chimeric polypeptides of embodiments Cl-Cl 00.

Embodiment Cl02. The composition of embodiment C101, wherein the composition is a pharmaceutical composition.

Embodiment Cl 03. A kit comprising at least one dose of the composition of embodiment C101 or Cl02.

Embodiment Cl04. A method of stimulating an immune cell, the method comprising:

contacting an immune cell with an effective amount of any of the multi-chain chimeric polypeptides of embodiments Cl-Cl00 or the composition of embodiment C101 or Cl02.

Embodiment Cl05. The method of embodiment Cl04, wherein the immune cell is contacted in vitro.

Embodiment Cl06. The method of embodiment Cl05, wherein the immune cell was previously obtained from a subject.

Embodiment Cl07. The method of embodiment Cl06, wherein the method further comprises obtaining the immune cell from the subject prior to the contacting step.

Embodiment C108. The method of embodiment C104, wherein the immune cell is contacted in vivo.

Embodiment Cl09. The method of any one of embodiments C104-C108, wherein the immune cell is selected from the group consisting of: an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, a Th 17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, and a natural killer cell.

Embodiment Cl 10. The method of any one of embodiments C104-C109, wherein the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor.

Embodiment CUE The method of any one of embodiments C104-C109, wherein the method further comprises, after the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor.

Embodiment Cl 12. The method of any one of embodiments C104-C111, wherein the method further comprises administering the immune cell to a subject in need thereof.

Embodiment Cl 13. The method of embodiment Cl 12, wherein the subject has been identified or diagnosed as having age-related disease or condition.

Embodiment Cl 14. The method of embodiment Cl 13, wherein the age-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in panceatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment Cl 15. The method of embodiment Cl 12, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment Cl 16. The method of embodiment Cl 15, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment Cl 17. The method of embodiment Cl 12, wherein the subject has been diagnosed or identified as having an infectious disease.

Embodiment Cl 18. The method of embodiment Cl 17, wherein the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Embodiment Cl 19. A method of inducing or increasing proliferation of an immune cell, the method comprising:

contacting an immune cell with an effective amount of any of the multi-chain chimeric polypeptides of embodiments Cl-Cl00 or the composition of embodiment C101 or Cl02.

Embodiment C120. The method of embodiment Cl 19, wherein the immune cell is contacted in vitro.

Embodiment C121. The method of embodiment Cl20, wherein the immune cell was previously obtained from a subject.

Embodiment C122. The method of embodiment C121, wherein the method further comprises obtaining the immune cell from the subject prior to the contacting step.

Embodiment C123. The method of embodiment Cl 19, wherein the immune cell is contacted in vivo.

Embodiment C124. The method of any one of embodiments Cl 19-C123, wherein the immune cell is selected from the group consisting of: an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, a Th 17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, and a natural killer cell.

Embodiment Cl25. The method of any one of embodiments Cl 19-C124, wherein the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor.

Embodiment C126. The method of any one of embodiments Cl 19-C124, wherein the method further comprises, after the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor.

Embodiment C127. The method of any one of embodiments Cl 19-C126, wherein the method further comprises administering the immune cell to a subject in need thereof.

Embodiment C128. The method of embodiment Cl27, wherein the subject has been identified or diagnosed as having an age-related disease or condition.

Embodiment C129. The method of embodiment C128, wherein the age-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in panceatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment C130. The method of embodiment C127, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment C131. The method of embodiment C130, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment C132. The method of embodiment C127, wherein the subject has been diagnosed or identified as having an infectious disease.

Embodiment Cl33. The method of embodiment Cl27, wherein the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Embodiment Cl34. A method of inducing differentiation of an immune cell into a memory or memory-like immune cell, the method comprising:

contacting an immune cell with an effective amount of any of the multi-chain chimeric polypeptides of embodiments Cl-Cl00 or the composition of embodiment C101 or Cl02.

Embodiment C135. The method of embodiment Cl34, wherein the immune cell is contacted in vitro.

Embodiment C136. The method of embodiment C135, wherein the immune cell was previously obtained from a subject.

Embodiment C137. The method of embodiment C136, wherein the method further comprises obtaining the immune cell from the subject prior to the contacting step.

Embodiment C138. The method of embodiment C134, wherein the immune cell is contacted in vivo.

Embodiment C139. The method of any one of embodiments C134-C138, wherein the immune cell is selected from the group consisting of: an immature thymocyte, a peripheral blood lymphocyte, a naive T cell, a pluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell, a Th 17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Thl cell, a Th3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, and a natural killer cell.

Embodiment C140. The method of any one of embodiments C134-C139, wherein the immune cell has previously been genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor.

Embodiment C141. The method of any one of embodiments C134-C139, wherein the method further comprises, after the contacting step, introducing into the immune cell a nucleic acid encoding a chimeric antigen-receptor or a recombinant T-cell receptor.

Embodiment C142. The method of any one of embodiments C134-C141, wherein the method further comprises administering the immune cell to a subject in need thereof.

Embodiment Cl43. The method of embodiment Cl42, wherein the subject has been identified or diagnosed as having an age-related disease or condition.

Embodiment C144. The method of embodiment C143, wherein the age-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in panceatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment Cl45. The method of embodiment Cl42, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment C146. The method of embodiment C145, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment C147. The method of embodiment C142, wherein the subject has been diagnosed or identified as having an infectious disease.

Embodiment Cl48. The method of embodiment Cl47, wherein the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Embodiment C149. A method of killing a cancer cell, an infected cell, or a senescent cell in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the multi-chain chimeric polypeptides of embodiments C1-C100 or the composition of embodiment C101 or C102.

Embodiment C150. The method of embodiment C149, wherein the subject has been identified or diagnosed as having a cancer.

Embodiment C151. The method of embodiment Cl50, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment C152. The method of embodiment C149, wherein the subject has been identified or diagnosed as having an aging-related disease or condition.

Embodiment Cl53. The method of embodiment Cl52, wherein the aging-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in panceatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment Cl 54. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the multi-chain chimeric polypeptides of embodiments Cl-Cl 00 or the composition of embodiment C101 or Cl02.

Embodiment Cl55. The method of embodiment Cl54, wherein the subject has been identified or diagnosed as having a cancer or infectious disease.

Embodiment Cl56. The method of embodiment Cl57, wherein the cancer is selected from the group consisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment C157. The method of embodiment C154, wherein the subject has been identified or diagnosed as having age-related disease or condition.

Embodiment C158. The method of embodiment C157, wherein the age-related disease or condition is selected from the group consisting of: Alzheimer’s disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson’s disease, age-associated loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington’s disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

Embodiment C159. The method of embodiment C154, wherein the subject has been diagnosed or identified as having an infectious disease.

Embodiment Cl60. The method of embodiment Cl59, wherein the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

Embodiment C161. Nucleic acid encoding any of the multi-chain chimeric polypeptides of any one of embodiments Cl-Cl00.

Embodiment Cl62. A vector comprising the nucleic acid of embodiment C161.

Embodiment Cl63. The vector of embodiment Cl62, wherein the vector is an expression vector.

Embodiment Cl64. A cell comprising the nucleic acid of embodiment C161 or the vector of embodiment C162 or C163.

Embodiment Cl65. A method of producing a multi-chain chimeric polypeptide, the method comprising:

culturing the cell of embodiment Cl64 in a culture medium under conditions sufficient to result in the production of the multi-chain chimeric polypeptide; and recovering the multi-chain chimeric polypeptide from the cell and/or the culture medium.

Embodiment Cl66. A multi-chain chimeric polypeptide produced by the method of embodiment Cl65.

Embodiment C167. The multi-chain chimeric polypeptide of embodiment C12, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 3.

Embodiment Cl68. The multi-chain chimeric polypeptide of embodiment Cl67, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 3.

Embodiment Cl69. The multi-chain chimeric polypeptide of embodiment Cl68, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 3.

Embodiment Cl70. The multi-chain chimeric polypeptide of embodiment Cl69, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO: 3.

Embodiment C171. The multi-chain chimeric polypeptide of embodiment C12, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 4.

Embodiment Cl72. The multi-chain chimeric polypeptide of embodiment C171, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 4.

Embodiment Cl73. The multi-chain chimeric polypeptide of embodiment Cl72, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 4.

Embodiment C174. The multi-chain chimeric polypeptide of embodiment C173, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO: 4.

D. Exemplary Embodiments

Embodiment DE A multi-chain chimeric polypeptide comprising:

(c) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and (iii) a first domain of a pair of affinity domains;

(d) a second chimeric polypeptide comprising:

(i) a second domain of a pair of affinity domains; and (ii) a second target-binding domain, wherein:

the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains;

the first target-binding domain and the second targeting-binding domain each independently bind specifically to a receptor of IL-21 or a receptor of IL-7.

Embodiment D2. The multi-chain chimeric polypeptide of embodiment DI, wherein the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide.

Embodiment D3. The multi-chain chimeric polypeptide of embodiment DI, wherein the first chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

Embodiment D4. The multi-chain chimeric polypeptide of any one of embodiments D1-D3, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide.

Embodiment D5. The multi-chain chimeric polypeptide of any one of embodiments D1-D3, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment D6. The multi-chain chimeric polypeptide of any one of embodiments D1-D5, wherein the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide.

Embodiment D7. The multi-chain chimeric polypeptide of any one of embodiments D1-D5, wherein second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

Embodiment D8. The multi-chain chimeric polypeptide of any one of embodiments D1-D7, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment D9. The multi-chain chimeric polypeptide of embodiment D8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1.

Embodiment D10. The multi-chain chimeric polypeptide of embodiment D9, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1.

Embodiment Dll. The multi-chain chimeric polypeptide of embodiment D10, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1.

Embodiment D12. The multi-chain chimeric polypeptide of embodiment Dll, wherein the soluble human tissue factor domain comprises SEQ ID NO: 1.

Embodiment D13. The multi-chain chimeric polypeptide of embodiment D8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 3.

Embodiment D14. The multi-chain chimeric polypeptide of embodiment D13, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 3.

Embodiment DI 5. The multi-chain chimeric polypeptide of embodiment DI4, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 3.

Embodiment DI6. The multi-chain chimeric polypeptide of embodiment DI 5, wherein the soluble human tissue factor domain comprises SEQ ID NO: 3.

Embodiment DI7. The multi-chain chimeric polypeptide of embodiment D8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 4.

Embodiment DI 8. The multi-chain chimeric polypeptide of embodiment DI7, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 4.

Embodiment DI9. The multi-chain chimeric polypeptide of embodiment DI 8, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 4.

Embodiment D20. The multi-chain chimeric polypeptide of embodiment DI9, wherein the soluble human tissue factor domain comprises SEQ ID NO: 4.

Embodiment D21. The multi-chain chimeric polypeptide of any one of embodiments D8-D11, D13-D15, and D17-D19, wherein the soluble human tissue factor domain does not comprise one or more of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment D22. The multi-chain chimeric polypeptide of embodiment D21, wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acid position 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acid position 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acid position 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to amino acid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acid position 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acid position 135 of mature wildtype human tissue factor protein; and a phenylalanine at an amino acid position that corresponds to amino acid position 140 of mature wildtype human tissue factor protein.

Embodiment D23. The multi-chain chimeric polypeptide of any one of embodiments D1-D22, wherein the soluble tissue factor domain is not capable of binding to Factor Vila.

Embodiment D24. The multi-chain chimeric polypeptide of any one of embodiments D1-D23, wherein the soluble tissue factor domain does not convert inactive Factor X into Factor Xa.

Embodiment D25. The multi-chain chimeric polypeptide of any one of embodiments D1-D24, wherein the multi-chain chimeric polypeptide does not stimulate coagulation in a mammal.

Embodiment D26. The multi-chain chimeric polypeptide of any one of embodiments D1-D25, wherein the first chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the first chimeric polypeptide.

Embodiment D27. The multi-chain chimeric polypeptide of any one of embodiments D1-D26, wherein the second chimeric polypeptide further comprises a peptide tag at the N-terminal end or the C-terminal end of the second chimeric polypeptide.

Embodiment D28. The multi-chain chimeric polypeptide of any one of embodiments D1-D27, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminal end.

Embodiment D29. The multi-chain chimeric polypeptide of embodiment D28, wherein the signal sequence comprises SEQ ID NO: 31.

Embodiment D30. The multi-chain chimeric polypeptide of embodiment D28, wherein the signal sequence is SEQ ID NO: 223.

Embodiment D31. The multi-chain chimeric polypeptide of any one of embodiments D1-D30, wherein the pair of affinity domains is a sushi domain from an alpha chain of human IL-15 receptor (IL15Ra) and a soluble IL-15.

Embodiment D32. The multi-chain chimeric polypeptide of embodiment D31, wherein the soluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment D33. The multi-chain chimeric polypeptide of embodiment D31, wherein the soluble IL-15 comprises a sequence that is at least 80% identical to SEQ ID NO: 14.

Embodiment D34. The multi-chain chimeric polypeptide of embodiment D33, wherein the soluble IL-15 comprises a sequence that is at least 90% identical to SEQ ID NO: 14.

Embodiment D35. The multi-chain chimeric polypeptide of embodiment D34, wherein the soluble IL-15 comprises a sequence that is at least 95% identical to SEQ ID NO: 14.

Embodiment D36. The multi-chain chimeric polypeptide of embodiment D35, wherein the soluble IL-15 comprises SEQ ID NO: 14.

Embodiment D37. The multi-chain chimeric polypeptide of any one of embodiments D31-D36, wherein the sushi domain of IL15Ra comprises a sushi domain from human IL15Ra.

Embodiment D38. The multi-chain chimeric polypeptide of embodiment D37, wherein the sushi domain from human IL15Ra comprises a sequence that is at least 80% identical to SEQ ID NO: 28.

Embodiment D39. The multi-chain chimeric polypeptide of embodiment D38, wherein the sushi domain from human IL15Ra comprises a sequence that is at least 90% identical to SEQ ID NO: 28.

Embodiment D40. The multi-chain chimeric polypeptide of embodiment D39, wherein the sushi domain from human IL15Ra comprises a sequence that is at least 95% identical to SEQ ID NO: 28.

Embodiment D41. The multi-chain chimeric polypeptide of embodiment D40, wherein the sushi domain from human IL15Ra comprises SEQ ID NO: 28.

Embodiment D42. The multi-chain chimeric polypeptide of embodiment D37, wherein the sushi domain from human IL15Ra is a mature full-length IL15Ra.

Embodiment D43. The multi-chain chimeric polypeptide of any one of embodiments D1-D30, wherein the pair of affinity domains is selected from the group consisting of: bamase and bamstar, a PKA and an AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25.

Embodiment D44. The multi-chain chimeric polypeptide of any one of embodiments D1-D43, wherein one or both of the first target-binding domain and the second target-binding domain is an agonistic antigen-binding domain.

Embodiment D45. The multi-chain chimeric polypeptide of embodiment D44, wherein the first target-binding domain and the second target-binding domain are each agonistic antigen-binding domains.

Embodiment D46. The multi-chain chimeric polypeptide of embodiment D44 or D45, wherein antigen-binding domain comprises a scFv or single-domain antibody.

Embodiment D47. The multi-chain chimeric polypeptide of any one of embodiments D1-D43, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble IL-21 or a soluble IL-7.

Embodiment D48. The multi-chain chimeric polypeptide of embodiment D47, wherein the first target-binding domain and the second target-binding domain are each independently a soluble IL-21 or a soluble IL-7.

Embodiment D49. The multi-chain chimeric polypeptide of any one of embodiments D1-D48, wherein the first target-binding domain and the second targetbinding domain both bind specifically to a receptor of IL-21 or a receptor of IL-7.

Embodiment D50. The multi-chain chimeric polypeptide of embodiment D49, wherein the first target-binding domain and the second target-binding domain bind specifically to the same epitope.

Embodiment D51. The multi-chain chimeric polypeptide of embodiment D50, wherein the first target-binding domain and the second target-binding domain comprise the same amino acid sequence.

Embodiment D52. The multi-chain chimeric polypeptide of any one of embodiments D1-D48, wherein the first target-binding domain binds specifically to a receptor for IL-21, and the second target-binding domain binds specifically to a receptor for IL-7.

Embodiment D53. The multi-chain chimeric polypeptide of any one of embodiments D1-D48, wherein the first target-binding domain binds specifically to a receptor for IL-7, and the second target-binding domain bind specifically to a receptor for IL-21.

Embodiment D54. The multi-chain chimeric polypeptide of embodiment D53, wherein the first target-binding domain comprises a soluble IL-21.

Embodiment D55. The multi-chain chimeric polypeptide of embodiment D54, wherein the soluble IL-21 is a soluble human IL-21.

Embodiment D56. The multi-chain chimeric polypeptide of embodiment D55, wherein the soluble human IL-21 comprises a sequence at least 80% identical to SEQ ID NO: 78.

Embodiment D57. The multi-chain chimeric polypeptide of embodiment D56, wherein the soluble human IL-21 comprises a sequence at least 90% identical to SEQ ID NO: 78.

Embodiment D58. The multi-chain chimeric polypeptide of embodiment D57, wherein the soluble human IL-21 comprises a sequence at least 95% identical to SEQ ID NO: 78.

Embodiment D59. The multi-chain chimeric polypeptide of embodiment D58, wherein the soluble human IL-21 comprises a sequence of SEQ ID NO: 78.

Embodiment D60. The multi-chain chimeric polypeptide of any one of embodiments D53-D59, wherein the second target-binding domain comprises a soluble IL-7.

Embodiment D61. The multi-chain chimeric polypeptide of embodiment D60, wherein the soluble IL-7 is a soluble human IL-7.

Embodiment D62. The multi-chain chimeric polypeptide of embodiment D61, wherein the soluble human IL-7 comprises a sequence at least 80% identical to SEQ ID NO: 11.

Embodiment D63. The multi-chain chimeric polypeptide of embodiment D62, wherein the soluble human IL-7 comprises a sequence at least 90% identical to SEQ ID NO: 11.

Embodiment D64. The multi-chain chimeric polypeptide of embodiment D63, wherein the soluble human IL-7 comprises a sequence at least 95% identical to SEQ ID NO: 11.

Embodiment D65. The multi-chain chimeric polypeptide of embodiment D64, wherein the soluble human IL-7 comprises a sequence of SEQ ID NO: 11.

Embodiment D66. The multi-chain chimeric polypeptide of embodiment DI, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 104.

Embodiment D67. The multi-chain chimeric polypeptide of embodiment D66, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 104.

Embodiment D68. The multi-chain chimeric polypeptide of embodiment D67, wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 104.

Embodiment D69. The multi-chain chimeric polypeptide of embodiment D68, wherein the first chimeric polypeptide comprises SEQ ID NO: 104.

Embodiment D70. The multi-chain chimeric polypeptide of embodiment D69, wherein the first chimeric polypeptide comprises SEQ ID NO: 106.

Embodiment D71. The multi-chain chimeric polypeptide of any one of embodiments DI and D66-D70, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 108.

Embodiment D72. The multi-chain chimeric polypeptide of embodiment D71, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 108.

Embodiment D73. The multi-chain chimeric polypeptide of embodiment D72, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 108.

Embodiment D74. The multi-chain chimeric polypeptide of embodiment D73, wherein the second chimeric polypeptide comprises SEQ ID NO: 108.

Embodiment D75. The multi-chain chimeric polypeptide of embodiment D74, wherein the second chimeric polypeptide comprises SEQ ID NO: 110.

Embodiment D76. The multi-chain chimeric polypeptide of embodiment DI, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 96.

Embodiment D77. The multi-chain chimeric polypeptide of embodiment D76, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 96.

Embodiment D78. The multi-chain chimeric polypeptide of embodiment D77, wherein the first chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 96.

Embodiment D79. The multi-chain chimeric polypeptide of embodiment D68, wherein the first chimeric polypeptide comprises SEQ ID NO: 96.

Embodiment D80. The multi-chain chimeric polypeptide of embodiment D69, wherein the first chimeric polypeptide comprises SEQ ID NO: 98.

Embodiment D81. The multi-chain chimeric polypeptide of any one of embodiments DI and D76-D80, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 100.

Embodiment D82. The multi-chain chimeric polypeptide of embodiment D81, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 100.

Embodiment D83. The multi-chain chimeric polypeptide of embodiment D82, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 100.

Embodiment D84. The multi-chain chimeric polypeptide of embodiment D83, wherein the second chimeric polypeptide comprises SEQ ID NO: 100.

Embodiment D85. The multi-chain chimeric polypeptide of embodiment D84, wherein the second chimeric polypeptide comprises SEQ ID NO: 106.

Embodiment D86. The multi-chain chimeric polypeptide of any one of embodiments D1-D65, wherein the first chimeric polypeptide further comprises one or more additional target-binding domain(s), where at least one of the one or more additional antigen-binding domain(s) is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment D87. The multi-chain chimeric polypeptide of embodiment D86, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the at least one of the one or more additional antigenbinding domain(s), and/or a linker sequence between the at least one of the one or more additional antigen-binding domain(s)and the first domain of the pair of affinity domains.

Embodiment D88. The multi-chain chimeric polypeptide of any one of embodiments D1-D65, wherein the first chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal and/or C-terminal end of the first chimeric polypeptide.

Embodiment D89. The multi-chain chimeric polypeptide of embodiment D88, wherein at least one of the one or more additional target-binding domains directly abuts the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment D90. The multi-chain chimeric polypeptide of embodiment D88, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first domain of the pair of affinity domains.

Embodiment D91. The multi-chain chimeric polypeptide of embodiment D88, wherein the at least one of the one or more additional target-binding domains directly abuts the first target-binding domain in the first chimeric polypeptide.

Embodiment D92. The multi-chain chimeric polypeptide of embodiment D88, wherein the first chimeric polypeptide further comprises a linker sequence between the at least one of the one or more additional target-binding domains and the first target-binding domain.

Embodiment D93. The multi-chain chimeric polypeptide of embodiment D88, wherein at least one of the one or more additional target-binding domains is disposed at the N- and/or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target-binding domains is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment D94. The multi-chain chimeric polypeptide of embodiment D93, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the N-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment D95. The multi-chain chimeric polypeptide of embodiment D93, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment D96. The multi-chain chimeric polypeptide of embodiment D93, wherein the at least one additional target-binding domain of the one or more additional target-binding domains disposed at the C-terminus directly abuts the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment D97. The multi-chain chimeric polypeptide of embodiment D93, wherein the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target-binding domain and the first target-binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment D98. The multi-chain chimeric polypeptide of embodiment D93, wherein the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, directly abuts the soluble tissue factor domain and/or the first domain of the pair of affinity domains.

Embodiment D99. The multi-chain chimeric polypeptide of embodiment D93, wherein the first chimeric polypeptide further comprises a linker sequence disposed (i) between the soluble tissue factor domain and the at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, and/or (ii) between the first domain of the pair of affinity domains and the at least one of the one or more additional targetbinding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment DI00. The multi-chain chimeric polypeptide of any one of embodiments D1-D65 and D86-D99, wherein the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminal end or the Cterminal end of the second chimeric polypeptide.

Embodiment D101. The multi-chain chimeric polypeptide of embodiment DI00, wherein at least one of the one or more additional target-binding domains directly abuts the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment DI02. The multi-chain chimeric polypeptide of embodiment DI00, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment DI03. The multi-chain chimeric polypeptide of embodiment DI00, wherein at least one of the one or more additional target-binding domains directly abuts the second target-binding domain in the second chimeric polypeptide.

Embodiment DI04. The multi-chain chimeric polypeptide of embodiment DI00, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains and the second targetbinding domain in the second chimeric polypeptide.

Embodiment DI05. The multi-chain chimeric polypeptide of any one of embodiments D86-D104, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same antigen.

Embodiment DI06. The multi-chain chimeric polypeptide of embodiment DI05, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains bind specifically to the same epitope.

Embodiment DI07. The multi-chain chimeric polypeptide of embodiment DI06, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains comprise the same amino acid sequence.

Embodiment DI08. The multi-chain chimeric polypeptide of any one of embodiments D86-D104, wherein the first target-binding domain, the second targetbinding domain, and the one or more additional target-binding domains bind specifically to different antigens.

Embodiment DI09. The multi-chain chimeric polypeptide of any one of embodiments D86-D108, wherein the one or more additional antigen-binding domains bind specifically to a target selected from the group consisting of: CD 16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-β receptor II (TGF^RII), a ligand of ΤΟΕ-βΚΙΙΙ, a ligand of DNAM-1, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD 155, and a receptor for CD28.

Embodiment DI 10. The multi-chain chimeric polypeptide of any one of embodiments D86-D108, wherein the one or more additional target-binding domains is a soluble interleukin or cytokine protein.

Embodiment Dill. The multi-chain chimeric polypeptide of embodiment DI 10, wherein the soluble interleukin, cytokine, or ligand protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L.

Embodiment DI 12. The multi-chain chimeric polypeptide of any one of embodiments D86-D108, wherein the one or more additional target-binding domains is a soluble interleukin or cytokine receptor.

Embodiment DI 13. The multi-chain chimeric polypeptide of embodiment DI 12, wherein the soluble receptor is a soluble TGF-β receptor II (TGF^RII), a soluble TGF βΚΙΙΙ, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-1, a scMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, or a soluble CD28.

Embodiment DI 14. A composition comprising any of the multi-chain chimeric polypeptides of embodiments Dl-Dl 13.

Embodiment DI 15. The composition of embodiment DI 14, wherein the composition is a pharmaceutical composition.