Pd-1-binding molecules and methods use thereof.

PD-1 BINDING MOLECULES AND METHODS OF USE THEREOF cnan Ln/zznz/E/YiAi

Cross Reference to Related Requests

[0001] This application claims priority to US Patent Applications Serial No. 62/198,867 (filed July 30, 2015; pending), 62/239,559 (filed October 9, 2015; pending), 62/ 255,140 (filed Nov. 13, 2015; pending), and 62/322,974 (filed April 15, 2016; pending), each of which applications is incorporated herein by reference in its entirety.

Reference to the Sequence List

[0002] This application includes one or more Sequence Listings in accordance with 37 CFR 1.821 et seq., which are described on machine-readable media (file name: 1301 0122PCT Sequence Listing ST25.txt, created July 1, 2016, and which has a size of 282,789 bytes), the file of which is incorporated herein by reference in its entirety.

Field of Invention

[0003] The present invention is directed to PD-1 binding molecules comprising the PD-1 binding domain of selected anti-PD-1 antibodies capable of binding both

Cynomolgus monkey PD-1 and human PD-1: PD-1 mAb 1; PD-1 mAb 2; PD-1 mAb 3; PD-1 mAb 4; PD-1 mAb 4; PD-1 mAb 5; PD-1 mAb 6; PD-1 mAb 7; PD-1 mAb 7; PD-1 mAb 8; PD-1 mAb 9; PD-1 mAb 10; PD-1 mAb 11; PD-1 mAb 12; PD-1 mAb 13; PD-1 mAb 14, or PD-1 mAb 15. The invention relates particularly to PD-1 binding molecules that are humanized or chimeric versions of such antibodies, or that comprise PD-1 binding fragments of such anti-PD-1 antibodies (specifically immunoconjugates, diabodies, BiTE, antibodies bispecific, etc.). The invention particularly relates to such PD-1 binding molecules that are additionally capable of binding to an epitope of a molecule involved in the regulation of an immune checkpoint presented on the surface of an immune cell. The present invention also pertains to methods of using such PD-1 binding molecules to detect PD-1 or stimulate an immune response. The present invention also pertains to methods of combination therapy in which a PD1 binding molecule comprising one or more PD-1 binding domains of such selected anti-PD1 antibodies is administered in combination with one or more additional molecules that They are effective in stimulating an immune response and/or in combination with one or more additional molecules that specifically bind to an anti-cancer antigen.

cnan ίη/ζζηζ/Ε/γίΛΐ cnan Ln/zznz/E/YiAi

Background of the Invention

I. Cell-Mediated Immune Responses

[0004] The immune system of humans and other mammals is responsible for providing protection against infections and diseases. Such protection is provided by both humoral immune response and cell-mediated immune response. The humoral response results in the production of antibodies and other biomolecules that are capable of recognizing and neutralizing foreign targets (antigens). In contrast, the cell-mediated immune response involves the activation of macrophages, natural killer (NK) cells, and antigen-specific cytotoxic T-lymphocytes by T cells, and the release of several cytokines in response to recognition of an antigen (Dong, C. et al. (2003) Immune Regulation by Novel Costlmulatory Molecules, Immunolog. Res. 28(1):39-48).

[0005] The ability of T cells to optimally mediate an immune response against an antigen requires two distinct signaling interactions (Viglietta, V. et al. (2007) Modulating Co-Stimulation Neurotherapeutics 4:666675; Korman, AJ et al. al (2007) Checkpoint Blockade in Cancer Immunotherapy Adv. Immunol. 90:297-339). First, the antigen that has been placed on the surface of the Antigen Presenting Cells (APC) must be presented to a T cell.

CD4⁺ without prior antigen-specific treatment. Such presentation supplies a signal through the T Cell Receptor (TCR) that instructs the T Cell to initiate an immune response that will be specific for the presented antigen. Second, a series of costimulatory and inhibitory signals, mediated through interactions between APCs and distinct T cell surface molecules, first trigger T cell activation and proliferation and finally their inhibition. In this way, the first signal confers specificity to the immune response while the second signal serves to determine the nature, magnitude and duration of the response.

[0006] the immune system is firmly controlled by co-stimulatory and coinhibitory ligands and receptors. These molecules provide the second signal for T cell activation and provide a balanced network of positive and negative signals to maximize immune responses against infection while limiting immunity to themselves (Wang, L. et al. (March 7, 2011 ) VISTA, A Novel Mouse Ig Superfamily Ligand That Negatively Regulates T-Cell Responsos, J. Exp. Med. 10.1084/jem.20100619:1-16; Lepenies, B. et al. (2008) The Role Of Negative Costimulators During Parasitic Infections, Endocrine, Metabolic & Immune

Disorders

drug

Targets 8:279-288). Of particular importance is the binding between the ligands B7.1 (CD80) and B7.2 (CD86) of the Antigen Presenting Cell and the CD28 and CTLA-4 receptors of the CD4 + T cell ( Sharpe, AH et al. (2002) The B7-CD28 Superfamily Nature Rev. Immunol. 2:116126; Dong, C. et al. (2003) Immune Regulation by Novel Costimulatory Molecules, Immunolog. Res. 28(l):39 -48; Lindley, PS et al. (2009) The Clinical Utility Of Inhibiting CD28-Mediated Costimulation, Immunol. Rev. 229:307-321). Binding of B7.1 or B7.2 to CD28 stimulates T cell activation; the binding of B7.1 or B7.2 to CTLA-4 inhibits such activation (Dong, C. et al. (2003) Immune Regulation by Novel Costimulatory Molecules, Immunolog. Res. 28(1):39-48; Lindley, PS et al. (2009) The Clinical Utility Of Inhibiting CD28-Mediated Costimulation, Immunol. Rev. 229:307-321; Greenwald, RJ et al. (2005) The B7 Family Revisited, Ann. Rev. Immunol. 23:515 -548). CD28 is constitutively expressed on the surface of T cells (Gross, J., et al. (1992) Identification And Distribution Of The Costimulatory Receptor CD28 In The Mouse, J. Immunol. 149:380-388), while CTLA-4 expression is rapidly upregulated following T cell activation (Linsley, P. et al. (1996) Intracellular Trafficking Of CTLA4 And Focal Localization Towards Sites Of TCR Engagement, Immunity 4:535-543). Since CTLA-4 is the cnan Ln/zznz/E/YiAi receptor with the highest affinity (Sharpe, AH et al. (2002) The cnan Ln/zznz/E/YiAi

B7-CD28 Superfamily, Nature Rev. Immunol. 2:116-126), binding first initiates T cell proliferation (via CD28) and then inhibits it (via nascent expression of CTLA-4), thereby diminishing the effect when proliferation is no longer necessary.

[0007] Additional investigations of CD28 receptor ligands have led to the identification and characterization of a set of related B7 molecules (the B7 Superfamily) (Coyle, AJ et al. (2001) The Expanding B7 Superfamily: Increasing Complexity In Costimulatory Signs Regulating T-Cell Function, Nature Immunol. 2 (3): 203-209; Sharpe, AH et al. (2002) The B7CD28 Superfamily, Nature Rev. Immunol. 2:116-126; Greenwald, RJ et al. (2005 ) The B7 Family Revisited, Ann. Rev. Immunol. 23:515-548; Collins, M. et al. (2005) The B7 Family Of Immune-Regulatory Ligands, Genome Biol. 6:223.1-223.7;

Locke, P. et al. (2004) Emerging Mechanisms Of Immune Regulation: The Extended B7 Family And Regulatory T-Cells. Arthritis Res. Ther. 6:208-214; Korman, A.J. et al. (2007) Checkpoint Blockade in Cancer Immunotherapy, Adv. Immunol. 90:297-339; Flies, D.B. et al. (2007) The New B7s: Playing a Bivotal Role in Tumor Immunity, J. Immunother. 30(3):251260; Agarwal, A. et al. (2008) The Role Of Positive Costimulatory Molecules In Transplantation And Tolerance, Curr. Opinion. Organ Transplant. 13:366-372; Lenschow, DJ et al. (1996) CD28/B7 System of T-Cell Costimulation, Ann.

Rev. Immunol. 14:233-258; Wang, S. et al. (2004) Cosignaling Molecules Of The B7-CD28 Family In Positive And Negative Regulation Of T Lymphocyte Responses, Microbes Infect. 6:759-766). Currently there are several known members of the family: B7.1 (CD80), B7.2 (CD86), inducible co-stimulatory ligand (ICOS-L), programmed death ligand 1 (PD-L1; B7-H1) , programmed death ligand 2 (PD-L2; B7-DC), B7-H3, B7-H4 and B7-H6 (Collins, M. et al. et al (2005) The B7 Family Of Immune-Regulatory Ligands, Genome Biol. 6:223.1-223.7; Flajnik, M.F. et al. (2012)

Evolution Of The B7 Family: Co-Evolution Of B7H6 And Nkp30, Identification Of A New B7 Family Member, B7H7, And Of B7' s Historical Relationship With The MHC, Immunogenetics epub doi.org/10.1007/s00251-012-0616-2 ).

cnan Ln/zznz/E/YiAi

II. Programmed Death-1 (PD-1)

[0008] Programmed death 1 (PD-1, also known as CD279) is an approximately 31 kD type I membrane protein member of the extended CD28/CTLA-4 family of T cell regulators that broadly and negatively regulate immune responses (Ishida, Y. et al. (1992) Induced Expression Of PD-1, A Novel Member Of The Immunoglobulin Gene Superfamily, Upon Programmed Cell Death, EMBO J. 11:3887-3895; United States Patent Application

United Publication No. 2007/0202100; 2008/0311117;

2009/00110667; United States Patent Nos. 6,808,710; 7,101,550; 7,488,802; 7,635,757; 7,722,868; PCT Publication No. WO 01/14557).

[0009] PD-1 is expressed on activated T cells, B cells and monocytes (Agata, Y. et al. (1996) Expression Of The PD-1 Antigen On The Surface Of Stimulated Mouse T And B Lymphocytes, Int. Immunol. 8(5):765-772; Yamazaki, T. et al. (2002) Expression Of Programmed Death 1 Ligands By Marine TCells And APC, J. Immunol. 169:5538-5545) and at low levels in natural killer T cells ( NK) (Nishimura, H. et al. (2000) Facilitation Of Beta Selection And Modification Of Positive Selection In The Thymus Of PD-l-Deficient Mice, J. Exp. Med. 191:891-898; Martin-Orozco, N. et al (2007) Inhibitory Costimulation And Anti-Tumor Immunity, Semin. Cancer Biol. 17 (4):288-298).

[0010] The extracellular region of PD-1 consists of a single immunoglobulin (Ig)V Domain with 23% identity with the equivalent Domain in CTLA-4 (Martin-Orozco, N. et al (2007) Inhibitory Costimulation And Anti -Tumor Immunity, Semin. Cancer Biol. 17(4):288-298). The extracellular IgV domain is followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located on a Tyrosine-based immunoreceptor cnan Ln/zznz/E/YiAi inhibitory motif and a Tyrosine-based immunoreceptor switch motif, suggesting that PD-1 negatively regulates phosphorylation signals. TCR (Ishida, Y. et al. (1992) Induced Expression Of PD-1, Ά Novel Member Of The Immunoglobulin Gene Superfamily, Upon Programmed Cell Death, EMBO J. 11:3887-3895; Blank, C. et al. (2006) Contribution Of The PD-L1/PD-1 Pathway To T-Cell Exhaustion: An Update On Implications For Chronic Infections And Tumor Evasion Cancer, Immunol. Immunother. 56 (5):739-745).

[0011] PD1 mediates its inhibition of the immune system by binding to B7-H1 and B7-DC (Flies, DB et al (2007) The New B7s: Playing a Pivotal Role in Tumor Immunity, J. Immunother. 30(3 ):251-260; United States Patent Nos. 6,803,192; 7,794,710; United States Patent Application Publications Nos. 2005/0059051; 2009/0055944; 2009/0274666; 2009/0313687; PCT Publications Nos. WO 01/39722; WO 02/086083).

[0012] B7-H1 and B7-DC are widely expressed on the surfaces of human and murine tissues, such as heart, placenta, muscle, fetal, liver, spleen, lymph nodes, and thymus as well as liver, lung, kidney, pancreatic islet cells and murine small intestine. (Martin-Orozco, N. et al. (2007) Inhibitory Costimulation And Anti-Tumor Immunity, Semin. Cancer Biol. 17(4):288-298). In humans, B7-H1 protein expression was found in endothelial cells (Chen, Y. et al. (2005) Expression of B7-H1 in

CbQb ίη/ΖΖΠΖ/Ε/ΥΙΛΙ

Inflammatory Renal Tubular Epithellal Cells, Nephron. Exp. Nephrol. 102:e81-e92; by Hai j, S. et al. (2005) Renal

Tubular Epithellal Cells Modulate T-Cell Responses Via ICOS-L And B7-H1 Kidney Int. 68:2091-2102; Mazanet, MM et al. (2002) B7-H1 Is Expressed By Human Endothelial Cells And

Suppresses T-Cell Cytokine Synthesis, J. Immunol. 169:35813588), myocardium (Brown, JA et al. (2003) Blockade Of Programmed Death-1 Ligands On Dendritic Cells Enhances T-Cell Activation And Cytokine Production, J. Immunol. 170:12571266), syncyciotrophoblasts (Petroff, MG et al. al. (2002) B7 Family Molecules: Novel Immunomodulators At The MaternalFetal Interface, Placenta 23:S95-S101). The molecules are also expressed by resident macrophages of some tissues, by macrophages that have been activated with interferon (IFN)- and or tumor necrosis factor (TNF)-a (Latchman, Y. et al. (2001) PD-L2 Is A Second Ligand For PD-1 And Inhibits TCell Activation, Nat. Immunol 2:261- 268), and in tumors (Dong, H. (2003) B7-H1 Pathway And Its Role In The Evasion

Of Tumor Immunity, J. Mol. Med. 81:281-287).

[0013] It has been found that the interaction between B7-H1 and PD-1 provides an important negative co-stimulatory signal to T and B cells (Martin-Orozco, N. et al. (2007) Inhibitory Costimulation And Anti-Tumor Immunity , Semin. Cancer Biol. 17(4):288-298) and functions as an inducer of cell death (Ishida, Y. et al. (1992) Induced Expression cnan Ln/zznz/E/YiAi

Of PD-1, A Novel Member Of The Immunoglobule in Gene Superfamily, Upon Programmed Cell Death, EMBO J. 1 1:3887-3895; Subudhi, S.K. et al. (2005) The Balance Of Immune Responses: Costimulation Verse Coinhibition, J. Molec. Med. 83:193cnan Ln/zznz/E/YiAi

202). More specifically, it has been found that the interaction between low concentrations of the PD-1 receptor and the B7-H1 ligand results in the transmission of an inhibitory signal that strongly inhibits the proliferation of antigen-specific CD8 + T cells. ; At higher concentrations, interactions with PD-1 do not inhibit T cell proliferation but markedly reduce the production of multiple cytokines (Sharpe, AH et al. (2002) The B7-CD28 Superfamily, Nature Rev. Immunol. 2:116-126). T cell proliferation and cytokine production by both resting T cells and previously activated CD8 T cells, and even naïve T cells from umbilical cord blood, have been found to be inhibited by B7-Fc fusion proteins. soluble (Freeman, GJ et al. (2000) Engagement Of The PD-1 Immunoinhibitory Receptor By A Novel B7 Family Member Leads To Negative Regulation Of Lymphocyte Activation, J. Exp. Med. 192:1-9; Latchman, Y. et al. (2001) PD-L2 Is A Second Ligand For PD-1 And Inhibits T-Cell Activation, Nature Immunol. 2:261-268; Carter, L. et al. (2002) PD-1'.PD-L

Inhibitory Pathway Affects Both CD4 (+) and CD8(+) T-cells And cnan Ln/zznz/E/YiAi

Is Overeóme By IL-2, Eur. J. Immunol. 32(3):634-643; Sharpe, A.H. et al. (2002) The B7-CD28 Superfamily, Nature Rev. Immunol. 2:116-126).

[0014] The role of B7-H1 and PD-1 in inhibiting T cell activation and proliferation has suggested that these biomolecules may serve as therapeutic targets for inflammation and cancer treatments. Thus, the use of anti-PD-1 antibodies to treat infections and tumors, and the modulation of an adaptive response, has been proposed (see, United States Patent Application Publication Nos. 2010/0040614; 2010/0028330 ;

2004/0241745; 2008/0311117; 2009/0217401; Patents of the

United States Nos. 7,521,051; 7,563,869; 7,595,048;

PCT Publications Nos. WO 2004/056875; WO 2008/083174). Antibodies capable of specifically binding to PD-1 have been reported by Agata, T. et al. (1996) Expression Of The PD-1 Antigen On The Surface Of Stimulated Mouse T And B Lymphocytes, Int. Immunol. 8(5):765-772; and Berger, R. et al. (2008) Phase I Safety And Pharmacokinetic Study Of CT011, A Humanized Antibody Interacting With PD-1, In Patients With Advanced Hematology Malignancies, Clin. Cancer Res. 14 (10):3044-3051 (see, also, United States Patent Nos. 8,008,449 and 8,552,154; United States Patent Publication Nos. 2007/0166281; 2012/0114648;

2012/0114649; 2013/0017199; 2013/0230514 and 2014/0044738; and

PCT Patent Publications Nos. WO 2003/099196; WO 2004/004771; WO 2004/056875; WO 2004/072286; WO 2006/121168; WO 2007/005874; WO 2008/083174; WO 2009/014708; WO 2009/073533; WO 2012/135408, WO 2012/145549; and WO 2013/014668).

[0015] However, despite all such prior advances, there remains a need for improved compositions capable of more vigorously directing the body's immune system to attack cancer cells or pathogen-infected cells, especially at lower therapeutic concentrations. . Although the adaptive immune system can be a powerful defense mechanism against cancer and disease, it is frequently hindered by immunosuppressive mechanisms in the tumor microenvironment, such as the expression of PD-1. Furthermore, coinhibitory molecules expressed by tumor cells, immune cells, and stromal cells in the tumor environment can dominantly attenuate T cell responses against cancer cells. Thus, there remains a need for potent PD-1 binding molecules. In particular, there is a need for potent PD1-binding molecules that have a desirable binding kinetic profile and that antagonize the PD-1/PDL1 axis by blocking the PD-1/PD-L1 interaction, which may provide therapeutic value. improved to patients suffering from cancer or other cnan Ln/zznz/E/YiAi diseases and conditions. The present invention is directed to cnan Ln/zznz/E/YiAi these and other objectives.

Compendium of the Invention:

[0016] The present invention is directed to PD-1 binding molecules comprising the PD-1 binding domain of selected anti-PD-1 antibodies capable of binding both cynomolgus monkey PD-1 and PD-1 from human: PD-1 mAb 1; PD-1 mAb 2; PD-1 mAb 3; PD-1 mAb 4; PD-1 mAb 4; PD-1 mAb 5; PD-1 mAb 6; PD-1 mAb 7; PD-1 mAb 7; PD-1 mAb 8; PD-1 mAb 9; PD-1 mAb 10; PD-1 mAb 11; PD-1 mAb 12; PD-1 mAb 13; PD-1 mAb 14, or PD-1 mAb 15. The invention relates particularly to PD-1 binding molecules that are humanized or chimeric versions of such antibodies, or that comprise PD1 binding fragments of such anti-PD-1 antibodies (specifically immunoconjugates, diabodies, BiTE, bispecific antibodies, etc.). The invention particularly relates to such PD-1 binding molecules that are additionally capable of binding to an epitope of a molecule involved in the regulation of an immune checkpoint presented on the surface of an immune cell. The present invention also pertains to methods of using such PD-1 binding molecules to detect PD-1 or stimulate an immune response. The present invention also pertains to methods of combination therapy in which a PD1 binding molecule comprising one or more PD-1 binding domains of such selected anti-PD1 antibodies is administered in combination with one or more additional molecules that They are effective in stimulating an immune response and/or in combination with one or more additional molecules that specifically bind to an anti-cancer antigen.

[0017] In detail, the invention provides an anti-human PD-1 binding molecule comprising the three Heavy Chain CDR Domains, CDRhI, CDRh2, and CDRh3 and the three Light Chain CDR domains, CDRlI, CDRl2, and CDRl3 , where:

(A) (1) The CDRrI Domain, CDRr2 Domain, and CDRh3 Domain are the Heavy Chain CDRs of PD-1 mAb 1, and respectively have the amino acid sequences: SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73; and (2) the CDRlI Domain, CDRl2 Domain, and CDRl3 Domain are the Light Chain CDRs of PD-1 mAb 1, and respectively have the amino acid sequences: SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78;

or (B) (1) The CDRrI Domain, CDRr2 Domain, and CDRr3 Domain are the Heavy Chain CDRs of PD-1 mAb 2, and respectively have the amino acid sequences: SEQ ID NO:85, SEQ ID NO:86 , and SEQ ID NO:87; and (2) the CDRlI Domain, CDRl2 Domain, and CDRl3 Domain cnan Ln/zznz/E/YiAi cnan Ln/zznz/E/YiAi are the Light Chain CDRs of PD-1 mAb 2, and respectively have the sequences of amino acids: SEQ ID NO:90, SEQ ID NO:91, and SEQ ID NO:92; or (C) (1) the CDRhI Domain, CDRr2 Domain, and

CDRh3 are the Heavy Chain CDRs of PD-1 mAb 3, and respectively have the amino acid sequences: SEQ ID NO:99, SEQ ID NO:100, and SEQ ID NO:101; and (2) the CDRlI Domain, CDRl2 Domain, and CDRl3 Domain are the Light Chain CDRs of PD-1 mAb 3, and respectively have the amino acid sequences: SEQ ID NO:104, SEQ ID NO:105, and SEQ ID NO:106;

or (D) (1) the CDRrI Domain, CDRr2 Domain, and

CDRh3 are the Heavy Chain CDRs of PD-1 mAb 4, and respectively have the amino acid sequences: SEQ ID NO:109, SEQ ID NO:110, and SEQ ID NO:111; and (2) the CDRlI Domain, CDRl2 Domain, and CDRl3 Domain are the Light Chain CDRs of PD-1 mAb 4, and respectively have the amino acid sequences: SEQ ID NO:114, SEQ ID NO:115, and SEQ ID NO:116;

or (E) (1) the CDRrI Domain, CDRr2 Domain, and CDRrI Domain

CDRh3 are the Heavy Chain CDRs of PD-1 mAb 5, and respectively have the amino acid sequences: SEQ ID NO:119, SEQ ID NO:120, and SEQ ID NO:121; and cnan Ln/zznz/E/YiAi (2) the CDRlI Domain, CDRl2 Domain, and CDRl3 Domain are the Light Chain CDRs of PD-1 mAb 5, and respectively have the amino acid sequences: SEQ ID NO:124, SEQ ID NO:125, and SEQ ID NO:126; or (F) (1) the CDRrI Domain, CDRh2 Domain, and CDRh2 Domain

CDRr3 are the Heavy Chain CDRs of PD-1 mAb 6, and respectively have the amino acid sequences: SEQ ID NO:129, SEQ ID NO:130, and SEQ ID NO:131; and (2) the CDRlI Domain, CDRl2 Domain, and CDRl3 Domain are the Light Chain CDRs of PD-1 mAb 6, and respectively have the amino acid sequences: SEQ ID NO:134, SEQ ID NO:135, and SEQ ID NO:136; or (G) (1) The CDRrI Domain, CDRr2 Domain, and

CDRr3 are the Heavy Chain CDRs of PD-1 mAb 7, and respectively have the amino acid sequences: SEQ ID NO:139, SEQ ID NO:140, and SEQ ID NO:141; and (2) the CDR Domain_l1, CDR Domain_l2, and CDR Domain_l3 They are the Light Chain CDRs of mAb 7 of PD-1, and respectively they have the amino acid sequences: SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; or (Η) (1) The CDRrI Domain, CDRr2 Domain, and

CDRr3 are the Heavy Chain CDRs of PD-1 mAb 8, and cnan Ln/zznz/E/YiAi respectively have the amino acid sequences: SEQ ID NO:161, SEQ ID NO:162, and SEQ ID NO:163; and (2) the CDRlI Domain, CDRl2 Domain, and CDRl3 Domain are the Light Chain CDRs of PD-1 mAb 8, and respectively have the amino acid sequences: SEQ ID NO:166, SEQ ID NO:167, and SEQ ID NO:168; or (I) (1) The CDRhI Domain, CDRh2 Domain, and Domain

CDRh3 are the Heavy Chain CDRs of PD-1 mAb 9, and respectively have the amino acid sequences: SEQ ID NO:171, SEQ ID NO:172, and SEQ ID NO:173; and (2) the CDRlI Domain, CDRl2 Domain, and CDRl3 Domain are the Light Chain CDRs of PD-1 mAb 9, and respectively have the amino acid sequences: SEQ ID NO:176, SEQ ID NO:177, and SEQ ID NO:178; or (J) (1) Domain CDRhI, Domain CDRh2, and Domain

CDRh3 are the Heavy Chain CDRs of PD-1 mAb 10, and respectively have the amino acid sequences: SEQ ID NO:192, SEQ ID NO:193, and SEQ ID NO:194; and (2) the CDRlI Domain, CDRl2 Domain, and CDRl3 Domain are the Light Chain CDRs of PD-1 mAb 10, and respectively have the amino acid sequences: SEQ ID NO:197, SEQ ID NO:198, and SEQ ID NO:199;

cnan ίη/ζζηζ/Ε/γίΛΐ (Κ) (1) Domain CDRhI, Domain CDRh2, and Domain

CDRh3 are the Heavy Chain CDRs of PD-1 mAb 11, and respectively have the amino acid sequences: SEQ ID NO:202, SEQ ID NO:203, and SEQ ID NO:204; and (2) the CDRlI Domain, CDRl2 Domain, and CDRl3 Domain are the Light Chain CDRs of PD-1 mAb 11, and respectively have the amino acid sequences: SEQ ID NO:207, SEQ ID NO:208, and SEQ ID NO:209;

or (L) (1) Domain CDRhI, Domain CDRh2, and Domain

CDRh3 are the Heavy Chain CDRs of PD-1 mAb 12, and respectively have the amino acid sequences: SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:214; and (2) the CDRlI Domain, CDRl2 Domain, and CDRl3 Domain are the Light Chain CDRs of PD-1 mAb 12, and respectively have the amino acid sequences: SEQ ID NO:217, SEQ ID NO:218, and SEQ ID NO:219;

or (Μ) (1) The CDR Domain_h1, CDR Domain_h2, and Domain

CDRh3 are the Heavy Chain CDRs of PD-1 mAb 13, and respectively have the amino acid sequences: SEQ ID NO:222, SEQ ID NO:223, and SEQ ID NO:224; and (2) the CDRlI Domain, CDRl2 Domain, and CDRl3 Domain are the Light Chain CDRs of PD-1 mAb 13, and respectively have the amino acid sequences: SEQ ID

NO:227, SEQ ID NO:228, and SEQ ID NO:229; or (N) (1) The CDRhI Domain, CDRr2 Domain, and

CDRh3 are the Heavy Chain CDRs of PD-1 mAb 14, and respectively have the amino acid sequences: SEQ ID NO:232, SEQ ID NO:233, and SEQ ID NO:234; and (2) the CDRlI Domain, CDRl2 Domain, and CDRl3 Domain are the Light Chain CDRs of PD-1 mAb 14, and respectively have the amino acid sequences: SEQ ID NO:237, SEQ ID NO:238, and SEQ ID NO:239; or (O) (1) The CDRrI Domain, CDRr2 Domain, and Domain

CDRh3 are the Heavy Chain CDRs of PD-1 mAb 15, and respectively have the amino acid sequences: SEQ ID NO:242, SEQ ID NO:243, and SEQ ID NO:244; and (2) the CDRlI Domain, CDRl2 Domain, and CDRl3 Domain are the Light Chain CDRs of PD-1 mAb 15, and respectively have the amino acid sequences: SEQ ID NO:247, SEQ ID NO:248, and SEQ ID NO:249; or (P) (1) The CDRrI Domain, CDRr2 Domain, and

CDRr3 are the Heavy Chain CDRs of mAb 7 (1.2) of hPD-1, and respectively have the amino acid sequences: SEQ ID NO:139, SEQ ID NO:140, and SEQ ID NO:141; and (2) The CDRlI Domain, CDRl2 Domain, and CDRl3 Domain

CbQb ίη/ΖΖΩΖ/Ε/ΥΙΛΙ cnan Ln/zznz/E/YiAi are the Light Chain CDRs of mAb 7(1.2) of hPD-1, and respectively have the amino acid sequences: SEQ ID NO:157, SEQ ID NO :145, and SEQ ID NO:146;

or (Q) (1) The CDRhI Domain, CDRh2 Domain, and CDRh3 Domain are the Heavy Chain CDRs of hPD-1 mAb 7(1.3), and respectively have the amino acid sequences: SEQ ID NO:139, SEQ ID NO:140, and SEQ ID NO:141; and (2) The CDRlI Domain, CDRl2 Domain, and CDRl3 Domain are the Light Chain CDRs of hPD-1 mAb 7(1.3), and respectively have the amino acid sequences: SEQ ID NO:157, SEQ ID NO:158 , and SEQ ID NO:146; or (R) (1) The CDRhI Domain, CDRh2 Domain, and CDRh3 Domain are the Heavy Chain CDRs of mAb 9 (2.2) of hPD-1, and respectively have the amino acid sequences: SEQ ID NO:183, SEQ ID NO:172, and SEQ ID NO:173; and (2) The CDRlI Domain, CDRl2 Domain, and CDRl3 Domain are the Light Chain CDRs of mAb 9 (2.2) of hPD-1, and respectively have the amino acid sequences: SEQ ID NO:188, SEQ ID NO:189 , and SEQ ID NO:178.

[0018] The invention further relates to embodiments of such anti-human PD-1 binding molecules wherein the molecule is an antibody, and especially where the molecule is a chimeric antibody or a humanized antibody.

[0019] The invention further relates to embodiments of such anti-human PD-1 binding molecules wherein the Heavy Chain Variable Domain has the amino acid sequence SEQ ID NO:79, SEQ ID NO:93, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:179, SEQ ID NO:181, or SEQ ID NO:250.

[0020] The invention further relates to embodiments of such anti-human PD-1 binding molecules wherein the Light Chain Variable Domain has the amino acid sequence of SEQ ID NO:81, SEQ ID NO:95, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:184, SEQ ID NO:186, or SEQ ID NO:251.

[0021] The invention further relates to embodiments wherein the anti-human PD-1 binding molecule is a bispecific binding molecule, capable of simultaneously binding to human PD-1 and a second epitope, and relates particularly to the embodiment wherein the second epitope is an epitope of a molecule involved in the regulation of an immune checkpoint present on the surface of an immune cell (especially where the second epitope is an epitope of B7-H3, B7-H4 , BTLA, CD40, CD40L, CD47, CD70, CD80, CD86, CD94, CD137, CD137L, CD226, CTLA-4, Galectin-9, GITR, GITRL, HHLA2, ICOS, ICOSL, KIR, LAG-3, LIGHT, MHC class I or II, NKG2a, NKG2d, 0X40, OX40L, PD1H, PD-1, PD-L1, PD-L2, PVR, SIRPa, TCR, TIGIT, TIM-3 or VISTA, and more particularly where the second epitope is a epitope of CD137, CTLA-4,

CbQb ίη/ΖΖΩΖ/Ε/ΥΙΛΙ cnan Ln/zznz/E/YiAi

LAG-3, 0X40, TIGIT, or TIM-3).

[0022] The invention further relates to embodiments wherein the anti-human PD-1 binding molecule is a bispecific molecule comprising a LAG-3 epitope binding site particularly wherein the LAG-3 epitope binding site 3 includes:

(A) (1) the CDRhI Domain, CDRh2 Domain, and CDRh3 Domain of the Heavy Chain Variable Regions of mAb 1 of LAG-3, which have the amino acid sequences: SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44 respectively; and (2) the CDRlI Domain, CDRl2 Domain, and CDRl3 Domain of the Light Chain Variable Regions of mAb 1 of LAG-3, which have the amino acid sequences: SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48, respectively;

or (B) (1) the CDRhI Domain, CDRr2 Domain, and CDRh3 Domain of the VH1 Heavy Chain Variable Regions of hLAG-3 mAb 1, having the amino acid sequences: SEQ ID NO:42, SEQ ID NO :43, and SEQ ID NO:44; respectively; AND (2) the CDRlI Domain, CDRl2 Domain, and CDRl3 Domain of the VL4 Light Chain Variable Regions of hLAG-3 mAb 1, which have the amino acid sequences: SEQ ID NO:55, SEQ ID NO:47, and SEQ ID NO:48, respectively;

cnan Ln/zznz/E/YiAi (C) (1) Domain CDRhI, Domain CDRh2, and Domain

CDRh3 of the Heavy Chain Variable Regions of mAb 6 of LAG-3, which have the amino acid sequences: SEQ ID

NO:57, SEQ ID NO:58, and SEQ ID NO:59, respectively; and (2) the CDRlI Domain, CDRl2 Domain, and CDRl3 Domain of the VH1 Light Chain Variable Regions of LAG-3 mAb 6, which have the amino acid sequences: SEQ ID NO:61, SEQ ID NO:62, and SEQ ID NO:63, respectively;

or (D) (1) the CDRhI Domain, CDRh2 Domain, and

CDRh3 of the Heavy Chain Variable Regions of mAb 6 of hLAG-3, which have the amino acid sequences: SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59, respectively; and (2) the CDRlI Domain, CDRl2 Domain, and CDRl3 Domain of the Light Chain Variable Regions of mAb 6 of LAG-3, which have the amino acid sequences: SEQ ID NO:298, SEQ ID NO:62, and SEQ ID NO:63, respectively;

[0023] The invention further relates to embodiments of such anti-human PD-1 binding molecules wherein the molecule is a diabody, and especially, wherein the diabody is a covalently linked complex comprising two, or three, or four, or five polypeptide chains. The invention further relates to embodiments of such anti-human PD-1 binding molecules wherein the molecule is a trivalent binding molecule, and especially where the trivalent binding molecule is a covalently linked complex comprising three, four, five or more than five polypeptide chains. The invention further relates to the embodiment of such anti-human PD-1 binding molecules wherein the molecule comprises an Fe Region. The invention further relates to the embodiment of such anti-human PD-1 binding molecules wherein the molecule comprises an albumin binding domain, and especially a deimmunized albumin binding domain. [0024] The invention further relates to embodiments of all such anti-human PD-1 binding molecules where the molecule comprises an Fe Region, and where the Fe Region is a Variant Fe Region comprising one or more amino acid modifications that reduce the affinity of the Variant Fe Region for an FcyR and/or improve serum half-life, and more particularly, wherein the modifications comprise at least one amino acid substitution selected from the group consisting of:

(1) L234A; L235A;

(2) L234A and L235A;

(3) M252Y; M252Y and S254T;

(4) M252Y and T256E;

(5) M252Y, S254T and T256E; or (6) K288D and H435K;

where the numbering is that of the EU index as in Kabat.

[0025] The invention further relates to embodiments in which any of the PD-1 binding molecules described above are used to stimulate a T cell-mediated immune response. The invention further relates to embodiments in which that any of the PD-1 binding molecules described above are used in the treatment of a disease or condition associated with a suppressed immune system, especially cancer or an infection.

[0026] The invention particularly relates to the use in the treatment or diagnosis or prognosis of cancer, wherein the cancer is characterized by the presence of a cancer cell selected from the group consisting of a cell of: an adrenal gland tumor, a cancer associated with AIDS, an alveolar soft tissue sarcoma, an astrocytic tumor, bladder cancer, bone cancer, a brain and spinal cord cancer, a metastatic brain tumor, a breast cancer, tumors carotid body, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, benign cutaneous fibrous histiocytoma, a desmoplastic small round cell tumor, an ependymoma, an Ewing tumor, an extraskeletal myxoid chondrosarcoma, a cnan Ln/zznz/E/YiAi bone fibrogenesis imperfecta, a fibrous dysplasia of bone, a gallbladder or bile duct cancer, gastric cancer , gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, leukemia, a lipoma/benign lipomatous tumor, a liposarcoma/malignant lipomatous tumor, a liver cancer, a lymphoma, a lung cancer, a medulloblastoma, a melanoma, a meningioma, a multiple endocrine neoplasia, a multiple myeloma, a myelodysplastic syndrome, a neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, a pituitary tumor, a prostate cancer, a posterior uveal melanoma, a rare hematological disorder, a metastatic renal cancer, a rhabdoid tumor, a rhabdomyosarcoma, a sarcoma, a skin cancer, a soft tissue sarcoma, a squamous cell cancer , stomach cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic thyroid cancer, and uterine cancer.

[0027] The invention particularly relates to the use in the treatment or diagnosis or prognosis of cancer, where the cancer is colorectal cancer, hepatocellular carcinoma, glioma, kidney cancer, breast cancer, multiple myeloma, cnan Ln/zznz/E /YiAi bladder cancer, neuroblastoma; sarcoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, rectal cancer, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), B-acute lymphoblastic leukemia (B-ALL), leukemia chronic lymphocytic lymphoma (CLL), hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin lymphomas (NHL), including mantle cell leukemia (MLC), and small lymphocytic lymphoma (SLL), Hodgkin's lymphoma, Systemic mastocytosis, or Burkitt lymphoma.

[0028] The invention further relates to embodiments in which any of the PD-1 binding molecules described above are detectably labeled and used in the detection of PD-1.

cnan Ln/zznz/E/YiAi

Brief Description of the Drawings

[0029] Figure 1 provides a schematic of a representative covalently linked diabody having two epitope binding sites composed of two polypeptide chains, each with an E-helix or K-helix Heterodimer Promoting Domain. A cysteine ​​residue can occur in a ligand and/or in the Heterodimer Promoting Domain as shown in Figure 3B. VL and VH Domains that recognize the same epitope are shown using the same shading or fill pattern.

[0030] Figure 2 provides a schematic of a representative covalently linked diabody molecule having two epitope binding sites composed of two polypeptide chains, each with a CH2 and CH3 Domain, such that the associated chains form all or part of an Fe Region. VL and VH Domains that recognize the same epitope are shown using the same shading or filling pattern.

[0031] Figures 3A-3C provide schemes showing representative tetravalent diabodies having four epitope binding sites composed of two pairs of polypeptide chains (i.e., four polypeptide chains in total). One polypeptide of each pair possesses a CH2 and CH3 Domain, so that the associated chains form all or part of an Fe Region. VL and VH Domains that recognize the same epitope are shown using the same shading or filling pattern. The two pairs of polypeptide chains may be the same. In such embodiments where the VL and VH Domains recognize different epitopes (as shown in Figures 3A-3C), the resulting molecule possesses four epitope binding sites and is bispecific and bivalent with respect to each bound epitope. In such embodiments where the VL and VH Domains recognize the same epitope (for example, the same VL Domain CDR and the same VH Domain CDR are used on both chains), the resulting molecule possesses four epitope binding sites and cnan Ln/zznz/E/YiAi cnan Ln/zznz/E/YiAi is monospecific and tetravalent with respect to a single epitope. Alternatively, the two pairs of polypeptides may be different. In such embodiments where the VL and VH Domains of each pair of polypeptides recognize different epitopes (as shown in Figures 3A-3C), the resulting molecule possesses four epitope binding sites and is tetraspecific and monovalent with respect to each epitope. United. Figure 3A shows an Fe diabody containing a peptide Heterodimer Promoting Domain comprising a cysteine ​​residue. Figure 3B shows a diabody containing an Fe Region, containing E-helix and K-helix Heterodimer Promoting Domains comprising a cysteine ​​residue and a ligand (with an optional cysteine ​​residue). Figure 3C shows a diabody containing an Fe Region, which contains CH1 and CL antibody domains.

[0032] Figures 4A and 4B provide schematics of a representative covalently linked antibody molecule having two epitope binding sites composed of three polypeptide chains. Two of the polypeptide chains possess a CH2 and CH3 Domain, such that the associated chains form all or part of an Fe Region. The polypeptide chains comprising the VL and VH domains further comprise a Heterodimer Promoting Domain. Domains VL and VH that recognize the same cnan Ln/zznz/E/YiAi epitope are shown using the same hatching or filling pattern.

[0033] Figure 5 provides schematics of a representative covalently linked antibody molecule having two epitope binding sites composed of three polypeptide chains. Two of the polypeptide chains possess a CH2 and CH3 Domain, so that the associated chains form an Fe Region comprising all or part of an Fe Region. The polypeptide chains comprising the linked VL and VH Domains further comprise a Domain Promoting Heterodimer. VL and VH Domains that recognize the same epitope are shown using the same shading or fill pattern.

[0034] Figures 6A-6F provide schemes of trivalent binding molecules containing representative Fe Regions which contain three epitope binding sites. Figures 6A and 6B, respectively, schematically illustrate the domains of trivalent binding molecules comprising two diabody binding domains to one Fab binding domain which have different domain orientations in which the diabody binding domains are N -terminal or C-terminal to an Fe Region. The molecules in Figures 6A and 6B comprise four chains. Figures 6C and 6D, respectively, schematically illustrate the trivalent binding molecule domains comprising two N-terminal diabody binding domains, and a Fab binding domain in which the light chain and heavy chain are linked by a spacer polypeptide, or a scFv-like binding domain. The trivalent binding molecules in Figures 6E and 6F respectively schematically illustrate the domains of trivalent binding molecules comprising two C-terminal diabody-like binding domains for an Fe Region, and an attached Fab-like binding domain, or a binding domain. scFv type in which the diabody-like binding domains are. The trivalent linker molecules in Figures 6C-6F comprise three chains. VL and VH Domains that recognize the same epitope are shown using the same shading or fill pattern.

[0035] Figures 7A-7D show that PD-1 anti-PD-1 mAb 1-15 antibodies bind to human PD-1. Binding curves for binding to shPD-l-His are shown in Figure 7A (PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 4, and PD1 mAb 9), Figure 7B ( PD-1 mAb 5, PD-1 mAb 6, and PD-1 mAb 7), and Figure 7C (PD-1 mAb 3, PD-1 mAb 8, PD-1 mAb 10, PD-1 mAb 11 of PD-1, mAb 12 of PD-1, mAb 13 of PD-1, mAb 14 of PD-1, and mAb 15 of PD-1). The binding curves for binding to human Fe shPD-1 are shown in Figure 7D (PD-1 mAb 3, PD-1 mAb 8, PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12 PD-1, PD-1 mAb 13, PD-1 mAb 14, and PDcnan mAb 15 Ln/zznz/E/YiAi

1) ·

[0036] Figures 8A-8C show that anti-PD-1 PD-1 mAb 1-15 antibodies bind to cynomolgus monkey PD-1. The binding curves for binding to scynoPD-1 are shown in Figure 8A (PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 4 and PD-1 mAb 5, PD-1 mAb 6 1, PD-1 mAb 7), Figure 8B (PD-1 mAb 9, and Figure 8C (PD-1 mAb 3, PD-1 mAb 8, PD-1 mAb 10, PD-1 mAb 11 PD-1, PD-1 mAb 12, PD-1 mAb 13, PD-1 mAb 14, and PD-1 mAb 15).

[0037] Figures 9A-9D show the ability of anti-PD-1 PD-1 mAb 1-15 antibodies to prevent the binding of human PD-L1 to human PD-1. The inhibition curves are shown in Figure 9A (PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 3, PD-1 mAb 15, and PD-1 mAb A), Figure 9B (PD-1 mAb 4), Figure 9C (PD-1 mAb 5, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb A), and Figure 9D (PD-1 mAb 3 of PD-1, PD-1 mAb 8, PD-1 mAb 10, PD-1 mAb 11, PD1 mAb 12, PD-1 mAb 13, PD-1 mAb 14, PD-1 mAb 1, and PD-1 mAb A).

[0038] Figures 10A-10B show the tissue specificity of the PD-1 anti-human PD-1 mAb 7 antibody. Figure 10A shows histological stains of a normal colon tissue (Panels i and vii), liver (Panels ii and viii), lung (Panels iii and ix), pancreas (Panels iv and x), kidney (Panels and xi) and from the heart (Panels vi and xii). cnan Ln/zznz/E/YiAi

Figure 1OA, Panels i-vi show results from tissue incubated with PD-1 labeled mAb 7 (0.313 pg/mL). Figure 10A, Panels i-vi show results from tissue incubated with labeled isotype control mAb (0.313 pg/mL). Figure 10B shows histological stains of skin (Panels i and iv), tonsils (Panels ii and v), and NSO cells expressing PD-1 (Panels iii and vi). Figure 10A, Panels i-vi show the results of tissue incubated with PD-1 labeled mAb 7 (0.313 pg/mL).

[0039] Figure 11 shows the binding profiles of humanized anti-human PD-1 antibodies hPD-1 mAb 2, hPD-1 mAb 2 7(1.1), hPD-1 mAb 7(1.2), mAb 9 (1.1) of hPD-1, and the reference anti-PD-1 antibodies PD-1 mAb A and PD-1 mAb B that have IgGl (AA) or IgG4 (P) to bind to PD-1 cell surface.

[0040] Figures 12A-12B show the capacity of humanized anti-human PD-1 antibodies hPD-1 mAb 2, hPD-1 mAb 7(1.1), hPD-1 mAb 7(1.2), mAb 9 ( 1.1) of hPD-1, and the reference anti-PD-1 antibodies PD-1 mAb A and PD-1 mAb B, which have IgGl (AA) or IgG4 (P) to block PD-L1 binding soluble human Figure 12A) and PD-L2 (Figure 12B) to cell surface PD-1.

[0041] Figure 13 shows the capacity of humanized anti-PD antibodies hPD-1 mAb 2, hPD-1 mAb 7(1.1), hPD-1 mAb 7(1.2), hPD-1 mAb 9 (1.1) 1, and the reference cnan Ln/zznz/E/YiAi cnan Ln/zznz/E/YiAi anti-PD-1 antibodies PD-1 mAb A and PD-1 mAb B, which have IgGl (AA) or IgG4 (P) to antagonize the PD-l/PDL1 axis by blocking the PD-1/PD-L1 interaction and preventing downregulation of T cell responses in a Jurkat-luc-NFAT/CHO-PD- luciferase reporter assay Lll.

[0042] Figure 14 shows that PD-1 mAb 2, PD-1 mAb 7, PD-1 mAb 9 and PD-1 mAb 15 are capable of stimulating cytokine production at levels comparable to or greater than those reference anti-PD-1 antibodies (PD-1 mAb A and PD-1 mAb B) and that treatment with PD-1 mAb 2, PD-1 mAb 7, PD-1 mAb 9 and PD-1 mAb PD-1 15 in combination with LAG-3 mAb 1 provided the greatest enhancement of cytokine release. The IFNy secretion profiles of Staphylococcal enterotoxin B (SEB)-stimulated PBMC treated with antiPD-1 and anti-LAG-3 antibodies alone and in combination.

[0043] Figures 15A-15B show the capacity of humanized anti-human PD-1 antibodies mAb 2 of hPD-1, mAb 7 (1.2) of hPD-1, mAb 9 (1.1) of hPD-1, and the antibodies reference anti-PD-1 PD-1 mAb A and PD-1 mAb B, which have IgGl (AA) or IgG4 (P) to stimulate cytokine production. The secretion profiles of IFNy (Figure 15A) and TNFα (Figure 15B), of PBMC stimulated with SEB treated with anti-PD-1 antibodies.

[0044] Figures 16A-16B show that the PD-1 x LAG-3 bispecific diabody constructs DART A, DART D, DART

E, DART F, DART G and DART H, are capable of stimulating cytokine production at levels comparable to or greater than those observed after administration of the combination of an anti-PD-1 mAb + an anti-LAG-3 mAb ( mAb from PD-1 A + mAb A from LAG-3), and that the PD-1 X LAG-3 bispecific diabody constructs DART A, DART D, DART E, DART F and DART G provided the greatest release enhancement of cytokine. The IFNy secretion profiles of PBMC stimulated with a low concentration of SEB (0.2 ng/mL treated with PD-1 x LAG-3 bispecific diabodies, or anti-PD-1 and anti-LAG-3 antibodies alone and in combination. The results using PBMCs from two representative donors are shown in Figure 16A and Figure 16B.

[0045] Figures 17A-17B show that the PD-1 x LAG-3 bispecific diabody constructs DART A, DART B and DART C are capable of stimulating cytokine production at levels greater than those observed after administration of the combination of an anti-PD-1 mAb + an anti-LAG-3 mAb (PD-1 mAb A + LAG-3 mAb A). The IFNy secretion profiles of PBMCs from two representative donors, stimulated with a high concentration of SEB (85 ng/mL) treated with bispecific PD-1 x LAG-3 diabodies, or anti-PD-1 and anti-PD-1 antibodies, are schematized. -LAG-3 alone and in combination. Results using PBMCs from two representative donors are shown in Figure 17A and cnan Ln/zznz/E/YiAi

Figure 17B.

[0046] Figures 18A-18B show that the PD-1 x LAG-3 bispecific diabody constructs DART A, DART B and DART C are capable of stimulating cytokine production at levels greater than those observed after administration of the combination of an anti-PD-1 mAb + an anti-LAG-3 mAb (mAb A PD-1 + mAb A LAG-3). The IFNy secretion profiles of the PBMCs of two representative donors are schematized, stimulated with a medium concentration of SEB (0.5 ng/mL) treated with bispecific PD-1 x LAG-3 diabodies, or anti-PD-1 and anti-PD-1 antibodies. -LAG-3 alone and in combination. The results using PBMCs from two representative donors are shown in Figure 18A and Figure 18B.

[0047] Figure 19 shows that the PD-1 x LAG-3 bispecific diabody constructs DART D and DART H are capable of stimulating cytokine production at levels comparable to or greater than those observed after administration of the combination of a mAb anti-PD-1 + an anti-LAG-3 mAb (PD-1 mAb A + LAG-3 mAb A) and that DART D provided the greatest enhancement of cytokine release. The IL2 secretion profiles of PBMCs from two representative donors, stimulated with a high concentration of SEB (85 ng/mL) treated with bispecific PD-1 x LAG-3 diabodies, or anti-PD-1 and anti-cann antibodies, are schematized. Ln/zznz/E/YiAi cnan Ln/zznz/E/YiAi

LAG-3 alone and in combination.

[0048] Figure 20 shows that the PD-1 x LAG-3 bispecific diabody constructs DART B and DART I are capable of stimulating cytokine production at levels comparable or greater than those observed after administration of the combination of a mAb anti-PD-1 + an anti-LAG-3 mAb (PD-1 mAb A + LAG-3 mAb A, hPD-1 mAb 7(1.2) + hLAG-3 mAb 1(1.4), mAb 7 (1.2) from hPD-1 + mAb 6(1.1) from hLAG-3). The IFNy secretion profiles of the PBMCs of two representative donors are schematized, stimulated with a medium concentration of SEB (0.5 ng/mL) treated with bispecific diabodies of PD-1 x LAG-3, or anti-PD-1 antibodies and anti-LAG-3 alone and in combination.

[0049] Figures 21A-21D show that the bispecific PD-1 x LAG-3 DART 1 diabody is capable of stimulating cytokine production at levels higher than those observed after administration of the combination of an anti-PD-1 mAb + an anti-LAG-3 mAb (PD-1 mAb + LAG-3 mAb A). The secretion profiles of IFNy (Figures 21A and 21C) and IL-2 (Figures 21B and 21D) of CD4 memory cells from two representative donors, stimulated with tetanus toxoid (5 (pg/mL) treated with the diabody) are schematized. bispecific PD-1 x LAG-3 DART-I, anti-PD-1 and anti-LAG-3 antibodies in combination, or an isotype control. Results at day 7 using CD4 memory T cells from two representative donors are shown in Figures 21A-B and Figures 21C-D.

[0050] Figure 22 shows that the pharmacokinetics of the PD-1 x LAG 3 bispecific molecule, DART I are comparable to those of the anti-PD-1 antibody, IgG4 of PD-1 mAb A (P) in cynomolgus monkey . Lines indicate mean serum concentration of DART I (solid) and PD-1 mAb A (dashed line). Individual values ​​for the male (filled) and female (open) monkey are plotted for DART I (triangles) and PDS-1 mAb A (circles).

[0051] Figures 23A-23C show serum antibody concentrations and percentage of PD-1 binding on the surface of CD4+ or CD8+ T cells over time in animals after treatment with different anti-PD-1 antibodies. The percentage of PD 1 binding on the surface of CD4+ or CD8+ T cells after anti-PD 1 mAb treatment is marked on the right y-axes; symbols represent % PD 1 binding in T cells for each animal individual and dashed lines represent mean values. Serological mAb concentrations are plotted on the left y-axes; symbols represent serological levels for each animal individual and solid lines represent non-linear entries of the data. Each panel represents data for animals (n=1/sex/group) administered 10 mg/kg IgG4 (P) hPD-1 mAb 7(1.2) (Figure

CbQb ίη/ΖΖΩΖ/Ε/ΥΙΛΙ

23Α), IgG4 (P) PD-1 mAb A (Figure 23B), or PD-1 IgG4 (P) mAb B (Figure 23B) by IV Infusion on Day 1.

cnan Ln/zznz/E/YiAi

Detailed description of the invention:

[0052] The present invention is directed to PD-1 binding molecules comprising the PD-1 binding domain of selected anti-PD-1 antibodies capable of binding both cynomolgus monkey PD-1 and PD-1. from human: PD-1 mAb 1; PD-1 mAb 2; PD-1 mAb 3; PD-1 mAb 4; PD-1 mAb 4; PD-1 mAb 5; PD-1 mAb 6; PD-1 mAb 7; PD-1 mAb 7; PD-1 mAb 8; PD-1 mAb 9; PD-1 mAb 10; PD-1 mAb 11; PD-1 mAb 12; PD-1 mAb 13; PD-1 mAb 14, or PD-1 mAb 15. The invention relates particularly to PD-1 binding molecules that are humanized or chimeric versions of such antibodies, or that comprise PD1 binding fragments of such anti-PD-1 antibodies (especially immunoconjugates, diabodies (including but not limited to DART-A, DART-B, DART-C, DART-D, DART-E, DART-F, DART-G, DARTH, DART-I, and DART-J), BiTEs, bispecific antibodies, etc.). The invention particularly relates to such PD-1 binding molecules that are additionally capable of binding to an epitope of a molecule involved in the regulation of an immune checkpoint presented on the surface of an immune cell. The present invention also pertains to methods of using such PD-1 binding molecules to detect PD-1 or stimulate an immune response. The present invention also pertains to methods of combination therapy in which a PD1 binding molecule comprising one or more PD-1 binding domains of such selected anti-PD1 antibodies is administered in combination with one or more additional molecules that They are effective in stimulating an immune response and/or in combination with one or more additional molecules that specifically bind to an anti-cancer antigen.

cnan Ln/zznz/E/YiAi

I. Antibodies and Their Binding Domains

[0053] The antibodies of the present invention are immunoglobulin molecules capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located on the Variable domain of the immunoglobulin molecule. As used herein, the terms antibody and antibodies refer to monoclonal antibodies, multispecies antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, camelized antibodies, single chain Fvs (scFv), single chain antibodies. chain, Fab fragments, F(ab') fragments, disulfide-linked bispecific Fvs (sdFv), intrabodies and epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, that is, molecules containing an antigen binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGi, IgG₂, IgGs, IgG4, IgAi and IgA₂) or subclass. In addition to their known diagnostic uses, antibodies have been shown to be useful as therapeutic agents. Antibodies are capable of binding immunospecifically to a polypeptide or protein or a non-protein molecule due to the presence in such molecule of a particular domain or portion or conformation (an epitope). A molecule containing an epitope may have immunogenic activity, such that it provokes an antibody production response in an animal; Such molecules are called antigens). Recent decades have seen a resurgence of interest in the therapeutic potential of antibodies and antibodies have become one of the leading classes of biotechnologically derived medicines (Chan, CE et al. (2009) The Use Of Antibodies In The Treatment Of Infectious Diseases, Singapore Med. J. 50 (7):663-666). More than 200 antibody-based drugs have been approved for use or are in development.

[0054] The term monoclonal antibody refers to a homogeneous population of antibody wherein the monoclonal antibody is composed of amino acids (naturally or unnaturally) that are involved in selective binding to an antigen. Monoclonal antibodies are highly specific, directing against a single epitope (or antigenic site). The term monoclonal antibody encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2 Fv), single-chain (scFv), mutants thereof. themselves, fusion proteins comprising an antibody moiety, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule comprising an antigen recognition site of the required specificity and capacity and the ability to bind to an antigen. It is not intended to be limited with reference to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes all immunoglobulins as well as the fragments, etc. , described above under the definition of antibody. Methods for creating monoclonal antibodies are known in the art. One method that could be used is the method of Kohler, G. et al. (1975) Continuous Cultures Of Fused Cells cnan Ln/zznz/E/YiAi

Secreting Antibody Of Predefinad Specificity Of Nature 256:495-497 or a modification thereof. Typically, monoclonal antibodies are developed in mice, rats or rabbits. Antibodies are produced by immunization of an animal with an immunogenic amount of cells, cell extracts, or protein preparations containing the desired epitope. The immunogen may be, but is not limited to, primary cells, cultured cell lines, cancer cells, proteins, peptides, nucleic acids or tissues. The cells used for immunization can be cultured for a period of time (for example, at least 24 hours) before use as an immunogen. The cells can be used as immunogens by themselves or in combination with a non-denaturing adjuvant, such as Ribi (see, for example, Jennings, VM (1995) Review of Selected Adjuvants Used in Antibody Production, ILAR J. 37(3):119125). In general, cells should be kept intact and preferably viable when used as immunogens. Intact cells may allow antigens to be better detected than cells disrupted by the immunized animal. The use of denaturing or aggressive adjuvants, for example, Freud's adjuvant, can disrupt cells and is therefore discouraged. The immunogen may be administered on multiple occasions at periodic intervals such as twice a week or weekly, or Ln/zznz/E/YiAi may be administered in such a manner as to maintain viability in the animal (e.g., in a recombinant tissue). Alternatively, existing monoclonal antibodies and any other equivalent antibodies that are immunospecific for a desired pathogenic epitope can be sequenced and produced recombinantly by any means known in the art. In one embodiment, such an antibody is subjected to sequencing and the polynucleotide sequence found is then cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector in a host cell and the host cell can then be expanded and frozen for future use. The polynucleotide sequence of the antibodies can be used for genetic manipulation to generate the monospecific or multispecific (e.g., bispecific, trispecific, or tetraspecific) molecules of the invention as well as an optimized affinity, a chimeric antibody, a humanized antibody, and/or a caninized antibody, to improve affinity, or other characteristics of the antibody. The general principle in humanizing an antibody involves retaining the basic sequence of the antigen-binding portion of the antibody, while exchanging the non-human remnant of the antibody for human antibody sequences.

[0055] Natural antibodies (such as antibodies

CbQb ίη/ΖΖΩΖ/Ε/ΥΙΛΙ

IgG) are composed of two Light Chains in complex with two Heavy Chains. Each light chain contains a Variable Domain (VL) and a Constant Domain (CL). Each heavy chain contains a Variable Domain (VH), three Constant Domains (CH1, CH2, and CH3), and a Hinge Domain located between the CH1 and CH2 domains. The basic structural unit of naturally occurring immunoglobulins (e.g., IgG) is thus a tetramer having two light chains and two heavy chains, usually expressed as a glycoprotein of around 150,000 Da. The amino-terminal (Nterminal) portion of each chain includes a Variable Domain of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal (C-terminal) portion of each chain defines a constant region, with light chains having a single Constant Domain and heavy chains usually having three Constant Domains and one Hinge Domain. Thus, the structure of the light chains of an IgG molecule is n-VL-CL-c and the structure of the heavy chains of IgG is n-VH-CHl-H CH2-CH3-C (where H is the hinge domain, y and c represent, respectively, the N-terminus and the C-terminus of the polypeptide). The Variable Domains of an IgG molecule consist of the complementary determining regions (CDR), which contain the residues in contact with the epitope, and the non-CDR segments, designated as segments of cnan Ln/zznz/E/YiAi structure (FR ), which generally maintain the structure and determine the placement of CDR loops to allow such contact (although certain structure residues can also contact antigen). In this way, Domains VL and VH have the structure n-FRl-CDRl-FR2-CDR2-FR3-CDR3-FR4-c. Polypeptides that are (or can serve as) the first, second, and third CDR of a Light Chain antibody are designated herein respectively as CDRlI Domain, CDRl2 Domain, and CDRl3 Domain. Similarly, polypeptides that are (or can serve as) the first, second and third CDR of a heavy chain antibody are designated herein respectively as CDRhI Domain, CDRh2 Domain and CDRh3 Domain. In this way, the terms CDRlI Domain, CDRl2 Domain, CDRl3 Domain, CDRhi Domain, CDRh2 Domain, and CDRh3 Domain refer to polypeptides that when incorporated into a protein make the protein capable of binding to a specific epitope regardless of whether such a protein is an antibody having light and heavy chains or a diabody or a single chain binding molecule (for example, a scFv, a BiTe, etc.), or is another type of protein. Accordingly, as used herein, the term "epitope-binding fragment" means a fragment of an antibody capable of immunospecifically binding to an epitope, and the term "epitope-binding site" refers to that portion of a cnan Ln/ molecule. zznz/E/YiAi comprising an epitope binding fragment that is responsible for epitope binding. An antigen binding fragment may contain 1, 2, 3, 4, 5 or all 6 of the CDR domains of such an antibody and, although capable of binding immunospecifically to such epitope, may exhibit immunospecificity, affinity or selectivity towards such epitope. which differs from that of such an antibody. Preferably, however, an epitope binding fragment will contain all 6 CDR Domains of such an antibody. An epitope-binding fragment of an antibody may be a single polypeptide chain (e.g., an scFv), or may comprise two or more polypeptide chains, each with an amino terminus and a carboxyl terminus (e.g., a diabody). , a Fab fragment, an F(ab')2 fragment, etc.).

[0056] The invention particularly encompasses light chain Variable Domain (scFv) fragments of the anti-PD-1 antibodies of this invention and multispecific binding molecules comprising the same. Light chain Variable Domain fragments are made by joining Light chain and/or Heavy chain Variable Domains using a short linker peptide. Bird et al. (1988) (Single-Chain Antigen-Binding Proteins, Science 242:423-426) describes an example of a peptide bond that binds approximately 3.5 nm between the carboxy terminus of one Variable Domain and the amino terminus of the other Variable Domain. Linkers from other cnan Ln/zznz/E/YiAi sequences have been designed and used (Bird et al. (1988) Single-Chain Antigen-Binding Proteins, Science 242:423426). The linkers can in turn be modified for additional functions, such as drug attachment or attachment to solid supports. Single chain variants can be produced either recombinantly or synthetically. For the synthetic production of scFv, an automated synthesizer can be used. For the recombinant production of scFv, a suitable plasmid with polynucleotide encoding the scFv can be introduced into a suitable host cell, whether eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic, such as E. coli. Polynucleotides encoding the scFvs of interest can be made by routine manipulations such as polynucleotide ligation. The resulting scFvs can be isolated using standard protein purification techniques known in the art.

[0057] The invention also particularly encompasses humanized variants of the anti-PD-1 antibodies of the invention and multispecific binding molecules comprising the same. The term humanized antibody refers to a chimeric molecule, generally prepared using recombinant techniques, that has an antigen-binding site from an immunoglobulin from a non-human species and a remnant immunoglobulin structure from the cnan Ln/zznz/E/YiAi molecule. which is based on the structure and/or sequence of a human immunoglobulin. Anti-human PD-1 antibodies of the present invention include humanized, chimeric or caninized variants of PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 3, PD-1 mAb 4, PD-1 mAb PD-1 5, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 8, PD-1 mAb 9, PD-1 mAb 10, PD-1 mAb 11, mAb 12 of PD-1, PD-1 mAb 13, PD-1 mAb 14, or PD-1 mAb 15. The polynucleotide sequence of the variable domains of such antibodies can be used to generate such derivatives and to improve the affinity, or other characteristics of such antibodies. The general principle in humanizing an antibody involves retaining the basic sequence of the antigen-binding portion of the antibody, while exchanging the non-human remnant of the antibody for human antibody sequences. There are four general steps to humanize a monoclonal antibody. These are: (1) determine the nucleotide and predict the amino acid sequence of the heavy and light variable domains of the initiating antibody (2) design the humanized antibody or the caninized antibody, that is, decide which region of antibody structure will be used during the humanization or canonization process (3) the current humanization or caninization methodologies/techniques and (4) the transfection and expression of the humanized antibody. See, for example, United States Patents cnan Ln/zznz/E/YiAi cnan Ln/zznz/E/YiAi Nos. 4,816,567; 5,807,715; 5,866,692; and 6,331,415.

[0058] The antigen binding site may comprise either an entire Variable Domain fused to a constant domain or only the complementarity determining regions (CDRs) of such Variable Domain grafted to suitable framework regions. The antigen binding sites can be wild type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but the possibility of an immune response to the foreign variable region remains (LoBuglio, AF et al. (1989) Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response Proc. Nati. Acad. Sci. (USA) 86:42204224). Another procedure focuses not only on providing human-derived constant regions, but on modifying the variable domains so as to reshape them as much as possible to the human form. Both the heavy and light chain variable domains are known to contain three CDR complementarity determining regions that can vary in response to the antigens in question and determine binding capacity, flanked by four framework regions (the FRs) that are relatively conserved. in a given species and that putatively provide a scaffold for CDRs. When non-human antibodies are prepared against a particular antigen, the variable domains can be reshaped or humanized by grafting the CDRs derived from a non-human antibody to the FRs present in the human antibody to be modified. Application of this approach to various antibodies has been reported by Sato, K. et al. (1993) Cancer Res 53:851- 856. Riechmann, L. et al (1988) Reshaping Human Antibodies for Therapy Nature 332:323-327; Verhoeyen, M. et al (1988) Reshaping Human Antibodies: Grafting An Antilysozyme Activity, Science 239:1534-1536; Kettleborough, CA et al (1991) Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation,” Protein Engineering 4:773-3783; Maeda, H. et al (1991) Construction Of Reshaped Human Antibodies With HIVNeutralizing Activity, Human Antibodies Hybridoma 2:124-134; Gorman, SD et al (1991) Reshaping A Therapeutic CD4 Antibody, Proc. Nati. Academic Sci. (USA) 88:4181-4185;

Tempest, PR et al (1991) Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in Vivo, Bio/Technology 9:266-271; Co, MS et al (1991) Humanized Antibodies For Antiviral Therapy, Proc.

Nati. Academic Sci. (USA) 88:2869-2873; Carter, P. et al (1992) Humanization Of An Anti-pl85her2 Antibody For Human Cancer Therapy, Proc. Nati. Academic Sci. (USA) 89:42854289; and Co, MS et al (1992) Chimeric And Humanized

CbQb ίη/ΖΖΠΖ/Ε/ΥΙΛΙ cnan Ln/zznz/E/YiAi

Antibodies With Specificity For The CD33 Antigenf J. Immunol. 148:1149-1154. In some embodiments, humanized antibodies retain all CDR sequences (for example, a humanized mouse antibody containing all 6 CDRs of mouse antibodies). In other embodiments, human antibodies having one or more CDRs (one, two, three, four, five, or six) that differ in sequence from the original antibody.

[0059] A number of humanized antibody molecules have been described that comprise an antigen binding site derived from a non-human immunoglobulin, including chimeric antibodies having rodent or modified rodent Variable Domains and their associated complementarity regions (the CDRs). ) fused to human Constant Domains (see, for example, Winter et al (1991) Man-made Antibodies Nature 349:293-299; Lobuglio et al. (1989) Mouse/Human Chimarle Monoclonal Antibody In Man: Kinetics And Immune Responso, Proc. Nati. Academic Sci. (USA) 86:4220-4224 (1989), Shaw et al (1987) Characterization Of A Mouse/Human Chimeric Monoclonal Antibody (17-1 A) To A Colon Cancer Tumor-Associated Antigen, J. Immunol. 138:4534-4538, and Brown et al (1987) Tumor-Specific Genetically Engineered Murine/Human Chimeric Monoclonal Antibody, Cancer Res. 47:3577-3583). Other references describe rodent CDRs grafted to human cnan Ln/zznz/E/YiAi supporting framework regions (FRs) prior to fusion with a human antibody Constant Domain (see, for example, Riechmann, L. et al ( 1988) Reshaping Human Antibodies for Therapy, Nature 332:323-327; Verhoeyen, M. et al (1988) Reshaping Human Antibodies: Grafting An Antilysozyme Activity, Science 239:1534-1536; and Jones et al (1986) Replacing The ComplementarityDetermining Regions In A Human Antibody With Those From A Mouse, Nature 321:522-525). Another reference describes rodent CDRs supported by recombinantly coated rodent framework regions. See, for example, European Patent Publication No. 519,596. These humanized molecules are designed to minimize unwanted immunological responses toward rodent anti-human antibody molecules, which limits the duration and effectiveness of therapeutic applications of these fractions in human recipients. Other methods to humanize antibodies that can also be used are described by Daugherty et al (1991) Polymerase Chain Reaction Facilitates The Cloning, CDR-Grafting, And Rapid Expression Of A Murine Monoclonal Antibody Directed Against The CD 18 Component Of Leukocyte Integrins, Nucí. Acids Res. 19:2471-2476 and in United States Patent Nos. 6,180,377; 6,054,297; 5,997,867; and 5,866,692.

II. Fcy receptors (FcyRs)

[0060] The CH2 and CH3 Domains of the two heavy chains interact to form the Fe Region, which is a domain that is recognized by cellular Fe Receptors, including but not limited to Fe gamma Receptors (the FcyRs). As used herein, the term Fe Region is used to define a C-terminal region of an IgG heavy chain. The amino acid sequence of the CH2-CH3 domain of an exemplary human IgGl is (SEQ ID NO:1):

CbQb ίη/ΖΖΠΖ/Ε/ΥΙΛΙ

<td>231 240 APELLGGPSV</td><td>250 FLFPPKPKDT</td><td>260 LMISRTPEVT</td><td>270 CVVVDVSHED</td><td>280 PEVKFNWYVD</td>

<td> 290</td><td> 300</td><td> 310</td><td> 320</td><td> 330</td>

<td>GVEVHNAKTK</td><td>PREEQYNSTY</td><td>RVVSVLTVLH</td><td>QDWLNGKEYK</td><td>CKVSNKALPA</td>

<td> 340</td><td> 350</td><td> 360</td><td> 370</td><td> 380</td>

<td>PIEKTISKAK</td><td>GQPREPQVYT</td><td>LPPSREEMTK</td><td>NQVSLTCLVK</td><td>GFYPSDIAVE</td>

<td> 390</td><td> 400</td><td> 410</td><td> 420</td><td> 430</td>

<td>WESNGQPENN</td><td>YKTTPPVLDS</td><td>DGSFFLYSKL</td><td>TVDKSRWQQG</td><td>NVFSCSVMHE</td>

<td> 440</td><td> 447</td><td></td><td></td><td></td>

<td>ALHNHYTQKS</td><td>LSLSPGX</td><td></td><td></td><td></td>

as numbered by the EU index according to Kabat, where, X is a Power Plant (K) or is absent.

[0061] The amino acid sequence of the CH2-CH3 domain of

<td colspan="3">An exemplary human IgG2 is (SEQ ID</td><td colspan="2">NO:2):</td>

<td>231 240 APPVA-GPSV</td><td>250 FLFPPKPKDT</td><td>260 LMISRTPEVT</td><td>270 CVVVDVSHED</td><td>280 PEVQFNWYVD</td>

<td> 290</td><td> 300</td><td> 310</td><td> 320</td><td> 330</td>

<td>GVEVHNAKTK</td><td>PREQFNSTF</td><td>RVVSVLTVVH</td><td>QDWLNGKEYK</td><td>CKVSNKGLPA</td>

<td> 340</td><td> 350</td><td> 360</td><td> 370</td><td> 38 0</td>

<td>PIEKTISKTK</td><td>GQPREPQVYT</td><td>LPPSREEMTK</td><td>NQVSLTCLVK</td><td>GFYPSDISVE</td>

cnan Ln/zznz/E/YiAi

390 400 410 420 430

WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE 440 447

ALHNHYTQKS LSLSPGX as numbered by the EU index according to Kabat, where, X is a Power Plant (K) or is absent.

[0062] The amino acid sequence of the CH2-CH3 domain of an exemplary human IgG3 is (SEQ ID NO:3):

<td> 231 240</td><td> 250</td><td> 260</td><td> 270</td><td> 280</td>

<td>APELLGGPSV</td><td>FLFPPKPKDT</td><td>LMISRTPEVT</td><td>CVVVDVSHED</td><td>PEVQFKWYVD</td>

<td> 290</td><td> 300</td><td> 310</td><td> 320</td><td> 330</td>

<td>GVEVHNAKTK</td><td>PREQYNSTF</td><td>RVVSVLTVLH</td><td>QDWLNGKEYK</td><td>CKVSNKALPA</td>

<td> 340</td><td> 350</td><td> 360</td><td> 370</td><td> 380</td>

<td>PIEKTISKTK</td><td>GQPREPQVYT</td><td>LPPSREEMTK</td><td>NQVSLTCLVK</td><td>GFYPSDIAVE</td>

<td> 390</td><td> 400</td><td> 410</td><td> 420</td><td> 430</td>

<td>WESSGQPENN</td><td>YNTTPPMLDS</td><td>DGSFFLYSKL</td><td>TVDKSRWQQG</td><td>NIFSCSVMHE</td>

<td> 440</td><td> 447</td><td></td><td></td><td></td>

<td>ALHNRFTQKS</td><td>LSLSPGX</td><td></td><td></td><td></td>

as numbered by the EU index according to Kabat, where, X is a lysine (K) or absent.

[0063] The amino acid sequence of the CH2-CH3 domain of an exemplary human IgG4 is (SEQ ID NO:4):

231 240 250 260 270280

APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD

290 300 310 320330

GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS

340 350 360 370380

SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE

CbQb ίη/ΖΖΩΖ/Ε/ΥΙΛΙ

390 400 410 420 430

WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE

440 447

ALHNHYTQKS LSLSLGX as numbered by the UE index according to Kabat, where, X is a Power Plant (K) or is absent.

[0064] Throughout the present specification, the numbering of residues in the constant region of an IgG heavy chain is that of the EU index according to Kabat et al., Sequences of Proteins of Immunological Interest 5^th Ed. Public Health Service, NH1, MD (1991) (Kabat), expressly incorporated herein by reference. The term EU index according to Kabat refers to the numbering of the human IgGl EU antibody. The amino acids of the Variable Domains of mature immunoglobulin heavy and light chains are designated by the position of an amino acid in the chain. Kabat described numerous amino acid sequences, identified as the consensus amino acid sequence for each subgroup, and assigned a residue number to each amino acid, and the CDRs are identified as defined by Kabat (CDRhl will be understood as defined by Chothia, C. &amp; Lesk, AM ((1987) Canonical structures for the hypervariable regions of immunoglobulins, J. Mol. Biol. 196:901-917) starts five residues early). The scheme of

Kabat's CbQb ίη/ΖΖΩΖ/Ε/ΥΙΛΙ numbering can be extended to antibodies not included in his compendium by aligning the antibody in question with one of the Kabat consensus sequences by reference to conserved amino acids. This method of assigning residual numbers has become standard in the field and readily identifies amino acids at equivalent positions in different antibodies, including chimeric or humanized variants. For example, an amino acid at position 50 of a human antibody light chain occupies the equivalent position of an amino acid at position 50 of a mouse antibody light chain.

[0065] Polymorphisms have been observed at a number of different positions within the constant regions of the antibody (for example, positions CH1, including but not limited to, positions 192, 193, and 214; positions Fe, including but not limited to are limited to, positions 270, 272, 312, 315, 356, and 358 as listed by the EU index as set out in Kabat), and thus, there may be few differences between the sequence presented and sequences in the art. former. Polymorphic forms of human immunoglobulins have been well characterized. Currently, 18 Gm allotypes are known: Glm (1, 2, 3, 17) or Glm (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (bl, c3, b3, bO, b3, b4, s, t, gl, c5, u, v, g5) al ., (Lefranc, et

The

Human IgG Subclasses: Molecular cnan Ln/zznz/E/YiAi

Analysis Of Structure, Function And Regulation Pergamon, Oxford, pp. 43-78 (1990); Lefranc, G. et al., 1979, Hum. Genet.: 50, 199-211). It is specifically contemplated that any allotype, isoalotype or haplotype of any immunoglobulin gene may be incorporated into the antibodies of the present invention, and is not limited to the allotype, isoalotype or haplotype of the sequences provided herein. Furthermore, in some expression systems, the C-terminal amino acid residue (in bold above) of the CH3 Domain can be removed post-translationally. Accordingly, the C-terminal residue of the CH3 domain is an optional amino acid residue in the PD-1 binding molecules of the invention. Specifically encompassed by the present invention are PD-1 binding molecules that lack the C-terminal residue of the CH3 Domain. Also specifically encompassed by the present invention are such constructs comprising the C-terminal lysine residue of the CH3 Domain.

[0066] Activating and inhibitory signals are transduced through the ligation of an Fe Region to a cellular Fe gamma Receptor (FcyR). The ability of such ligation to result in diametrically opposite functions results from structural differences between the different FcyRs. Two distinct domains within the cytoplasmic signaling domains of the receptor called tyrosine-based immunoreceptor activation motifs (the ITAMs) and tyrosine-based immunoreceptor inhibition motifs (the ITIMs) account for the different responses. The selection of different cytoplasmic enzymes in these structures dictates the outcome of FcyR-mediated cellular responses. ITAM-containing FcyR complexes include FcyRI, FcyRIIA, FcyRIIIA, while ITIM-containing complexes only include FcyRIIB. Human neutrophils express the FcyRIIA gene. The clustering of FcyRIIA by immune complexes or specific antibody cross-linking serves to aggregate the ITAMs together with receptor-associated kinases which facilitates the phosphorylation of the ITAMs. Phosphorylation of ITAMs serves as a docking site for Syk kinase, of which its activation results in the activation of downstream substrates (e.g., PI3K). Cellular activation leads to the release of proinflammatory mediators. The FcyRIIB gene is expressed in B lymphocytes; its extracellular domain is 96% identical to FcyRIIA and it binds IgG complexes in an indistinguishable manner. The presence of an ITIM in the cytoplasmic domain of FcyRIIB defines this FcyR inhibitory subclass. Recently, the molecular basis for this inhibition was established. When co-ligated together with an activating FcyR, the ITIM in FcyRIIB is phosphorylated and attracts the SH2 domain of inositol polyphosphate 5' phosphatase (SHIP), which hydrolyzes the released cnan Ln/zznz/E/YiAi phosphoinositol messengers as consequence of ITAM-containing FcyR-mediated tyrosine kinase activation, which consequently prevents Ca influx⁺⁺ intracellular. In this way, FcyRIIB cross-linking regulates the activation response to FcyR ligation and inhibits cellular sensitivity. B cell activation, B cell proliferation, and antibody secretion are thus aborted.

cnan Ln/zznz/E/YiAi

III. Bispecific Antibodies, Multispecific Diabodies and DART® Diabodies

[0067] The ability of an antibody to bind an epitope of an antigen depends on the presence and amino acid sequence of the VL and VH Domains of the antibody. The interaction of an antibody light chain and an antibody heavy chain and, in particular, the interaction of their VL and VH domains forms one of the two epitope-binding sites of a natural antibody. Natural antibodies are capable of binding to only one species of epitope (i.e., they are munospecific), although they can bind to multiple copies of that species (i.e., exhibiting bivalency or multivalency).

[0068] The binding domains of the present invention bind to epitopes in an immunospecific manner. As used herein, an antibody, diabody, or other epitope-binding molecule is said to immunospecifically bind to a region of another molecule (i.e., an epitope) if it reacts or associates more frequently, more rapidly, with longer duration and/or with greater affinity with the epitope in relation to alternative epitopes. For example, an antibody that immunospecifically binds to a viral epitope is an antibody that binds to this viral epitope with greater affinity, avidity, faster, and/or for a longer duration than if it immunospecifically bound to other viral epitopes or to non-viral epitopes. viral. It is also understood by reading this definition that, for example, an antibody (or moiety or epitope) that immunospecifically binds to a first target may or may not bind specifically or preferentially to a second target. As such, immunospecific binding does not necessarily require (although may include) exclusive binding. Generally, but not necessarily, the reference to union means specific union. Two molecules are said to be capable of binding to each other in a physiospecific manner if such binding shows the specificity with which the receptors bind to their respective ligands.

[0069] The functionality of antibodies can be improved by generating multispecific antibody-based molecules that can simultaneously bind to two separate and distinct antigens (or different epitopes of the same antigen) and/or by generating a cnan-based molecule Ln/zznz/ E/YiAi cnan Ln/zznz/E/YiAi antibody that has higher valence (i.e., more than two binding sites) for the same epitope and/or antigen.

[0070] To provide molecules that have greater capacity than natural antibodies, a wide variety of recombinant bispecific antibody formats have been developed (see, for example, PCT Publications Nos.

WO 2008/003116, WO 2009/132876, WO 2008/003103, WO 2007/146968, WO 2009/018386, WO 2012/009544, WO 2013/070565), of which the majority use linker peptides either to fuse a fragment of additional epitope binding (e.g., a scFv, VL, VH, etc.) on, or within the core of the antibody (IgA, IgD, IgE, IgG or IgM), or to fuse multiple epitope binding fragments (e.g. example, two Fab or scFv fragments). Alternative formats use linker peptides to fuse an epitope binding fragment (e.g., a scFv, VL, VH, etc.) to a dimerization domain such as the CH2-CH3 Domain or alternative polypeptides (WO 2005/070966, WO 2006 /107786A WO 2006/107617A, WO 2007/046893). Typically, such procedures involve compromises and trade-offs. For example, PCT Publications Nos. WO 2013/174873, WO 2011/133886 and WO 2010/136172 describe that the use of linkers can cause problems in therapeutic settings and teach a trispecific antibody in which the CL and CHI Domains are changed from their respective natural positions and

4 Domains VL and VH have been diversified (WO 2008/027236; WO 2010/108127) to allow them to join more than one ancient. In this way, the molecules described in these documents exchange binding specificity for the ability to bind additional antigen species. PCT Publications Nos. WO 2013/163427 and WO 2013/119903 describe modifying the CH2 Domain to contain a fusion protein adduct comprising a binding domain. The paper indicates that the CH2 Domain probably plays only a minimal role in mediating effector function. PCT Publications Nos. WO 2010/028797, WO 2010028796 and WO 2010/028795 describe recombinant antibodies whose Fe Regions have been replaced with additional VL and VH Domains, to form trivalent binding molecules. PCT Publications Nos. WO 2003/025018 and W0 2003012069 describe recombinant diabodies whose individual chains contain scFv domains. PCT Publication No. WO 2013/006544 describes multivalent Fab molecules that are synthesized as a single polypeptide chain and then undergo proteolysis to produce heterodimeric structures. In this way, the molecules described in these documents exchange all or part of the ability to mediate effector function for the ability to bind additional antigen species. PCT Publications Nos. WO 2014/022540, WO 2013/003652, WO 2012/162583, WO

CbQb ίη/ΖΖΩΖ/Ε/ΥΙΛΙ

2012/156430, WO 2011/086091, WO 2008/024188, WO 2007/024715, WO 2007/075270, WO 1998/002463, WO 1992/022583 and WO 1991/003493 describe adding additional binding domains or functional groups to an antibody or to an antibody portion (for example, adding a diabody to the antibody light chain, or adding additional VL and VH Domains to the antibody light and heavy chains, or adding a heterologous fusion protein or chaining multiple Fab Domains together) . In this way, the molecules described in these documents exchange native antibody structure for the ability to bind additional antigen species.

[0071] The art has further indicated the ability to produce diabodies that differ from such natural antibodies in that they are capable of binding two or more different epitope species (i.e., display bispecificity or multispecificity in addition to bivalence or multivalency) (see, for example, Holliger et al. (1993) 'Diabodies' Small Bivalent And Bispecific Antibody Fragmenta, Proc. Nati. Acad. Sci. (USA) 90:6444-6448; US 2004/0058400 (Hollinger et al.); US 2004/0220388 / WO 02/02781 (Mertens et al.); Alt et al. (1999) FEES Lett. 454 (1-2): 90-94; Lu, D. et al. (2005) A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor cnan Ln/zznz/E/YiAi

Activity, J. Biol. Chem. 280(20): 19665-19672; WO 02/02781 (Mertens et al.); Olafsen, T. et al. (2004) Covalent

Disulfide-Llnked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting

Applications, Protein Eng. Des. Sel. 17(l):21-27; Wu, A. et al. (2001) Multimerization Of A Chimeric Anti-CD20 Single Chain Fv-Fv Fusion Protein Is Mediated Through Variable Domain Exchange, Protein Engineering 14(2) : 1025-1033; Asano et al. (2004) 'Ά Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fe Domain, Abstract 3P-683, J. Biochem. 76(8):992; Takemura, S. et al.

(2000) Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, Protein Eng. 13 (8): 583-588; Baeuerle, P.A. et al. (2009) Bispecific TCell Engaging Antibodies For Cancer Therapy, Cancer Res. 69 (12):4941-4944).

[0072] The design of a diabody is based on the antibody derivative known as single-chain Variable Domain fragment (scFv). Such molecules are made by linking Light and/or Heavy chain Variable Domains using a short linking peptide. Bird et al. (1988) (Single-Chain Antigen-Binding Proteins, Science 242:423-426) describes an example for binding peptides that connect approximately 3.5 nm between the carboxy-terminus of one Variable Domain and the amino-terminus of the other Variable Domain. The linkers of cnan Ln/zznz/E/YiAi cnan Ln/zznz/E/YiAi other sequences have been designed and used (Bird et al. (1988) Single-Chain Antigen-Binding Proteins, Science 242:423-426). The linkers can in turn be modified for additional functions, such as drug attachment or attachment to solid supports. Single chain variants can be produced either recombinantly or synthetically. For the synthetic production of scFv, an automated synthesizer can be used. For the recombinant production of scFv, a suitable plasmid containing polynucleotide encoding the scFv can be introduced into a suitable host cell, whether eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic, such as E. coli. . Polynucleotides encoding the scFvs of interest can be made by routine manipulations such as polynucleotide ligation. The resulting scFvs can be isolated using standard protein purification techniques known in the art.

[0073] The provision of non-monospecific diabodies provides a significant advantage over antibodies, including but not limited to, the ability to co-ligate and co-localize cells expressing different epitopes. Bispecific diabodies in this manner have wide-ranging applications including therapy and immunodiagnosis. Bispecificity allows greater flexibility in the model and cnan Ln/zznz/E/YiAi diabody design in various applications, providing improved avidity to multimeric antigens, cross-linking to differentiate antigens, and targeting to specific cell types that are based in the presence of both target antigens. Due to their increased valence, low dissociation rates and rapid release from circulation (for small sized diabodies, at or below ~50 kDa), diabody molecules known in the art have also shown particular use in the field of tumor imaging (Fitzgerald et al (1997) Improved Tumor Targeting By Dísulphlde Stabíllzed Diabodies Expressed In Pichia pastoris, Protein Eng. 10:1221).

[0074] The bispecificity of diabodies has led to their use to co-ligate differentiated cells, for example, cross-linking of cytotoxic T cells to tumor cells (Staerz et al (1985) Hybrld Antibodies Can Target Sites For Attack By T Cells, Nature 314:628-631, and Holliger et al (1996) Specific Killing Of Lymphoma Cells By Cytotoxic TCells Mediated By A Bispecific Diabody, Protein Eng. 9:299305; Marvin et al (2005) Recombinant Approaches To IgG-Like Bispecific Antibodies Acta Pharmacol. Without. 26:649-658). Alternatively or additionally, bispecific diabodies can be used to co-ligate receptors on the surface of different cells or on a single cell. The co-ligation of different cells and/or receptors is useful for effector functions of modulation and/or signaling of immune cells. Multispecific molecules (e.g., bispecific diabodies) comprising epitope binding sites can be targeted to a surface determinant of any immune cell such as B7-H3 (CD276), B7-H4 (VTCN1), BTLA (CD272), CD3, CD8, CD16, CD27, CD32, CD40, CD40L, CD47, CD64, CD70 (CD27L), CD80 (B7-1), CD86 (B7-2), CD94 (KLRD1), CD137 (4-IBB), CD137L (4-1BBL), CD226, CTLA-4 (CD152), Galectin-9, GITR, GITRL, HHLA2, ICOS (CD278), ICOSL (CD275), Killer Activating Receptor (KIR), LAG-3 (CD223), LIGHT (TNFSF14, CD258), MHC class I or II, NKG2a, NKG2d, 0X40 (CD134), OX40L (CD134L), PD1H, PD-1 (CD279) , PD-L1 (B7-H1, CD274), PD-L2 (B7-CD, CD273), PVR (NECL5, CD155), SIRPa, TCR, TIGIT, TIM-3 (HAVCR2), and/or VISTA (PD- 1H), which are expressed in T lymphocytes, natural killer (NK) cells, Antigen-presenting cells or other mononuclear cells. In particular, epitope binding sites targeting a cell surface receptor that is involved in regulating an immune checkpoint (or the ligand thereof) are useful in the generation of bispecific or multispecific binding molecules that antagonize or block signaling. inhibitor of immune checkpoint molecules and therefore stimulate, positively regulate or improve the immune response in a subject. Molecules involved in the regulation of immune checkpoints include, but are not limited to, B7-H3, B7-H4, BTLA, CD40, CD40L, CD47. , CD70, CD80, CD86, CD94, CD137, CD137L, CD226, CTLA-4, Galectin-9, GITR, GITRL, HHLA2, ICOS, ICOSL, KIR, LAG-3, LIGHT, MHC class I or II, NKG2a, NKG2d , 0X40, OX40L, PD1H, PD-1, PD-L1, PD-L2, PVR, SIRPa, TCR, TIGIT, TIM-3 and/or VISTA.

[0075] However, the above advantages come at a significant cost. The formation of such non-monospecific diabodies requires the successful assembly of two or more distinct and different polypeptides (i.e., such formation requires that the diabodies be formed through the heterodimerization of different polypeptide chain species). This is in contrast to monospecific diabodies, which are formed through homodimerization of identical polypeptide chains. Because at least two different polypeptides (i.e., two polypeptide species) must be provided to form a non-monospecific diabody, and because homodimerization of such polypeptides leads to inactive molecules (Takemura, S. et al (2000) Construction Of Ά Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, Protein Eng. 13(8):583-588), the production of such polypeptides must be achieved in such a way that covalent bonding between polypeptides of the same species is avoided (i.e., to avoid homodimerization) (Takemura, S. et al (2000 ) Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A

Refolding System Protein Eng. 13(8):583-588). The art has so far taught non-covalent association of such polypeptides (see, for example, Olafsen et al (2004) Covalent Disulfide-Linked Anti-CEA Diabody Allows SiteSpecific Conjugation And Radiolabeling For Tumor Targeting Applications, Prot. Engr. Des. Sel 17:21-27; Asano et al (2004) A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fe Domain Abstract 3P-683, J. Biochem. 76(8):992; Takemura, S. et al (2000) Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, Protein Eng. 13(8):583-588; Lu, D. et al. (2005) A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity J. Biol. Chem. 280(20): 19665-19672).

[0076] However, the art has recognized that bispecific diabodies composed of non-covalently associated polypeptides are unstable and readily dissociate into non-functional monomers (see, for example, Lu, D. et al. (2005) 'Ά Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity J. Biol. Chem. 280(20) : 19665-19672).

[0077] In the face of this challenge, the technique has been successful in developing non-monospecific heterodimeric diabodies cnan Ln/zznz/E/YiAi

Stable covalently linked CbQb ίη/ΖΖΩΖ/Ε/ΥΙΛΙ, called DART® diabodies (Dual Affinity Re-Targeting Reagents); see, for example. United States Patent Publications Nos. 2013-0295121; 2010-0174053 and 2009-0060910; European Patent Publications Nos. EP 2714079; EP 2601216; EP 2376109; EP 2158221 and PCT Publications Nos. WO 2012/162068; WO 2012/018687; WO 2010/080538; and Sloan, D.D. et al (2015) Targeting HIV Reservoir in Infected CD4 T Cells by DualAffínlty Re-targeting Molecules (DARTs) that Blnd HIV Envelope and Recruit Cytotoxic T Cells PLoS Pathog. 11 (11) :el005233. doi:10.1371/journal.ppat. 1005233; To the

Hussaini, M. et al. (2015) Targeting CD 123 In AML Using A T-Cell Directed Dual-Affinity Re-Targeting (DART®) Platform, Blood pii: blood-2014-05-575704; Chichili, GR et al (2015) A CD3xCD123 Bispecific DART For Redirecting Host T Cells To Myelogenous Leukemia: Preclinical Activity And Safety In Nonhuman Primates, Sci. Transí. Med. 7(289) :289ra82; Moore, PA et al (2011) Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of BCell Lymphoma, Blood 117 (17):4542-4551; Veri, MC et al. (2010) Therapeutic Control Of B Cell Activation Via Recruitment Of Fcgamma Receptor Ilb (CD32B) Inhibitory Function With A Novel Bispecific Antibody Scaffold, Arthritis Rheum. 62(7): 1933-1943; Johnson, S. et al. (2010) Effector Cell Recruitment With Novel Fv-Based Dual-Affinity cnan Ln/zznz/E/YiAi

Re-Targeting Protein Leads To Potent Tumor Cytolysis And in vivo B-Cell Depletion)' J. Mol. Biol. 399(3):436-449). Such diabodies comprise two or more covalently composed polypeptides and involve designing one or more cysteine ​​residues in each of the polypeptide species used that allows disulfide bonds to form and therefore covalently link two polypeptide chains. For example, the addition of a cysteine ​​residue to the C-terminus of such constructs has been shown to allow disulfide bonding between polypeptide chains, stabilizing the resulting heterodimer without interfering with the binding characteristics of the bivalent molecule.

[0078] Each of the two polypeptides of the simplest bispecific DART® diabody comprises three domains. The first polypeptide comprises (in the N-terminal to C-terminal direction): (i) a First Domain comprising a binding region of a Light Chain Variable Domain of a first immunoglobulin (VL1), (ii) a Second Domain comprising a binding region of a Heavy Chain Variable Domain of a second immunoglobulin (VH2), and (iii) a Third Domain containing a cysteine ​​residue (or a cysteine-containing domain) and a Heterodimer Promoting domain that serves to promote heterodimerization with the second polypeptide of the diabody and to covalently link the first and second diabody polypeptides with each other. The second polypeptide contains (in the N-terminal to C-terminal direction): (i) a First Domain comprising a binding region of a Variable Light Chain Domain of a second immunoglobulin (VL2), (ii) a Second Domain comprising a binding region of a Heavy Chain Variable Domain of the first immunoglobulin (VH1), and (iii) a Third Domain containing a cysteine ​​residue (or a cysteine-containing domain) and a complementary Heterodimer-Promoting Domain that forms complexes with the Heterodimer-Promoting Domain of the first polypeptide chain to promote heterodimerization with the first polypeptide chain. The cysteine ​​residue (or a cysteine-containing domain) of the third domain of the second polypeptide chain serves to promote covalent bonding of the second polypeptide chain to the first polypeptide chain of the diabody. Such molecules are stable, potent and have the ability to simultaneously bind to two or more antigens. In one embodiment, the Third Domains of the first and second polypeptides each contain a cysteine ​​residue, which serves to link the polypeptides together via a disulfide bond. Figure 1 provides a schematic of such a diabody, which uses domains promoting the E-helix/Khelix Heterodimer and a cysteine-containing linker for covalent cnan Ln/zznz/E/YiAi linkage. As provided in Figure 2 and Figures 3A-3C, one or both of the polypeptides may additionally possess the sequence of a CH2-CH3 Domain, such that forming complexes between the two diabody polypeptides forms an Fe Region that is capable of binding to the Fe receptor of cells (such as B lymphocytes, dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils and mast cells). As provided in greater detail below, the CH2 and/or CH3 Domains of such polypeptide chains need not be identical in sequence, and are advantageously modified to promote complex formation between the two polypeptide chains. [0079] Many variations of such molecules have been described (see, for example, United States Patent Publications Nos. 2015/0175697; 2014/0255407; 2014/0099318; 2013/0295121; 2010/0174053 and 2009/0060910; European Patent Publications Nos. EP 2714079; EP 2601216; EP 2376109; EP 2158221 and PCT Publications Nos. WO 2012/162068; WO 2012/018687; WO 2010/080538). These Fe Region-containing DART® diabodies may comprise two pairs of polypeptide chains. The first polypeptide chain comprises (in the N-terminal to C-terminal direction): (i) a First Domain comprising a binding region of a Variable Light Chain Domain of a first immunoglobulin (VL1), (ii) a Second Domain comprising cnan Ln/zznz/E/YiAi cnan Ln/zznz/E/ YiAi a binding region of a Heavy Chain Variable Domain of a second immunoglobulin (VH2), and (iii) a Third Domain that contains a cysterna residue (or a cysteine-containing domain) and that serves to promote heterodimerization with the second polypeptide of the diabody and to covalently link the first and second polypeptides to each other, and ( iv) a CH2-CH3 Domain. The second polypeptide contains (in the N-terminal to C-terminal direction): (i) a First Domain comprising a binding region of a Variable Light Chain Domain of a second immunoglobulin (VL2), (ii) a Second Domain comprising a binding region of a Heavy Chain Variable Domain of the first immunoglobulin (VH1), and (iii) a Third Domain containing a cysteine ​​residue (or a cysteine-containing domain) and a Heterodimer-Promoting Domain that promotes heterodimerization with the first polypeptide chain. Here two first polypeptides complex with each other to form an Fe Region. Figures 3A-3C provide schemes of three variations of such diabodies using different Heterodimer Promoting Domains.

[0080] Other DART® diabodies containing the Fe Region may comprise three polypeptide chains. The first polypeptide of such DART® diabodies contains three domains: (i) a VL1-containing Domain, (ii) a

CbQb ίη/ΖΖΠΖ/Ε/ΥΙΛΙ

VH2 and (iii) a Domain containing a CH2-CH3 sequence. The second polypeptide of such DART® diabodies contains: (i) a VL2-containing Domain, (ii) a VH1-containing Domain and (iii) a Domain that promotes heterodimerization and covalent bonding with the first polypeptide chain of the diabody. The third polypeptide of such DART® diabodies comprises a CH2-CH3 sequence. In this way, the first and second polypeptide chains of such DART® diabodies associate together to form a VL1/VH1 binding site that is capable of binding to the epitope, as well as a VL2/VH2 binding site that is capable of binding to the second epitope. Such more complex DART® molecules also possess cysterna-containing domains that function to form a covalently linked complex. In this way, the first and second polypeptides are linked to each other through a disulfide bond involving cysternal residues in their respective Third Domains. Notably, the first and third polypeptide chains complex with each other to form an Fe region that is stabilized by a disulfide bond. Figures 4A-4B provide schemes of such diabodies comprising three polypeptide chains.

[0081] Still other DART® diabodies containing the Fe Region may comprise five polypeptide chains that may comprise the binding regions of the Ln/zznz/E/YiAi cnan Domains

Light and Heavy Chain Variables of up to three different immunoglobulins (denoted as VL1/VH1, VL2/VH2 and VL3/VH3). For example, the first polypeptide chain of such diabodies may contain: (i) a Domain containing VH1, (ii) a Domain containing CH1 and (iii) a Domain containing a CH2-CH3 sequence. The second and fifth polypeptide chains of such diabodies may contain: (i) a VL1-containing domain, and (ii) a CL-containing domain. The third polypeptide chain of such diabodies may contain: (i) a domain containing VH1, (ii) a domain containing CH1, (iii) a domain containing a CH2-CH3 sequence, (iv) a domain containing VL2 , (v) a VH3-containing domain and (vi) a Heterodimer-Promoting Domain, where the Heterodimer-Promoting Domains promote dimerization of the third strand with the fourth strand. The fourth polypeptide chain of such diabodies may contain: (i) a VL3-containing Domain, (ii) a VH2-containing Domain, and (iii) a Domain that promotes heterodimerization and covalent bonding with the third polypeptide chain of the diabody. Here the first and third polypeptides form complexes with each other to form an Fe Region. Such more complex DART® molecules also possess cysteine-containing domains that function to form covalently linked complexes such that each polypeptide chain is linked into at least one addition of cnan Ln/zznz/E/YiAi polypeptide chain through a disulfide bond involving cysteine ​​residues. Preferably, such domains are arranged in the N-terminal to C-terminal direction. Figure 5 provides schemes of such diabodies comprising five polypeptide chains.

[0082] Alternative constructs are known in the art for applications where a tetravalent molecule is desirable but an Fe is not required including, but not limited to, tetravalent tandem antibodies, also referred to as TandAbs (see, for example, Patent Publications of United States Nos. 2005-0079170, 2007-0031436, 20100099853, 2011-020667, 2013-0189263; European Patent Publications Nos. EP 1078004, EP 2371866, EP 2361936 and EP 1293514; PCT Publications Nos. WO 1999/057150, WO 2003/025018, and WO 2013/013700) which are formed by the homodimerization of two identical chains each possessing a VH1, VL2, VH2, and VL2 Domain.

[0083] Recently, trivalent structures incorporating two diabody-like binding domains and one non-diabody-like domain and an Fe Region have been described (see, for example, PCT Application No: PCT/US15/33076, entitled Tri-Specific Binding Molecules and Methods of Use Thereof, filed May 29, 2015; and PCT/US15/33081, titled Tri-Specific Binding Molecules That Specifically Bind to Multiple Cancer Antigens and Methods of Use Thereof, filed May 29, 2015) . Such trivalent molecules can be used to generate monospecific, bispecific or trispecific molecules. Figures 6A-6F provide schemes of such trivalent molecules comprising 3 or 4 polypeptide chains.

cnan Ln/zznz/E/YiAi

IV. The Anti-Human PD-1 Binding Molecules of the Present Invention

[0084] PD-1 binding molecules of the present invention include antibodies, diabodies, BiTE, etc., and are capable of binding to a continuous or discontinuous (e.g., conformational) portion (epitope) of PD-1 of human (CD279). The PD-1 binding molecules of the present invention will preferably also exhibit the ability to bind PD-1 molecules of one or more non-human species, in particular, primate species (and especially a primate species, such as cynomolgus monkey). A representative human PD-1 polypeptide (NCBI Sequence NP_005009.2; including a residual signal sequence of 20 amino acids (shown underlined) and the residual mature protein of 268 amino acids) has the amino acid sequence (SEQ ID NO:68 ):

MQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS LVLLVWVLAV ICSRAAR GTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL cnan Ln/zznz/E/YiAi

[0085] In certain embodiments, the anti-human PD-1 binding molecules of the invention are characterized by any (one or more) of the following criteria:

(1) specifically binds to human PD-1 as endogenously expressed on the surface of a stimulated human T cell;

(2) binds specifically to human PD-1 with an equilibrium binding constant (Kd) of 40 nM or less;

(3) binds specifically to human PD-1 with an equilibrium binding constant (Kd) of 5 nM or less;

(4) specifically binds to human PD-1 with an association rate (k_to) 1.5 x 10⁴ M“¹min~¹ or more;

(5) specifically binds to human PD-1 with an association rate (k_to) of 90.0 x 10⁴ M^min^-1 or more;

(6) binds specifically to human PD-1 with a dissociation rate (ka) of 7 x 10^-4 M^_1min^_1 or less;

(7) binds specifically to human PD-1 with a dissociation rate (ka) of 2 x 10~⁴ min^-1 or less;

(8) specifically binds to non-human primate PD-1 (e.g., cynomolgus monkey PD-1);

(9) inhibits (i.e., blocks or interferes with) the binding/inhibitory activity) of PD-1 ligand (PD-L1/PD-L2) on PD-1;

(10) stimulates an immune response; and/or cnan Ln/zznz/E/YiAi

<td rowspan="2"> 1;</td><td rowspan="2"> (2) (3)</td><td rowspan="2">the the</td><td rowspan="2">three CDRls of the three CDRhs of the</td><td rowspan="2">VL Domain VH Domain</td><td rowspan="2">of of</td><td rowspan="2">mAb mAb</td><td rowspan="2"> 3 3</td><td rowspan="2">of of</td><td colspan="2">PD-1;</td>

<td>PD-1</td><td>and</td>

<td>the</td><td>three CDRls</td><td>of the</td><td>VL Domain</td><td colspan="2">PD-1 mAb 3</td><td>F</td><td></td><td></td><td></td><td></td>

<td></td><td colspan="3">(D) (1) the three CDRhs ।</td><td>of the Domain</td><td>V.H.</td><td>of :</td><td colspan="2">mAb 4</td><td>from PD</td><td> -</td>

<td> 1;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> (2)</td><td>the</td><td>three CDRls of the</td><td>VL Domain</td><td>of</td><td>mAb</td><td> 4</td><td>of</td><td>PD-1;</td><td></td>

<td></td><td> (3)</td><td>the</td><td>three CDRhs of the</td><td>VH Domain</td><td>of</td><td>mAb</td><td> 4</td><td>of</td><td>PD-1</td><td>and</td>

<td>the</td><td>three CDRls</td><td>of the</td><td>VL Domain</td><td colspan="2">PD-1 mAb 4</td><td>F</td><td></td><td></td><td></td><td></td>

<td></td><td>(E) (</td><td colspan="2">|1) the three CDRhs¹</td><td>of the Domain</td><td>V.H.</td><td>of :</td><td colspan="2">mAb 5</td><td>from PD</td><td> -</td>

<td> 1;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> (2)</td><td>the</td><td>three CDRls of the</td><td>VL Domain</td><td>of</td><td>mAb</td><td> 5</td><td>of</td><td>PD-1;</td><td></td>

<td></td><td> (3)</td><td>the</td><td>three CDRhs of the</td><td>VH Domain</td><td>of</td><td>mAb</td><td> 5</td><td>of</td><td>PD-1</td><td>and</td>

<td>the</td><td>three CDRls</td><td>of the</td><td>VL Domain</td><td colspan="2">PD-1 mAb 5</td><td>Λ</td><td></td><td></td><td></td><td></td>

(F)(1) the three hCDRs of the VH Domain of PD1 mAb 6;

(2) the three CDRls of the VL Domain of PD-1 mAb 6;

(3) the three CDRhs of the VH Domain of PD-1 mAb 6 and the three CDRls of the VL Domain of PD-1 mAb 6;

(G) (1) the three hCDRs of the VH Domain of PD1 mAb 7;

(3) the three CDRls of Domain VL of PD-1 mAb 7, or hPD-1 mAb 7 VL2, or hPD-1 mAb 7 VL3;

(3) the three CDRhs of the VH Domain of PD-1 mAb 7 and the three CDRls of the VL Domain of PD-1 mAb 7; or hPD-1 mAb 7 VL2, hPD-1 mAb 7 VL3.

(4) the VH Domain of hPD-1 mAb 7 VH1, or mAb7

hPD-1 VH2;

(5) the VL Domain of mAb 7 VL1 of hPD-1, or mAb7

hPD-1 VL2, or hPD-1 mAb 7 VL3;

(6) the VH and VL Domains of mAb 7(1.1) of hPD-1, OR mAb 7(1.2) of hPD-1, or mAb 7(1.3) of hPD-1, or mAb 7 (2.1) of hPD- 1, or hPD-1 mAb 7 (2.2), or hPD-1 mAb 7 (2.3).

(Η) (1) the three hCDRs of the VH Domain of mAb 8 of PD1;

(2) the three CDRls of the VL Domain of PD-1 mAb 8;

(3) the three CDRhs of the VH Domain of PD-1 mAb 8 and the three CDRls of the VL Domain of PD-1 mAb 8;

(I) (1) the three CDRls of the VH Domain of mAb 9 of PD1, or mAb 9 VH2 of hPD-1;

(2) the three CDRls of Domain VL of mAb 9 of PD-1, or mAb 9 VL2 of hPD-1;

(3) the three CDRhs of Domain VH of mAb 9 of PD-1, or mAb 9 VH2 of hPD-1 and the three CDRls of Domain VL of mAb 9 of PD-1, or mAb 9 VL2 of hPD-1;

(4) the VH Domain of hPD-1 mAb 9 VH1, or hPD-1 mAb 9 VH2;

(5) the VL Domain of hPD-1 mAb 9 VL1, or hPD-1 mAb 9 VL2;

(6) the VH and VL Domains of mAb 9 (1.1) of hPD-1, or cnan Ln/zznz/E/YiAi mAb 9 (1.2) of hPD-1, or mAb 9 (2.1) of hPD-1, or hPD-1 mAb 9 (2.2);

(J) (1) the three hCDRs of the VH Domain of PD-1 mAb 10;

(2) the three CDRls of the VL Domain of PD-1 mAb 10;

(3) the three CDRhs of the VH Domain of PD-1 mAb 10 and the three CDRls of the VL Domain of PD-1 mAb 10;

(K) (1) the three hCDRs of the VH Domain of PD-1 mAb 11;

(2) the three CDRls of the VL Domain of PD-1 mAb 11;

(3) the three CDRhs of the VH Domain of PD-1 mAb 11 and the three CDRls of the VL Domain of PD-1 mAb 11;

(L) (1) the three hCDRs of the VH Domain of PD-1 mAb 12;

(2) the three CDRls of the VL Domain of PD-1 mAb 12;

(3) the three CDRhs of the VH Domain of PD-1 mAb 12 and the three CDRls of the VL Domain of PD-1 mAb 12;

(Μ) (1) the three hCDRs of the VH Domain of PD-1 mAb 13;

(2) the three CDRls of the VL Domain of PD-1 mAb 13;

(3) the three CDRhs of the VH Domain of PD-1 mAb 13 and the three CDRls of the VL Domain of PD-1 mAb 13;

(N) (1) the three hCDRs of the VH Domain of PD-1 mAb 14;

cnan Ln/zznz/E/YiAi (2) the three CDRls of Domain VL of mAb 14 of PD-1;

cnan Ln/zznz/E/YiAi (3) the three CDRhs of Domain VH of mAb 14 of PD-1 and the three CDRls of Domain VL of mAb 14 of PD-1;

(O) (1) the three hCDRs of the VH Domain of PD-1 mAb 15;

(2) the three CDRls of the VL Domain of PD-1 mAb 15;

(3) the three CDRhs of the VH Domain of PD-1 mAb 15 and the three CDRls of the VL Domain of PD-1 mAb 15;

(4) the VH Domain of hPD-1 mAb VH1;

(5) the VL Domain of hPD-1 mAb VL1;

(6) Domains VH and VL of hPD-1 mAb 15;

or that binds, or competes to bind with, the same epitope as PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 3, PD-1 mAb 4, PD-1 mAb 5, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 8, PD-1 mAb 9, PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12

1, PD-1 mAb 13, PD-1 mAb 14, or PD-1 mAb 15.

A. Anti-Human PD-1 Antibody PD-1 mAb 1. Anti-Human PD-1 Antibody PD-1 mAb 1

Murine Human

[0089] The amino acid sequence of the VH Domain of PD-1 mAb 1 (SEQ ID NO: 69) is shown in the following (the CDR residues_h are shown underlined).

DVQLQESGPG RVKPSQSLSL TCTVTGFSIT NDYAWNWIRQ FPGNKLEWMG HITYSGSTSY NPSLKSRTST TRDTSKNHFF LQLSSVTPED TATYYCARDY GSGYPYTLDY WGQGTSVTVS S cnan Ln/zznz/E/YiAi

PD-1 mAb 1 CDRhI (SEQ ID NO:71): NDYAWN

CDR_h2 PD-1 mAb 1 (SEQ ID NO:72): HITYSGSTSYNPSLKS

CDR_h3 PD-1 mAb 1 (SEQ ID NO:73): DYGSGYPYTLDY

[0090] An exemplary polynucleotide encoding the Domain

VH of PD-1 mAb 1 is SEQ ID NO: 70 (nucleotides encoding the CDRh residues are shown underlined):

cagatccagt gatgtgcagc ttcaggagtc gggacctggc cgggtgaaac cttctcagtc tctgtccctc acctgcactg tcactggctt ctcaatcacc aatgattatg cctggaactg gatccgacag tttccaggaa acaaactgga gtggatgggc cacataacct acagtggcag cactagctac a acccatctc tcaaaagtcg aatctctatc actcgggaca catccaagaa ccacttcttc ctgcagttga gttctgtgac tcctgaggac acagccacat attactgtgc aa gagattac ggtagtggct acccctatac tttggactac tggggtcaag gtacctcagt caccgtctcc tcc

[0091] The amino acid sequence of the VL Domain of the PD-1 mAb (SEQ ID NO:74) is shown below (CDRl residues are shown underlined).

QIVLTQSPAL MSASPGEKVT MTCSATSIVS YVYWYQQKPG SSPQPWIYLT SNLASGVPAR FSGSGSGTSY SLTISSMEAE DAATYYCQQW SDNPYTFGGG TKLEIK

CDR_l1 PD-1 mAb 1 (SEQ ID NO:76): SATSIVSYVY

PD-1 mAb 1 CDRl 2 (SEQ ID NO:77): LTSNLAS

CDR_l PD-1 mAb 1 (SEQ ID NO:78): QQWSDNPYT

[0092] An exemplary polynucleotide encoding the Domain

VL of PD-1 mAb 1 is SEQ ID NO: 75 (nucleotides encoding CDRl residues are shown underlined):

caaattgttc tcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc atgacctgca gtgccacctc aattgtaagt tacgtttact ggtaccagca gaagcctgga tcctcccccc aaccctggat ttatctcaca tccaacctgg cttctggagt ccctgctcgc ttcagtggca gtggg tctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa gatgctgcca cttattactg ccagcagtgg agtgataacc cgtacacgtt cggagggggg accaagctgg aaataaaa

2. Humanization of Anti-Human PD-1 Antibody PD-1 mAb 1 to Form hPD-1 mAb 1

[0093] The murine anti-human PD-1 antibody PD-1 mAb 1 described above was humanized and further deimmunized when antigenic epitopes were identified in order to demonstrate the ability to humanize an anti-human PD-1 antibody. human to reduce its antigenicity after administration in a human recipient. Humanization produced a humanized VH Domain, designated herein as hPD-1 VH1 mAb 1, and a humanized VL Domain designated herein as hPD-1 VL1 mAb 1. Accordingly, an antibody comprising the humanized VL domains in pairs with the humanized VH Domain is referred to as hPD-1 mAb 1.

[0094] The amino acid sequence of the VH Domain of hPD-1 mAb 1 (SEQ ID NO: 79) is shown below (hCDR residues are shown underlined).

DVQLQESGPG LVKPSQTLSL TCTVSGFSIS NDYAWNWIRQ PPGKGLEWIG HITYSGSTSY NPSLKSRLTI TRDTSKNQFV LTMTNMDPVD TATYYCARDY GSGYPYTLDY WGQGTTVTVS S

[0095] An exemplary polynucleotide encoding hPD-1 mAb 1 VH1 is SEQ ID NO: 80 (nucleotides encoding hCDR residues are shown underlined):

cnan Ln/zznz/E/YiAi gacgtacagc tccaggaaag tggcccaggt ctggtgaagc catcccagac actgagcctg acttgcaccg tgagtggctt ctccatctca aatgactacg cctggaattg gattaggcag cctcccggta aagggctgga gtggatcggc cacatcacat acagcggctc cacatcatat aatcc cagtc tgaagagecg tcttaccatt actcgcgaca ctagtaagaa ccagtttgtt ctgaccatga ccaacatgga ccctgtggat actgcaacat actattgtgc tegagattat ggttctggtt acccttatac actcgactac tggggacagg gaaccactgt gaccgtgagc tcc

[0096] The amino acid sequence of the VL Domain of hPD-1 mAb 1 VL1 (SEQ ID NO:81) is shown below (hCDR residues are shown underlined).

EIVLTQSPAT LSVSPGEKVT ITCSATSIVS YVYWYQQKPG QAPQPLIYLT SNLASGIPAR FSGSGSGTDF TLTISSLEAE DAATYYCQQW SDNPYTFGGG TKVEIK

[0097] An exemplary polynucleotide encoding hPD-1 mAb 1 VL1 is SEQ ID NO: 82 (nucleotides encoding hCDR residues are shown underlined):

gaaatcgttc tgacccagag cccagcaacc ctgtctgtct cccccggaga aaaggtcacc attacttgct ctgctacttc tatcgtgtcc tacgtgtact ggtatcagca gaagcccggt caggctcccc agccattgat atatctgacc agcaacctgg cttctggtat cccagctcgt ttttccggta gcgggtccgg gactgatttc actttgacta tcagctctct ggaggcagaa gacgccgcca cctattattg tcaacagtgg tcagacaatc catacacttt tggcggtggc accaaagtcg aaataaag

B. Anti-Human PD-1 Antibody PD-1 mAb 2

1. Murine Anti-Human PD-1 Antibody PD-1 mAb 2

[0098] The amino acid sequence of the VH Domain of PD-1 mAb 2 (SEQ ID NO:83) is shown in the following (the CDR residues_h are shown underlined).

cnan Ln/zznz/E/YiAi

DVQLVESGGG LVQPGGSRKL SCAASGFVFS SFGMHWVRQA PEKGLEWVAY ISSGSMSISY ADTVKGRFTV TRDNAKNTLF LQMTSLRSED TAIYYCASLS DYFDYWGQGT TLTVSS

PD-1 mAb 2 CDRhI (SEQ ID NO:85): SFGMH

CDR_h2 PD-1 mAb 2 (SEQ ID NO:86): YISSGSMSISYADTVKG

CDR_h3 PD-1 mAb 2 (SEQ ID NO:87): LSDYFDY

[0099] An exemplary polynucleotide encoding the Domain

PD-1 mAb 2 VH is SEQ ID NO: 84 (nucleotides encoding hCDR residues are shown underlined):

gatgtgcagc tcgtggagtc tgggggaggc ttagtgcagc ctggagggtc ccggaaactc tcctgtgcag cctctggatt cgttttcagt agctttggaa tgcactqqgt tcgtcaggct ccagagaagg ggctggagtg ggtcgcatac atcagtagtg gcagtatgag cattt cctat gcagacacag tgaagggccg attcaccgtc accagagaca atgccaagaa caccctgttc ctgcaaatga ccagtctaag gtctgaggac acggccattt attactgtgc atccctgagt gactactttg actactggqg ccaaggcacc actctcacag tctcctcc

[00100] The amino acid sequence of the VL Domain of PD-1 mAb 2 (SEQ ID NO:88) is shown in the following (the CDR residues_l are shown underlined).

cnan Ln/zznz/E/YiAi

DVVMSQTPLS LPVSLGDQAS ISCRSSQSLV HSTGNTYLHW YLQKPGQSPK LLIYRVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV FFCSQTTHVP WTFGGGTKLE IK

CDRlI of PD-1 mAb 2 (SEQ ID NO:90): RSSQSLVHSTGNTYLH

PD-1 mAb 2 CDRl 2 (SEQ ID NO:91): RVSNRFS

CDR_l PD-1 mAb 2 3 (SEQ ID NO:92): SQTTHVPWT

[00101] An exemplary polynucleotide encoding the Domain

VL of PD-1 mAb 2 is SEQ ID NO: 89 (nucleotides encoding CDRl residues are shown underlined):

gatgtugtga tgtcccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc atctcttgca gatctagtca gagccttgtt cacagtactg gaaacaccta tttacattgq nacctgcaga agccaggcca gtctccaaag ctcctgatct acagggtttc taaccgattt tctggggtcc ccgaca ggtt cagtggcagt ggatcaggga cagatttcac actcaagatc agtagagtgg aggctgagga tctgggagtt tttttctgct ctcaaactac acatgttccg tggacgttcq gtggaggcac caagctggaa atcaaa

2. Humanization of Anti-Human PD-1 Antibody PD-1 mAb 2 to Form hPD-1 mAb 2

[00102] The murine anti-human PD-1 antibody PD-1 mAb 2 described above was humanized and further deimmunized when antigenic epitopes were identified in order to demonstrate the ability to humanize an anti-human PD-1 antibody. human to reduce its antigenicity after administration in a human recipient. Humanization produced a humanized VH Domain, herein designated as mAb

hPD-1 VH1 and a humanized VL Domain designated herein as hPD-1 VL1 mAb 1. Therefore, any antibody comprising the humanized VL Domains in pairs with the humanized VH Domain is referred to as hPD-1 mAb 2.

[00103] The amino acid sequence of the VH1 Domain of the PD-1 mAb (SEQ ID NO: 93) is shown below (hCDR residues are shown underlined).

EVQLVESGGG LVQPGGSLRL SCAASGFVFS SFGMHWVRQA PGKGLEWVAY ISSGSMSISY ADTVKGRFTI SRDNAKNTLY LQMNSLRTED TALYYCASLS DYFDYWGQGT TVTVSS

[00104] An exemplary polynucleotide encoding hPD-1 mAb 2 VH1 is SEQ ID NO: 94 (nucleotides encoding hCDR residues are shown underlined):

gaagtgcaat tggttgagag tggtggtggc ctggtgcagc caggtggaag tctgcggttg tcctgtgcag caagcggatt tgtgttcagc tcttttggga tgcattqgqt gcgccaggct cccggcaagg gtctcgagtg ggtagcatac atctccagcg ggtccatgtc tattagttat g ccgacacag tgaaaggcag gtttactatc tcccgtgaca atgcaaaaaa cacactgtac ctgcaaatga atagcctgcg caccgaggac accgccttgt actactgcgc ttccctgtct gattacttcg actactgggg tcagggcaca actgtgacag tttcttcc

[00105] The amino acid sequence of the VL1 Domain of the hPD-1 mAb (SEQ ID NO: 95) is shown below (hCDR residues are shown underlined).

DVVMTQSPLS LPVTEGQPAS TSCRSSQSLV HSTGNTYLHW YLQKPGQSPQ LLIYRVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCSQTTHVP WTFGQGTKLE IK cnan Ln/zznz/E/YiAi

[00106] An exemplary polynucleotide encoding hPD-1 mAb 2 VL1 is SEQ ID NO: 96 (nucleotides encoding hCDR residues are shown underlined):

cnan Ln/zznz/E/YiAi gacgttgtga tgacacagtc accactgagt ctgccagtta ccctgggcca gccagccagt atttcttgte ggagttcaca gagtctggta cattccacag gaaatacata tctccattgg tacctgcaaa aaccagggca gagcccccag ctgctgattt atagagtgtc taat cgattt tctgqcqtgc cagatcggtt cagcggcagc gggtctggca ctgatttcac actgaaaatc tctagggtgg aggcagagga cgtaggcgtt tactactqta gtcagaccac ccatgtaccc tggacttttq gccaaggtac taagctggaa atcaag

C. Murine Anti-Human PD-1 Antibody PD-1 mAb 3

[00107] The amino acid sequence of the VL Domain of PD-1 mAb 3 (SEQ ID NO: 97) is shown below (hCDR residues are shown underlined).

QVQLQQSGAE LVRPGASVTL SCKASGYTFT DYVMHWVKQT PVHGLEWIGT IDPETGGTAY NQKFKGKAIL TADKSSNTAY MELRSLTSED SAVYYFTREK ITTIVEGTYW YFDVWGTGTT VTVSS

PD-1 mAb 3 CDRhI (SEQ ID NO:99): DYVMH

PD-1 mAb 3 CDRh2 (SEQ ID NO:100): TIDPETGGTAYNQKFKG CDR_h3 of PD-1 mAb 3 (SEQ ID NO: 101): EKITTIVEGTYWYFDV [00108] An exemplary polynucleotide encoding the VH Domain of PD-1 mAb 3 is SEQ ID NO: 98 (the nucleotides encoding the CDRh residues are show underlined):

cnan Ln/zznz/E/YiAi caggttcaac tgcaacagtc tggggctgag ctggtgaggc ctggggcttc agtgacgctg tcctgcaagg cttcgggcta cacatttact gactatgtaa tgcactqgqt gaagcagaca cctgtgcatg gcctggaatg gattggaact attgatcctg aaactgg tgg tactgcctac aatcagaagt tcaagggcaa ggccatactg actgcagaca agtcctccaa cacagcctac atggagctcc gcagcctgac atctgaggac tctgccgtct attactttac aagagagaag attactacga tagtagaggg gacatactgg tacttcgatg tctggggcac agggaccacg gtcaccgtct cctca

[00109] The amino acid sequence of the VL Domain of PD-1 mAb 3 (SEQ ID NO: 102) is shown below (CDRl residues are shown underlined).

DVLLTQTPLS LPVSLGDQAS ISCRSSQNIV HSNGDTYLEW YLQKPGQSPK

LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHLP

YTFGGGTKLE IK

CDR_l1 PD-1 mAb 3 (SEQ ID NO:104): RSSQNIVHSNGDTYLE

PD-1 mAb 3 CDRl 2 (SEQ ID NO:105): KVSNRFS

CDR_l PD-1 mAb 3 (SEQ ID NO:106): FQGSHLPYT

[00110] An exemplary polynucleotide encoding the Domain

VL of PD-1 mAb 3 is SEQ ID NO: 103 (nucleotides encoding CDRl residues are shown underlined): gatgttttgc tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc atctcttgca gatctagtca gaacattgta catagtaatg gagacaccta tttggaatgg tacctgcaga aaccagg cca gtctccaaag ctcctgatct ataaagtttc caaccgattt tctggggtcc cagacaggtt cagtggcagt gggtcaggga cagattttac actcaaaatc agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatcttccg tacacgttcg gaggggggac caagctggaa ataaaa

D. Anti-Human PD-1 Antibody PD-1 mAb 4

Murino

[00111] The amino acid sequence of the VH Domain of PD-1 mAb 4 (SEQ ID NO: 107) is shown in the following (hCDR residues are shown underlined):

cnan Ln/zznz/E/YiAi

DVQLVESGGG LVQPGGSRKL SCAASGFVFS SFGMHWVRQA PEKGLEWVAY ISSGSMSISY ADTVKGRFTV TRDNAKNTLF LQMTSLRSED TAIYYCASLT DYFDYWGQGT TLTVSS

PD-1 mAb 4 CDRhI (SEQ ID NO:109): SFGMH

CDR_h2 PD-1 mAb 4 (SEQ ID NO:110): YISSGSMSISYADTVKG

CDR_h PD-1 mAb 4 3 (SEQ ID NO:111): LTDYFDY

[00112] An exemplary polynucleotide encoding the Domain

VH of PD-1 mAb 4 is SEQ ID NO: 108 (nucleotides encoding the CDRh residues are shown underlined): gatgtgcagc tcgtggagtc tgggggaggc ttagtgcagc ctggagggtc ccggaaactc tcctgtgcag cctctggatt cgttttcagt agctttggaa tgcactgggt tcgt caggct ccagagaagg ggctggagtg ggtcgcatat attagtagtg gcagtatgag tatttcctat gcagacacag tgaagggccg attcaccgtc accagagaca atgccaagaa caccctgttc ctgcaaatga ccagtctaag gtctgaggac acggccattt attactgtgc atccctgact gactactttg actactgggg ccaaggcacc actctcacag tctcctca

[00113] The amino acid sequence of the VL Domain of PD-1 mAb 4 (SEQ ID NO: 112) is shown below (CDRl residues are shown underlined).

DVVMSQTPLS LPVSLGDQAS ISCRSSQSLV HSTGNTYFHW YLQKPGQSPK LLIYRVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFCSQTTHVP WTFGGGTKLE IK

CDRlI of PD-1 mAb 4 (SEQ ID NO:114): RSSQSLVHSTGNTYFH

PD-1 mAb 4 CDRl 2 (SEQ ID NO:115): RVSNRFS

CDR_l PD-1 mAb 4 (SEQ ID NO:116): SQTTHVPWT cnan Ln/zznz/E/YiAi

[00114] An exemplary polynucleotide encoding the Domain

VL of PD-1 mAb 4 is SEQ ID NO: 113 (nucleotides encoding CDRl residues are shown underlined):

gatgttgtga tgtcccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc atctcctgca gatctagtca gagccttgtt cacagtactg gaaacaccta tttccattgg tacctgcaga agccaggcca gtctccaaag ctcctgatct acagggtttc taaccgattt tctggggtcc ccga caggtt cagtggcagt ggatcaggga cagatttcac actcaagatc agcagagtgg aggctgagga tctgggagtt tatttctqct ctcaaactac acatgttccg tggacgttcg gtggaggcac caagctggaa atcaaa

E. Murine Anti-Human PD-1 Antibody PD-1 mAb 5

[00115] The amino acid sequence of the VH Domain of PD-1 mAb 5 (SEQ ID NO: 117) is shown in the following (the CDR residues_h are shown underlined):

QVQLQQPGVE LVRPGASVKL SCKASGYSFT AYWMNWMKQR PGQGLEWIGV IHPSDSETWL NQKFKDKATL TVDKSSSTAY MQLISPTSED SAVYYCAREH YGSSPFAYWG QGTLVTVSA

PD-1 mAb 5 CDRhI (SEQ ID NO:119): AYWMN

CDR_h2 PD-1 mAb 5 (SEQ ID NO:120): HIVPSDSETWLNQKFKD

CDR_h3 PD-1 mAb 5 (SEQ ID NO:121): EHYGSSPFAY

[00116] An exemplary polynucleotide encoding the Domain

PD-1 mAb 5 VH is SEQ ID NO: 118 (nucleotides encoding hCDR residues are shown underlined):

cnan Ln/zznz/E/YiAi caggtccaac tgcagcagcc tggggttgaa ctggtgaggc ctggagcttc agtgaagctg tcctgcaagg cttctggcta ctccttcacc gcctactgga tgaactgqat gaaacagagg cctggacaag gccttgagtg qattqqcgtg attcatcctt ccgatag tga aacttggtta aatcagaagt tcaagga ca a ggccacattg actgtagaca aatcctccag cacagcctac atgcaactca tcagcccgac atctgaggac tctgcggtct attactgtgc aaqagagcac tacggtagta gcccgtttgc ttactggggc caagggactc tggtcactgt ctctgca

[00117] The amino acid sequence of the VL Domain of PD-1 mAb 5 (SEQ ID NO: 122) is shown in the following (CDRl residues are shown underlined):

DIVLTQSPAS LAVSLGQRAT ISGRANESVD NYGMSFMNWF QQKPGQPPKL LIYAASNQGS GVPARFSGSG SGTDFSLNIH PMEEDDTAMY FCQQSKEVPY TFGGGTKLEI K

CDR_l1 PD-1 mAb 5 (SEQ ID NO:124): RANESVDNYGMSFMN

CDR_l PD-1 mAb 5 (SEQ ID NO:125): AASNQGS

CDR_l PD-1 mAb 5 (SEQ ID NO:126): QQSKEVPYT

[00118] An exemplary polynucleotide encoding the Domain

VL of PD-1 mAb 5 is SEQ ID NO: 123 (nucleotides encoding CDRl residues are shown underlined): gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctcuagggca gagggccacc atctcctgca gagccaacga aagtgttgat aattatggca tgagttttat gaactggttc caacagaaac caggacag cc acccaaactc ctcatctatg ctgcatccaa ccaaggatcc gqggtccctq ccaggtttag tggcagtggg tctgggacag atttcagcct caacatccat cctatggagg aggatgatac tgcaatgtat ttctqtcagc aaagtaagga ggttccgtac acgttcqgaq gggggaccaa gctggaaata aaa

F. Murine Anti-Human PD-1 Antibody PD-1 mAb 6 [00119] The amino acid sequence of the VH Domain of PD-1 mAb 6 (SEQ ID NO: 127) is shown in the following (the residues of CDRh are underlined).

EVKLVESGGG LVNPGGSLKL SCAASGFTFS SYGMSWVRQT PEKRLEWVAT ISGGGSDTYY PDSVKGRFTI SRDNAKNNLY LQMSSLRSED TALYYCARQK ATTWFAYWGQ GTLVTVST cnan Ln/zznz/E/YiAi

PD-1 mAb 6 CDRhI (SEQ ID NO:129): SYGMS

CDR_h2 PD-1 mAb 6(SEQ ID NO:130): TISGGGSDTYYPDSVKG

CDR_h3 of PD-1 mAb 6(SEQ ID NO:131): QKATTWFAY

[00120] An exemplary polynucleotide encoding the Domain

VH of PD-1 mAb 6 is SEQ ID NO: 128 (nucleotides encoding hCDR residues are shown underlined):

gaaatcgtac tcacccagtc acctgcaacc ctttctctga gccccggtga acgtgccact ctcagctgca gagcaagtga gagtgtggac aattacggca tgtccttcat gaactqgttt cagcagaagc ctgggcagcc acctaagctg ctcatccacg ccgcctctaa ccgcggatct ggggtg cctt cacgtttttc tggatcagga agtggcactg acttcaccct tacaatcagc tctctggagc cagaggactt tgccgtctat ttctgccagc aatctaaaga ggtgccctat acttttggtg gcgggaccaa ggttgagatc aaa

[00121] The amino acid sequence of the VL Domain of PD-1 mAb 6 (SEQ ID NO: 132) is shown in the following (CDRl residues are shown underlined):

DIVLTQSPAS LAVSLGQRAT ISCRASESVD NYGISFMNWF QQKPGQPPKL LIYPASNQGS GVPARFSGSG SGTDFSLNIH PMEEDDAAMY FCQQSKEVPW TFGGGTKLEI K

100

PD-1 mAb 6 CDRlI (SEQ ID NO:134): RASESVDNYGISFMN cnan Ln/zznz/E/YiAi

PD-1 mAb 6 CDRl 2 (SEQ ID NO:135): PASNQGS

PD-1 mAb 6 CDRl 3 (SEQ ID NO:136): QQSKEVPWT

[00122] An exemplary polynucleotide encoding the Domain

VL of PD-1 mAb 6 is SEQ ID NO: 133 (nucleotides encoding CDRl residues are shown underlined): gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc atctcctgca gagccagcga aagtgttgat aattatggca ttagttttat gaactggttc caacagaaac caggacag cc acccaaactc ctcatctatc ctgcatccaa ccaaggatcc ggggtccctg ccaggtttag tggcagtggg tctgggacag acttcagcct caacatccat cctatggagg aggatgatgc tgcaatgtat ttctgtcagc aaagtaagga ggttccgtgg acgttcggtg gaggcaccaa gctggaaatc aaa

G. Anti-Human PD-1 Antibody PD-1 mAb 7

1. Murine Anti-Human PD-1 Antibody PD-1 mAb 7

[00123] The amino acid sequence of the VH Domain of PD-1 mAb 7 (SEQ ID NO: 137) is shown below (hCDR residues are shown underlined).

QVQLQQPGAE LVRPGASVKL SCKASGYSFT SYWMNWVKQR PGQGLEWIGV IHPSDSETWL DQKFKDKATL TVDKSSTTAY MQLISPTSED SAVYYCAREH YGTSPFAYWG QGTLVTVSS

PD-1 mAb 7 CDRhI (SEQ ID NO:139): SYWMN

CDR_h2 PD-1 mAb 7 (SEQ ID NO:140): HIVPSDSETWLDQKFKD

PD-1 mAb 7 CDRh 3 (SEQ ID NO:141): EHYGTSPFAY

[00124] An exemplary polynucleotide encoding the Domain

101

VH of PD-1 mAb 7 is SEQ ID NO: 138 (nucleotides encoding hCDR residues are shown underlined):

gaggtccaac tgcagcagcc tggggctgaa ctggtgaggc ctggagcttc agtgaagctg tcctgcaagg cttctggcta ctccttcacc agctactgga tgaactgggt gaagcagagg cctggacaag gccttgagtg gattggcgtg attcatcctt ccgatagtga aacttggtta gatcagaagt tcaagga caa ggccacattg actgtagaca aatcctccac cacagcctac atgcaactca tcagcccgac atctgaggac tctgcggtct attactgtgc aagggagcac tacggtacta gcccgtttgc ttactqqqqc caagggactc tggtcactgt gtcttcc

[00125] The amino acid sequence of the VL Domain of PD-1 mAb 7 (SEQ ID NO: 142) is shown below (CDRl residues are shown underlined).

DIVLTQSPAS LAVSLGQRAT ISCRANESVD NYGMSFMNWF QQKPGQPPKL

LIHAASNQGS GVPARFSGSG FGTDFSLNIH PMEEDDAAMY FCQQSKEVPY TFGGGTKLEI K

CDRlI of PD-1 mAb 7 (SEQ ID NO:144): RANESVDNYGMSFMN

CDR_l2 PD-1 mAb 7 (SEQ ID NO:145): AASNQGS

PD-1 mAb 7 CDRl 3 (SEQ ID NO: 146): QQSKEVPYT

[00126] An exemplary polynucleotide encoding the Domain

VL of PD-1 mAb 7 is SEQ ID NO: 143 (nucleotides encoding CDRl residues are shown underlined):

gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc atctcctgca gagccaacga aagtgttgat aattatggca tgagttttat. gaactqqttc caacagaaac caggacagcc acccaaactc ctcatccatg ctgcatccaa ccaaggatcc qqqqtccctq ccaggtttag tggcagtggg tttgggacag acttcagcct caacatccat cctatggagg aggatgatgc tgcaatgtat ttctgtcagc aaagtaagga ggttccgtac cnan L n/zznz/E/YiAi acgttcqqag gggggaccaa gctggaaata aaa

102

2. Humanization of Anti-Human PD-1 Antibody PD-1 mAb 7 to Form hPD-1 mAb 7

[00127] The murine anti-human PD-1 antibody PD-1 mAb 7 described above was humanized and further deimmunized when antigenic epitopes were identified in order to demonstrate the ability to humanize an anti-human PD-1 antibody. human to reduce its antigenicity after administration in a human recipient. Humanization produced two humanized VH Domains, designated herein as hPD-1 mAb 7 VH1 and hPD-1 mAb 7 VH2, and three humanized VL Domains designated herein as hPD-1 mAb 7 VL1, mAb 7 VL2 hPD-1 and hPD-1 mAb 7 VL3. Any of the humanized VL Domains can be paired with any of the humanized VH Domains. Accordingly, any antibody comprising one of the humanized VL Domains in pairs with the humanized VH Domain is generically referred to as hPD-1 mAb 7, and particular combinations of humanized VH/VL Domains are referred to for reference in VH/VL Domains. specific, for example, a humanized antibody comprising hPD-1 mAb 7 VH1 and hPD-1 mAb 1 VL2 are specifically referred to as hPD-1 mAb 7(1.2).

[00128] The amino acid sequence of the VH Domain of hPD-1 mAb 7 (SEQ ID NO:147) is shown below (hCDR residues are shown underlined).

cnan Ln/zznz/E/YiAi

103 cnan Ln/zznz/E/YiAi

QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWIGV IHPSDSETWL DQKFKDRVTI TVDKSTSTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSS

[00129] An exemplary polynucleotide encoding hPD-1 mAb 7 VH1 is SEQ ID NO:148 (nucleotides encoding hCDR residues are shown underlined):

caagttcaat tggtacagag cggggcagag gtgaagaaac ccggcgccag tgttaaggtg tcctgcaaag ccagcggtta cagctttaca agctattgga tgaattgggt gcgtcaagca ccagggcagg gtctggaatg gattggggtgattackatcctt ctgacagcga aacatggttg gaccagaaat ttaaagatcg tgtgacaatt acagtcgata agtccacaag cactgcttac atggaactct ccagcttgcq gtccgaggac accgctgtgt attattgcgc cagagagcac tacggcacat caccttttgc atactggggc cagggaactc tcgtaaccgt atcctcc

[00130] The amino acid sequence of the VH Domain of mAb 7

VH2 of hPD-1 (SEQ ID NO: 149) is shown in the following (hCDR residues are shown underlined):

QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWAGV IHPSDSETWL DQKFKDRVTI TVDKSTSTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSS

[00131] An exemplary polynucleotide encoding hPD-1 mAb 7 VH2 is SEQ ID NO: 150 (nucleotides encoding hCDR residues are shown underlined):

104 cnan Ln/zznz/E/YiAi caagttcaat tggtacagag cggggcagag gtgaagaaac ccggcgccag tgttaaggtg tcctgcaaag ccagcggtta cagctttaca agctattgga tgaattgqqt gcgtcaagca ccagggcagg gtctggaatg qqctqqqgtgattackatcctt ctgacagcga aacatggttg gaccagaaat ttaaagatcg tgtgacaatt acagtcgata agtccacaag cactgcttac atggaactct ccagcttgcg gtccgaggac accgctgtgt attattgcgc cagagagcac tacggcacat caccttttgc atactggggc cagggaactc tcgtaaccgt atcctcc

[00132] The amino acid sequence of the VL Domain of mAb 7

VL1 of hPD-1 (SEQ ID NO: 151) is shown in the following (hCDR residues are shown underlined):

EIVLTQSPAT LSLSPGERAT LSCRANESVD NYGMSFMNWF QQKPGQPPKT, LIHAASNQGS CVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI K

[00133] An exemplary polynucleotide encoding hPD-1 mAb 7 VL1 is SEQ ID NO: 152 (nucleotides encoding hCDR residues are shown underlined):

gaaatcgtac tcacccagtc acctgcaacc ctttctctga gccccggtga acgtgccact ctcagctgca gagcaaatga gagtgtggac aattacggca tgtccttcat gaactggttt cagcagaagc ctgggcagcc acctaagctg ctcatccacg ccgcctctaa ccagggatct qgggtgcct t cacgtttttc tggatcagga agtggcactg acttcaccct tacaatcagc tctctggagc cagaggactt tgccgtctat ttctgccagc aatctaaaga ggtgccctat acttttggtg gcgggaccaa ggttgagatc aaa

[00134] The amino acid sequence of the VL Domain of mAb 7

VL2 of hPD-1 (SEQ ID NO: 153) is shown in the following (hCDR residues are shown underlined):

105 cnan Ln/zznz/E/YiAi

EIVLTQSPAT LSLSPGERAT LSGRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI K

[00135] An exemplary polynucleotide encoding hPD-1 mAb 7 VL2 is SEQ ID NO: 154 (nucleotides encoding hCDR residues are shown underlined):

gaaatcgtac tcacccagtc acctgcaacc ctttctctga gccccggtga acgtgccact ctcagctgca gagcaagtga gagtgtggac aattacggca tgtccttcat gaactgqttt cagcagaagc ctgggcagcc acctaagctg ctcatccacg ccgcctctaa ccagggatct gqggtgcc tt cacgtttttc tggatcagga agtggcactg acttcaccct tacaatcagc tctctggagc cagaggactt tgccgtctat ttctgccagc aatctaaaga ggtgccctat acttttggtg gcgggaccaa ggttgagatc aaa

[00136] The amino acid sequence of the VL Domain of the mAb

VL3 of hPD-1 (SEQ ID NO: 155) is shown in the following (the CDR residues_h are shown underlined).

EIVLTQSPAT LSLSPGERAT LSGRASESVD NYGMSFMNWF QQKPGQPPKL T.THAASNRGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI K

[00137] An exemplary polynucleotide encoding hPD-1 mAb 7 VL3 is SEQ ID NO: 156 (nucleotides encoding hCDR residues are shown underlined):

gaaatcgtac tcacccagtc acctgcaacc ctttctctga gccccggtga acgtgccact ctcagctgca gagcaagtga gagtgtggac aattacggca tgtccttcat gaactgqttt cagcagaagc ctgggcagcc acctaagctg ctcatccacg ccgcctctaa ccgcggatct ggggtg cctt cacgtttttc tggatcagga agtggcactg acttcaccct tacaatcagc tctctggagc cagaggactt tgccgtctat ttctgccagc aatctaaaga ggtgccctat acttttggtg gcgggaccaa ggttgagatc aaa

106

[00138] The CDRlI of Domain VL of mAb 7 VL2 of hPD- and mAb 7 VL3 of hPD-Ι comprises an amino acid substitution from asparagine to serine and has the amino acid sequence: RASESVDNYGMSFMN ((SEQ ID NO: 157), the substituted serine is shown underlined). It is contemplated that a similar substitution may be incorporated into any of the CDRIII Domains of PD-1 mAb 7 described above.

[00139] Furthermore, the CDRl2 of the VL Domain of hPD-Ι mAb 7 VL3 comprises an amino acid substitution from glutamine to arginine and has the amino acid sequence: AASNRGS ((SEQ ID NO: 158), the substituted arginine is shown underlined ). It is contemplated that a similar substitution can be incorporated into any of the CDRl2 Domains of PD-1 mAb 7 described above.

H. Murine Anti-Human PD-1 Antibody PD-1 mAb 8

[00140] The amino acid sequence of the VH Domain of PD-1 mAb 8 (SEQ ID NO: 159) is shown below (hCDR residues are shown underlined).

EGQLQQSGPE LVKPGASVKI SCKASGYTFT DYYMNWVKQN HGKSLEWIGD INPKNGDTHY NQKFKGEATL TVDKSSTTAY MELRSLTSED SAVYYCASDF DYWGQGTTLT VSS

PD-1 mAb 8 CDRhI (SEQ ID NO:161): DYYMN

CDR_h2 PD-1 mAb 8 (SEQ ID NO:162): DINPKNGDTHYNQKFKG

CbQb Ln/Zznz/E/YIAI

107

CDR_h3 PD-1 mAb 8 (SEQ ID NO:163): DFDY

[00141] An exemplary polynucleotide encoding the Domain

VH of PD-1 mAb 8 is SEQ ID NO:160 (the nucleotides that cnan Ln/zznz/E/YiAi encode the CDRh residues are shown underlined):

gagggccagc tgcaacaatc tggacctgag ctggtgaagc ctggggcttc agtgaagata tcctgtaagg cttctggata cacgttcact gactactaca tgaactqgqt gaagcagaac catggaaaga gccttgagtg qattggagat attaatccta aaaatggtga cactcactac aaccagaagt tcaagggcga ggcca cattg actgtagaca agtcctccac cacagcctac atggagctcc gcagcctgac atctgaggac tctgcagtct attactgtgc gaqcgatttt gactactggg gccaaggcac cactctcaca gtctcctcc

[00142] The amino acid sequence of the VL Domain of PD-1 mAb 8 (SEQ ID NO: 164) is shown below (CDRl residues are shown underlined).

DVVMTQTPLS LPVGLGDQAS ISCRSSQTLV YSNGNTYLNW FLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFCSQSTHVP FTFGSGTKLE IK

CDRlI of PD-1 mAb 8 (SEQ ID NO:166): RSSQTLVYSNGNTYLN

CDR_l2 PD-1 mAb 8 (SEQ ID NO:77): 167): KVSNRFS

PD-1 mAb 8 CDRl 3 (SEQ ID NO:168): SQSTHVPFT

[00143] An exemplary polynucleotide encoding the Domain

VL of PD-1 mAb 8 is SEQ ID NO: 165 (nucleotides encoding CDRl residues are shown underlined):

108 gatgttgtga tgacccaaac tccactctcc ctgcctgtcg gtcttggaga tcaagcctcc atctcttgca gatctagtca gacccttgta tatagtaatg gaaacaccta tttaaattgg ttcctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt tctgqqqtcc cagacagg tt cagtggcagt ggatcaggga cagatttcac actcaagatc agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttcca ttcacgttcg gctcggggac aaagttggaa ataaaa

CbQb ίη/ΖΖΩΖ/Ε/ΥΙΛΙ

I. Anti-Human PD-1 Antibody PD-1 mAb 9

1. Murine Anti-Human PD-1 Antibody PD-1 mAb 9

[00144] The amino acid sequence of the VH Domain of PD-1 mAb 9 (SEQ ID NO: 169) is shown below (hCDR residues are shown underlined).

EVMLVESGGG LVKPGGSLKL SCAASGTFFS SYLVSWVRQT PEKRLEWVAT ISGGGGNTYY SDSVKGRFTI SRDNAKNTLY LQTSSLRSED TALYYCARYG FDGAWFAYWG QGTLVTVSS

PD-1 mAb 9 CDRhI (SEQ ID NO:171): SYLVS

CDR_h2 PD-1 mAb 9 (SEQ ID NO:172): TISGGGGNTYYSDSVKG

CDR_h3 PD-1 mAb 9 (SEQ ID NO:173): YGFDGAWFAY

[00145] An exemplary polynucleotide encoding the Domain

VH of PD-1 mAb 9 is SEQ ID NO: 170 (nucleotides encoding the hCDR residues are shown underlined):

109 GAAGTGTGCC TGGTGGGTC TGGGGGGCC TTAGTGAAGC CTGGAGGGTC CCTGGAACTC TCCTGTGCAGG CCTCTGTTTT CACTTTTCAGGT AGTTATTTTTTG TGTTGGGT TCGCCAGACT CCGGAGAGAAGA tagtggtg gtggtggtaa cacctatat tcagacagtg tgaagggtcg cnan ln/zznz/e/yiai attcaccatcatcatc tccagagaca atgccaagaa caccctgtc ctgcaaat gcagtgtgagg gtctgagggc acggcctgtgt attactgtgc aaggtatggggggggggggggggggg TTCGACGGCG CCTGGTTGCC TACTGGGGC CAAGGGACTC TGGTCACTGT CTCTTCC

[00146] The amino acid sequence of the VL Domain of PD-1 mAb 9 (SEQ ID NO: 174) is shown in the following (the CDR residues_l are shown underlined):

DIQMTQSPAS LSASVGDIVT ITCRASENIY SYLAWYQQKQ EKSPQLLVYN AKTLAAGVPS RFSGSGSGTQ FSLTINSLQP EDFGNYYCQH HYAVPWTFGG GTRLEIT

PD-1 mAb 9 CDRlI (SEQ ID NO:176): RASENIYSYLA

CDR_l2 PD-1 mAb 9 (SEQ ID NO:177): NAKTLAA

CDR_l3 PD-1 mAb 9 (SEQ ID NO:178): QHHYAVPWT

[00147] An exemplary polynucleotide encoding the Domain

VL of PD-1 mAb 9 is SEQ ID NO: 175 (nucleotides encoding CDRl residues are shown underlined): gacatccaga tgactcagtc tccagcctcc ctatctgcat ctgtgggaga tattgtcacc atcacatgtc gagcaagtga gaatatttac agttatttag catggtatca gcagaaacag gaaaaatctc ctcagctcct ggtctataat gcaaaaacct tggcagcagg tgtgccatca aggttcagtg gcagtggatc aggcacacag ttttctctga ccatcaacag cctgcagcct gaagattttg ggaattatta ctgtcagcat cattatgctg ttccgtggac gttcggtgga ggcaccagac tggaaatcac a

2. Humanization of Antibody PD-1 mAb 9

110

Anti-human PD-1 to Form hPD-1 mAb 9

[00148] The murine anti-human PD-1 antibody PD-1 mAb 9 described above is human and further deimmunized when antigenic epitopes were identified in order to demonstrate the ability to humanize an anti-human PD-1 antibody. -human to reduce its antigenicity after administration in a human recipient. Humanization produced two humanized VH Domains, designated herein as hPD-1 mAb 9 VH1 and hPD-1 mAb 9 VH2, and two humanized VL Domains, designated herein as hPD1 mAb 9 VL1 and hPD-1 mAb 9 VL2. 1. Any of the humanized VL Domains may be paired with the humanized VH Domains. Accordingly, any antibody comprising one of the humanized VL Domains in pairs with the humanized VH Domain is generically referred to as hPD-lmAb 9, and particular combinations of humanized VH/VL Domains are mentioned for reference in Specific VH/VL Domains, for example, a humanized antibody comprising hPD-1 mAb 9 VH1 and hPD-1 mAb 9 VL2 is specifically known as hPD-1 Ab 9 (1.,2). [00149] The amino acid sequence of the VL domain of mAb 9 VH1 of hPD-1 (SEQ ID NO: 179) is shown in the following (the CDR residues_h are shown underlined):

EVQLVESGGG LVRPGGSLKL SCAASGFTFS SYLVSWVRQA PGKGLEWVAT ISGGGGNTYY SDSVKGRFTI SRDNAKNSLY LQMNSLRAED TATYYCARYG FDGAWFAYWG QGTLVTVSS cnan Ln/zznz/E/YiAi

111

[00150] An exemplary polynucleotide encoding hPD-1 mAb 9 VH1 is SEQ ID NO:180 (nucleotides encoding hCDR residues are shown underlined):

gaggtgcagc tggtggaaag tgggggcggc ctggtgcgac ccgggggaag cnan Ln/zznz/E/YiAi tctgaaactg tcctgtgcag catcaggatt tactttttca tcttatctcg tgtcttgggt aagacaagca cccggaaaag gcttggaatg ggtggccact atctccggtg gaggt ggcaa cacctactat agcgacagtg tcaagggaaq atttaccatc agtcgcgaca acgctaagaa tagcctgtac ctccagatga actccctgcg cgccgaggac accgccacct attactgtgc acgctatgga tttgacggcg catggtttgc ctactqgqqa cagggcacat tggtaaccgt tagctcc

[00151] The amino acid sequence of the VH Domain of hPD-1 VH2 mAb 9 (SEQ ID NO: 181) is shown in the following (CDRh residues are shown underlined):

EVQLVESGGG LARPGGSLKL SCAASGFTFS SYLVGWVRQA PGKGLEWTAT ISGGGGNTYY SDSVKGRFTI SRDNAKNSLY LQMNSARAED TATYYCARYG FDGAWFAYWG QGTLVTVSS

[00152] An exemplary polynucleotide encoding hPD-1 mAb 9 VH2 is SEQ ID NO: 182 (nucleotides encoding hCDR residues are shown underlined):

gaggtgcagc tggtggaaag tgggggcggc ctggcgcgac ccgggggaag tctgaaactg tcctgtgcag catcaggatt tactttttca tcttatctcg tgggctgggt aagacaagca cccggaaaag gcttggaatg gacggccact atctccggtg gaggtggcaa cacctactat agcgacagtg t caagggaag atttaccatc agtcgcgaca acgctaagaa tagcctgtac ctccagatga actccgcacg cgccgaggac accgccacct attactgtgc acgctatgga tttgacggcg catggtttgc ctactgqgga cagggcacat tggtaaccgt tagctcc

[00153] The CDRhI of the VH Domain of hPD-1 VH2 mAb 9

112 It comprises a serine in the glycine amino acid substitution and has the amino acid sequence: SYLVG ((SEQ ID NO: 183), the substituted glycine is shown underlined). It is contemplated that a similar substitution may be incorporated into any of the CDRhI Domains of PD-1 mAb 9 described above.

[00154] The amino acid sequence of the VL domain of hPD-1 VL1 mAb 9 (SEQ ID NO: 184) is shown in the following (the CDR residues_h are shown underlined):

DLQMTQSPSS LSASVGDRVT ITCRASENIY SYLAWYQQKP GKAPKLLIYN AKTLAAGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQH HYAVPWTFGQ GTKLEIK cnan Ln/zznz/E/YiAi

[00155] An exemplary polynucleotide encoding hPD-1 mAb 9 VL1 is SEQ ID NO:185 (nucleotides encoding hCDR residues are shown underlined):

gacattcaga tgactcagtc tcccagcagt ctgtccgcat ccgtggggga tcgggtcacc atcacctgcc gtgcctcaga aaacatctat tcatacctcg cctggtatca acagaaacct ggtaaagccc caaaattgct catttacaac gccaagaccc tcgcagctgg cgtgccaagt aggttctcag gcagc ggctc agggacagat ttcaccctca ccatatcctc actgcagccc gaggattttg ccacttacta ctgccagcat cattacgcag tgccctggac cttcggacaa ggcactaagc tcgagatcaa a

[00156] The amino acid sequence of the VL Domain of hPD-1 VL2 mAb 9 (SEQ ID NO: 186) is shown in the following (the CDR residues_h are shown underlined):

113 cnan Ln/zznz/E/YiAi

DIQMTQSPSS LSASVGDRVT ITCRASENIY NYLAWYQQKP GKAPKLLTYD AKTLAAGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQH HYAVPWTFGQ GTKLEIK

[00157] An exemplary polynucleotide encoding mAb 9 VL2 of hPD-1 is SEQ ID NO: 187 (the nucleotides encoding the CDR residues_h are shown underlined):

gacattcaga tgactcagtc tcccagcagt ctgtccgcat ccgtggggga tcgggtcacc atcacctgcc gtgcctcaga aaacatctat aactacctcg cctggtatca acagaaacct ggtaaagccc caaaattgct catttacgac gccaagaccc tcgcagctgg cgtgccaagt aggttctcag gcag cggctc agggacagat ttcaccctca ccatatcctc actgcagccc gaggattttg ccacttacta ctgccagcat cattacgcag tgccctggac cttcggacaa ggcactaagc tcgagatcaa a

[00158] The CDRlI of Domain VL of mAb 9 of VL2 of hPD-1 comprises a serine in the asparagine amino acid substitution and has the amino acid sequence: RASENIYNYLA (SEQ ID NO: 188), 1a substituted asparagine is shown underlined) . It is contemplated that a similar substitution may be incorporated into any of the CDRIII Domains of PD-1 mAb 9 described above.

[00159] CDRl2 of the VL Domain of hPD-1 VL2 mAb 9 comprises an asparagine in the aspartate amino acid substitution and has the amino acid sequence: DAKTLAA ((SEQ ID NO: 189), the substituted aspartate is shown underlined ). It is contemplated that a similar substitution can be incorporated into any of the CDRl2 Domains of PD-1 mAb 7

114 described above.

cnan Ln/zznz/E/YiAi

J. Murine Anti-Human PD-1 Antibody PD-1 mAb 10

[00160] The amino acid sequence of the VH Domain of PD-1 mAb 10 (SEQ ID NO: 190) is shown in the following (hCDR residues are shown underlined):

EVILVESGGG LVKPGGSLKL SCAASGFTFS NYLMSWVRQT PEKRLEWVAS ISGGGSNIYY PDSVKGRFTI SRDNAKNTLY LQMNSLRSED TALYYCARQE LAFDYWGQGT TLTVSS

PD-1 mAb 10 CDRhI (SEQ ID NO:192): NYLMS

CDR_h2 of PD-1 mAb 10 (SEQ ID NO:193): SISGGGSNIYYPDSVKG

CDRh3 of PD-1 mAb 10 (SEQ ID NO: 194): QELAFDY

[00161] An exemplary polynucleotide encoding the Domain

PD-1 mAb 10 VH is SEQ ID NO: 191 (nucleotides encoding hCDR residues are shown underlined):

gaagtgatac tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc tcctgtgcag cctctggatt cactttcagt aactatctca tgtcttgqgt tcgccagact ccggagaaga ggctggagtg gqtcgcaagt attagtggtg gtggtagtaa tatctactat ccagacagtg tgaag ggtcg attcaccata tccagggaca atgccaagaa caccctgtac ctgcaaatga acagtctgag gtctgaggac acggccttgt attactgtgc aagacaagaa ctggcttttg actactgqqq ccaaggcacc actctcacag tctcctcc

[00162] The amino acid sequence of the VL Domain of PD-1 mAb 10 (SEQ ID NO: 195) is shown below (CDRh residues are shown underlined).

115 cnan Ln/zznz/E/YiAi

DIQMTQTTSS LSASLGDRVT ISCRTSQDIS NFLNWYQQKP DGTIKLLIYY TSRLHSGVPS RFSGSGSGTD YSLTISNLEQ EDIATYFCQQ GSTLPWTFGG GTKLEII

PD-1 mAb 10 CDRl 1 (SEQ ID NO:197): RTSQDISNFLN

PD-1 mAb 10 CDRl 2 (SEQ ID NO:198): YTSRLHS

PD-1 mAb 10 CDRl 3 (SEQ ID NO: 199): QQGSTLPWT

[00163] An exemplary polynucleotide encoding the Domain

VL of PD-1 mAb 10 is SEQ ID NO: 196 (nucleotides encoding CDRl residues are shown underlined):

gatatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc atcagttgca ggacaagtca ggacattagc aattttttaa actggtatca gcagaaacca gatggaacta ttaaactcct gatctactac acatcaagat tacactcagq agtcccatca aggttcagtg gcagtgggtc tgga acagat tattctctca ccattagcaa cctggagcaa gaagatattg ccacttactt ttgccaacag ggtagtacgc ttccgtggac gttcggtgga ggcaccaagc tggaaatcat a

K. Anti-Human Murine PD-1 Antibody PD-1 mAb 11 [00164] The amino acid sequence of the VH Domain of PD-1 mAb 11 (SEQ ID NO: 200) is shown in the following (residues of CDRh are shown underlined):

EVQLQQSGTV LARPGASVKM SCKTSGYTFT GYWMHWVKQR PGQGLKWMGA IYPGNSDTHY NQKFKGKAKL TAVTSASTAY MELSSLTNED SAIYYCTTGT YSYFDVWGTG TTVTVSS

PD-1 mAb 11 CDRhI (SEQ ID NO:202): GYWMH

116

CDR_h2 PD-1 mAb 11 (SEQ ID NO:203): AIYPGNSDTHYNQKFKG

CDR_h3 PD-1 mAb 11 (SEQ ID NO:204): GTYSYFDV

[00165] An exemplary polynucleotide encoding the Domain

VH of PD-1 mAb 11 is SEQ ID NO: 201 (nucleotides encoding hCDR residues are shown underlined):

gaggttcagc tccagcagtc tgggactgtg ctggcaaggc ctggggcttc agtgaagatg tcctgcaaga cttctggcta cacatttacc ggctactgga tgcactqqqt aaaacagagg cctggacagg gtctgaaatg gatqqqqgct atttatcctg gaaatagtga tactcactac aaccagaagt tca agggcaa ggccaaactg actgcagtca catccgccag cactgcctac atggagctca gcagcctgac aaatgaqgac tctqcqatct attactgtac tactgggacc tactcgtact tcgatgtctq gggcacaggg accacggtca ccgtctcctc a

[00166] The amino acid sequence of the VL Domain of PD-1 mAb 11 (SEQ ID NO:205) is shown below (CDRl residues are shown underlined).

cnan Ln/zznz/E/YiAi

DILLTQSPAI LSVSPGERVS FSCRASQSIG TSIHWYQHRT NGSPRLLIKY

ASESISGIPS RFSGSGSGTD FTLSINSVES EDIADYYCQQ SNSWLTFGAG

TKLELK

CDRlI of PD-1 mAb 11 (SEQ ID NO:207): RASQSIGTSIH

CDR_l2 of PD-1 mAb 11 (SEQ ID NO:208): YASESIS

CDR_l3 of PD-1 mAb 11 (SEQ ID NO:209): QQSNSWLT

[00167] An exemplary polynucleotide encoding the Domain

VL of PD-1 mAb 11 is SEQ ID NO: 206 (nucleotides encoding CDRl residues are shown underlined):

117 gacatcttgc tgactcagtc tccagccatc ctgtctgtga gtccaggaga aagagtcagt ttctcctgca gggccagtca gagcattggc acaagcatac actggtatca gcacagaaca aatggttctc caaggcttct cataaagtat gcttctgagt ctatctctgg gatcccttcc aggtttagtg gcagtggatc agggactgat tttactctta gcatcaacag tgtggagtct gaagatattg cagattatta ctqtcaacaa agtaatagct ggctcacgtt cggtgctggg accaagctgg agctgaaa cnan Ln/zznz/E/YiAi

L. Murine Anti-Human PD-1 Antibody PD-1 mAb 12 [00168] The amino acid sequence of the VL Domain of PD-1 mAb 12 (SEQ ID NO:210) is shown in the following (the residues of CDRh are underlined).

QGHLQQSGAE LVRPGASVTL SCKASGFTFT DYEMHWVKQT PVHGLEWIGT IDPETGGTAY NQKFKGKAIL TVDKSSTTTY MELRSLTSED SAVFYCSRER ITTWEGAYW YFDVWGTGTT VTVSS

PD-1 mAb 12 CDRhI (SEQ ID NO:212): DYEMH

CDR_h2 PD-1 mAb 12 (SEQ ID NO:213): TIDPETGGTAYNQKFKG

CDR_h3 of PD-1 mAb 12 (SEQ ID NO:214):ERITTWEGAYWYFDV

[00169] An exemplary polynucleotide encoding the Domain

PD-1 mAb 12 VH is SEQ ID NO:211 (nucleotides encoding hCDR residues are shown underlined):

cagggtcacc tgcagcagtc tggggctgag ctggtgaggc ctggggcttc agtgacgctg tcctgcaagg cttcgggctt cacatttact gactatgaga tgcactgqqt gaaacagaca cctgtgcatg gcctggaatg gattqqgact attgatcctg aaactggtgg tactgcctac aatcagaagt t caagggcaa ggccatactg acagtagaca aatcttccac tacaacctac atggagctcc gcagcctgac atctgaggac tctgccgtct tttattgttc aagagagagg attactacgg ttgttgaggg ggcatactgg tacttcgatg tetggggcac agggaccacg gtcaccgtct cctca

118

[00170] The amino acid sequence of the VL Domain of PD-1 mAb 4 (SEQ ID NO:215) is shown in the following (CDRl residues are shown underlined):

DVLMTQTPLS LPVSLGDQAS ISCRSSQNIV HSNGNTYLEW YLQKPGQSPK LLICKVSTRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP YTFGGGTKLE IK cnan Ln/zznz/E/YiAi

CDRlI of PD-1 mAb 12 (SEQ ID NO:217): RSSQNIVHSNGNTYLE

CDR_l PD-1 mAb 12 (SEQ ID NO:218): KVSTRFS

PD-1 mAb 12 CDRl 3 (SEQ ID NO:219): FQGSHVPYT

[00171] An exemplary polynucleotide encoding the Domain

VL of PD-1 mAb 12 is SEQ ID NO:216 (nucleotides encoding CDR1 residues are shown underlined):

gatgttttga tgacccagac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc atctcttgca gatctagtca gaacattgta catagtaatg gaaacaccta tttagaatgg tacctgcaga aaccaggcca gtctccaaag ctcctgatct gcaaagtttc cacccgattt tctggggtcc cagacaggtt cagt ggcagt ggatcaggga cagatttcac actcaagatc agcagagtgg aggctgagga tctgggagtt tattattgct ttcaaggttc acatgttccg tacacgttcg gaggggggac caagctggaa ataaaa

M. Murine Anti-Human PD-1 Antibody PD-1 mAb 13

[00172] The amino acid sequence of the VL Domain of PD-1 mAb 13 (SEQ ID NO:220) is shown below (hCDR residues are shown underlined).

EVMLVESGGG LVKPGGSLKL SCAASGTFFS SHTMSWVRQT PEKRLEWVAT ISGGGSNIYY PDSVKGRFTI SRDNAKNTLY LQMSSLRSED TALYYCARQA YYGNYWYFDV WGTGTTVTVS S

119

PD-1 mAb 13 CDRhI (SEQ ID NO:222): SHTMS

CDR_h2 PD-1 mAb 13 (SEQ ID NO:223): TISGGGSNIYYPDSVKG

CDR_h3 PD-1 mAb 13 (SEQ ID NO:224): QAYYGNYWYFDV

[00173] An exemplary polynucleotide encoding the Domain

PD-1 mAb 13 VH is SEQ ID NO:221 (nucleotides encoding hCDR residues are shown underlined):

CbQb ίη/ΖΖΩΖ/Ε/ΥΙΛΙ gaagtgatgc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc tcctgtgcag cctctggatt cactttcagt agccatacca tgtcttgqgt tcgccagact ccggagaaga ggctggagtg gqt cgcaacc attagtggtg gtggttctaa tatctactat ccagacagtg tgaagggtcg attcaccatc tccagagaca atgccaagaa caccctgtac ctgcaaatga gcagtctgag gtctgaggac acggccttgt attactgtgc aagacaagct tactacggta attactggta cttcgatgtc tggggcacag g gaccacggt caccgtctcc tcc

[00174] The amino acid sequence of Domain VL of PD-1 mAb 13 (SEQ ID NO:225) is shown below (CDRl residues are shown underlined).

DIQMTQSPAT QSASLGESVT ITCLASQTIG TWLAWYQQKP GKSPQLLIYA ATSLADGVPS RFSGSGSGTK FSFKISSLQA EDFVSYYCQQ LDSIPWTFGG GTKLEIK

CDRlI of PD-1 mAb 13 (SEQ ID NO:227): LASQTIGTWLA

PD-1 mAb 13 CDRl 2 (SEQ ID NO:228): AATSLAD

PD-1 mAb 13 CDRl 3 (SEQ ID NO:229): QQLDSIPWT

[00175] An exemplary polynucleotide encoding the Domain

VL of PD-1 mAb 13 is SEQ ID NO:226 (nucleotides encoding CDR1 residues are shown underlined):

120 gacattcaga tgacccagtc tcctgccacc cagtctgcat ctctgggaga aagtgtcacc atcacgtgcc tggcaagtca gaccattggt acatggttag catggtatca gcagaaacca gggaaatctc ctcagctcct qatttatgct gcaaccagct tggcagatgq ggtcccatca aggttcagtg gtagtggatc tggcacaaaa ttttctttca agatcagcag cctacaggct gaagattttg taagttatta ctgtcaacaa cttgacagta ttccgtggac gttcggtgga ggcaccaagc tggaaatcaa a cnan Ln/zznz/E/YiAi

N. Murine Anti-Human PD-1 Antibody PD-1 mAb 14 [00176] The amino acid sequence of the VL Domain of PD-1 mAb 14 (SEQ ID NO:230) is shown in the following (the residues of CDRh are underlined).

QVQLQQPGAE LVKPGASVKM SCKASGYNFI SYWITWVKQR PGQGLQWIGN IYPGTDGTTY NEKFKSKATL TVDTSSSTAY MHLSRLTSED SAVYYCATGL HWYFDVWGTG TTVTVSS

PD-1 mAb 14 CDRhI (SEQ ID NO:232): SYWIT

CDR_h2 PD-1 mAb 14 (SEQ ID NO:233): NIYPGTDGTTYNEKFKS

CDR_h3 PD-1 mAb 14 (SEQ ID NO:234): GLHWYFDV

[00177] An exemplary polynucleotide encoding the Domain

PD-1 mAb 14 VH is SEQ ID NO:231 (nucleotides encoding hCDR residues are shown underlined): caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagatg tcctgcaagg cttctggcta caacttcatc agctactgga taacctgggt gaaacagagg cctggacaag gccttcagtg gattggaaat atttatcctg gtactgatgg tactacctac aatgagaagt tcaagagcaa ggccacactg actgtagaca catcctccag cacagcctac atgcacctca gtcgcctgac atctgaggac tctgcggtct attactgtgc aactgggcta cactggtact tcgatgtctg gggcacaggg accacggtca ccgtctcctc c

121

[00178] The amino acid sequence of the VL Domain of PD-1 mAb 14 (SEQ ID NO:235) is shown below (CDRl residues are shown underlined).

CbQb Ln/Zznz/E/YIAI

DIVMTQSQKF MSTSVGDRVS VTCKASQSVG TNVAWYQQKP GQSPKALIYS ASSRFSGVPD RFTGSGSGTD FTLTISNVQS EDLAEYFCQQ YNSYPYTFGG GTKLEIK

CDRlI of PD-1 mAb 14 (SEQ ID NO:237): KASQSVGTNVA

CDR_l2 PD-1 mAb 14 (SEQ ID NO:238): SASSRFS

CDR_l3 PD-1 mAb 14 (SEQ ID NO:239): QQYNSYPYT

[00179] An exemplary polynucleotide encoding the Domain

VL of PD-1 mAb 14 is SEQ ID NO:236 (nucleotides encoding CDRl residues are shown underlined):

gacattgtga tgacccagtc tcaaaaattc atgtccacat cagtaggaga cagggtcagt gtcacctgca aggccagtca gagtgtgggt actaatgtag cctggtatca acagaagccc ggtcaatctc ctaaagcact gatttactcg gcatcctccc gattcagtgg cgtccctgat cgcttcacag gcagt ggatc tgggacagat ttcactctca ccatcagtaa tgtgcagtct gaagacttgg cagagtattt ctgtcagcaa tataacagct atccgtacac gttcggaggg gggaccaagc tggaaataaa a

O. Anti-human PD-1 Antibody PD-1 mAb 15

1. Murine Anti-Human PD-1 Antibody PD-1 mAb 15

[00180] The amino acid sequence of the VL Domain of PD-1 mAb 15 (SEQ ID NO:240) is shown below (hCDR residues are shown underlined).

122 cnan Ln/zznz/E/YiAi

EVMLVESGGG LVKPGGSLKL SCAASGFIFS SYLISWVRQT PEKRLEWVAA ISGGGADTYY ADSVKGRFTI SRDNAKNTLY LQMSSLRSED TALYYCTRRG TYAMDYWGQG TSVTVSS

[00181] An exemplary polynucleotide encoding the Domain

PD-1 mAb 15 VH is SEQ ID NO:241 (nucleotides encoding hCDR residues are shown underlined):

gaagtgatgc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc tcctgtgcag cctctggatt cattttcagt agctatctca tctcttgggt tcgccagact ccggagaaga ggctggagtg gqtcgctgcc attagtggtg gtggtgctga cacctactat gccgacagtg t gaagggtcg attcaccatc tccagagaca atgccaagaa caccctgtat ctgcaaatga gcagtctgag gtctgaggac acggccttat attactgtac aaqacgaggg acctatgcta tggactactg gggtcaagga acctcagtca ccgtctcctc c

[00182] The amino acid sequence of the VL Domain of PD-1 mAb 15 (SEQ ID NO:245) is shown in the following (the CDR residues_l are shown underlined).

DIQMTQSPAS QSASLGESVT ITCLASQTIG TWLAWYQQKP GKSPQLLIYA ATSLADGVPS RFSGSGSGTK FSFKISSLQA EDFVNYYCQQ LYSIPWTFGG GTKLEIK

CDRlI of PD-1 mAb 15 (SEQ ID NO:247): LASQTIGTWLA

CDR_l PD-1 mAb 15 (SEQ ID NO:248): AATSLAD

CDR_l PD-1 mAb 15 (SEQ ID NO:249): QQLYSIPWT

[00183] An exemplary polynucleotide encoding the Domain

VL of PD-1 mAb 15 is SEQ ID NO:246 (nucleotides encoding CDR1 residues are shown underlined):

123

CbQb ίη/77Ω7/Β/ΥΙΛΙ gacattcaga tgacccagtc tcccgcctcc cagtctgcat ctctgggaga aagtgtcacc atcacatgcc tggcaagtca gaccattggt acatggttag catggtatca gcagaaacca gggaaatctc ctcagctcct gatttatgct gcaaccagct t ggcagatgg ggtcccatca aggttcagtg gtagtggatc tggcacaaaa ttttctttca agatcagcag cctacaggct gaagattttg taaattatta ctgtcaacaa ctttacagta ttccgtggac gttcggtgga ggcaccaagc tggaaatcaa a

2. Humanization of Anti-human PD-1 Antibody PD-1 mAb 15 to Form hPD-1 mAb 15

[00184] The murine anti-human PD-1 antibody PD-1 mAb 15 described above was humanized and further deimmunized when antigenic epitopes were identified in order to demonstrate the ability to humanize an anti-human PD-1 antibody. human to reduce its antigenicity after administration in a human recipient. Humanization produced a humanized VH Domain, designated herein as hPD-1 VH1 mAb 2, and a humanized VL Domain designated herein as hPD-1 VL1 mAb 1. An antibody comprising the humanized VL Domain in pairs with the humanized VH Domain is referred to as hPD-1 mAb.

[00185] The amino acid sequence of the VH Domain of hPD-1 mAb 15 (SEQ ID NO:250) is shown below (hCDR residues are shown underlined).

[00186] An exemplary polynucleotide encoding hPD-1 mAb 15 VH1 is SEQ ID NO:251 (nucleotides encoding hCDR residues are shown underlined):

124 cnan Ln/zznz/E/YiAi gaagtgcaac tggttgaaag tggcggcggg ctggtgcggc caggtggttc actcagactg tcttgtgcag cttcaggctt tacattctcc tcttatctta tctcttgggt gcgccaagcc ccaggtaagg gccttgaatg ggtcgccgcc attagtgggg gtggt gccga tacatattat gccgacagcg tcaagggacg tttcaccatc agcagggaca acgccaagaa tagccttrac ctgcagatga actcacttag agctgaagac accgctactt attactgcgc ccqgcgcggg acttacgcta tggactattg gggccagggc accttggtca ctgtctcatc c

[00187] The amino acid sequence of the VH Domain of PD-1 mAb 15 VL1 (SEQ ID NO:252) is shown in the following (the CDR residues_h are shown underlined).

DIQMTQSPSS LSASVGDRVT ITCLASQTIG TWLAWYQQKP GKAPKLLIYA ATSLADGVPS RFSGSGSGTD FTFTISSLQP EDFATYYCQQ LYSIPWTFGQ GTKLEIK

[00188] An exemplary polynucleotide encoding hPD-1 mAb 15 VL1 is SEQ ID NO:253 (nucleotides encoding hCDR residues are shown underlined):

v. Anti-Human PD-1 Antibodies PD-1 mAb 1-15, and Their Derivatives Having an Engineered Fe Region [00189] In traditional immune function, the interaction of antibody-antigen complexes with cells of the immune system immune response results in a wide array of responses, varying from effector functions such as antibody-dependent cytotoxicity, mast cell degranulation, and phagocytosis to immunomodulatory signals such as regulating lymphocyte proliferation and antibody secretion. All of these interactions are initiated through

125 the binding of the Fe Region of antibodies or immune complexes to specialized cell surface receptors on hematopoietic cells. The diversity of cellular responses activated by antibodies and immune complexes results from the structural heterogeneity of the three Fe receptors: FcyRI (CD64), FcyRII (CD32), and FcyRIII (CD 16). FcyRI (CD64), FcyRII A (CD32A), and FcyRIII (CD16) are activation (i.e., immune system enhancement) receptors; FcyRIIB (CD32B) is a receptor for inhibition (i.e. regulation of the immune system). Furthermore, interaction with the neonatal Fe Receptor (FcRn) mediates the recycling of IgG molecules from the endosome to the cell surface and releases them into the blood. The amino acid sequence of exemplary wild-type igGl (SEQ ID NO:1), IgG2 (SEQ ID NO:2), IgG3 (SEQ ID NO:3), and IgG4 (SEQ ID NO:4) are presented below. former.

[00190] Modification of the Fe Region typically leads to an altered phenotype, for example, altered serum half-life, altered stability, altered susceptibility to cellular enzymes, or altered effector function. It may be desired to modify an antibody or other binding molecule of the present invention with respect to effector function, for example, to improve the effectiveness of such a molecule in treating cancer. Reduction or elimination of effector function is desirable in certain cases, by cnan Ln/zznz/E/YiAi

126 For example, in the case of antibodies whose mechanism of action involves blocking or antagonism, but not the extermination of cells carrying a target antigen. Increased effector function is generally desirable when targeting undesirable cells, such as tumor and foreign cells, where FcyRs are expressed at low levels, e.g., tumor-specific B cells with low levels of FcyRIIB (e.g., lymphoma of non Hodgkin, CLL, and Burkitt lymphoma). In such embodiments, the molecules of the invention with conferred or altered effector function activity are useful for the treatment and/or prevention of a disease, disorder or infection where improved efficacy of effector function activity is desired.

[00191] In certain embodiments, the PD-1 binding molecules of the present invention comprise an Fe region that possesses one or more modifications (e.g., substitutions, deletions or insertions) in the amino acid sequence of an Fe region. wild type (e.g., SEQ ID NO:1), which reduces the affinity and avidity of the Fe Region and, thus, the molecule of the invention, for one or more FcyR receptors. In other embodiments, the molecules of the invention comprise an Fe Region that possesses one or more modifications to the amino acids of the wild-type Fe Region, which increases the affinity and avidity of the Fe Region and, thus , the cnan molecule Ln/zznz/E/YiAi

127 invention, for one or more FcyR receptors. In other embodiments, the molecules comprise a variant Fe Region wherein such variant confers or mediates increased antibody-dependent cell-mediated cytotoxicity (ADCC) activity and/or increased binding to FcyRIIA, relative to a molecule that does not comprise the Faith Region or comprising a wild-type Faith Region. In alternative embodiments, the molecules comprise a variant Fe Region where such variant confers or mediates decreased ADCC activity (or other effector function) and/or increased binding to FcyRIIB, relative to a molecule that does not comprise the Fe Region. Fe or comprising a wild-type Fe Region. In some embodiments, the invention encompasses PD-1 binding molecules comprising a variant Fe Region, which variant Fe region does not show detectable binding to any FcyR, relative to a comparable molecule comprising the type Fe Region. wild. In other embodiments, the invention encompasses PD-1 binding molecules comprising a variant Fe Region, which variant Fe Region only binds to a single FcyR, preferably one of FcyRIIA, FcyRIIB, or FcyRIIIA. Any increased affinity and/or avidity is preferably assessed by measuring in vitro the degree of binding to FcyR or FcyR-related activity in cells expressing low levels of the FcyR when Ln/zznz/E/YiAi cnan Ln/zznz/E/YiAi

128 binding activity of the original molecule (without the modified Fe Region) cannot be detected in cells, or in cells expressing target antigens without FcyR receptor at a density of 30,000 to 20,000 molecules/cell, at a density of 20,000 to 10,000 molecules/cell, at a density of 10,000 to 5,000 molecules/cell, at a density of 5,000 to 1,000 molecules/cell, at a density of 1,000 to 200 molecules/cell or at a density of 200 molecules/cell or less (but at least 10, 50, 100 or 150 molecules/cell).

[00192] The PD-1 binding molecules of the present invention may comprise a variant Fe Region that has altered affinities for an activating and/or inhibitory Fcy receptor. In one embodiment, the PD-1 binding molecule comprises a variant Fe Region that has increased affinity for FcyRIIB and decreased affinity for FcyRIIIA and/or FcyRIIA, relative to a molecule comparable to a wild-type Fe Region. In another embodiment, the PD-1 binding molecules of the present invention comprise a variant Fe Region, which has decreased affinity for FcyRIIB and increased affinity for FcyRIIIA and/or FcyRIIA, relative to a comparable molecule with an Fe Region. wild type. In yet another embodiment, the PD-1 binding molecules of the present invention comprise a variant Fe Region that has decreased affinity for FcyRIIB and decreased affinity for FcyRIIIA and/or FcyRIIA, with

131 mast cells, calcium mobilization, degranulation, cytokine production, or serotonin release.

[00196] In certain embodiments, the molecules comprise an Fe Region comprising regions of two or more IgG isotypes (e.g., IgGl, IgG2, IgG3 and IgG4). As used herein, an Fe Region is said to be of a particular IgG isotype if its amino acid sequence is most homologous to that of the isotype relative to other IgG isotypes. The various IgG isotypes display different physical and functional properties including serum half-life, complement fixation, FcyR binding affinities, and effector function activities (e.g., ADCC, CDC, etc.) due to differences in sequences. of amino acids from their Hinge and/or Fe Regions, for example, as described in Flesch and Neppert (1999) J. Clin. Anal Lab. 14:141-156; Chappel et al. (1993) J Biol Chem 33:25124-25131; Chappel et al. (1991) Proc. Nati. Academic Sci. (USA) 88:9036-9040; or Brüggemann et al. (1987) J Exp Med 166:1351-1361. This type of variant Fe Region can be used alone, or in combination with an amino acid modification, to affect Fe-mediated effector function and/or binding activity. In combination, amino acid modification and the Hinge/Fe Region of IgG may show similar functionality (e.g., increased affinity for FcyRIIA) and may act additively or, in greater cnan Ln/zznz/E/YiAi

132 preferably, synergistically to modify the effector functionality in the molecule of the invention, relative to a molecule of the invention comprising a wild-type Fe Region. In other embodiments, the modification of amino acids and the Fe Region of IgG may display opposite functionality (e.g., increased and decreased affinity for FcyRIIA, respectively) and may act to selectively moderate or reduce a specific functionality in the molecule of the invention, in relation to a molecule of the invention that does not comprise an Fe Region or that comprises a wild type Fe Region of the same isotype.

[00197] In a specific preferred embodiment, the PD-1 binding molecules of the present invention comprise a variant Fe Region, wherein such variant Fe Region comprises at least one amino acid modification relative to a Fe Region of wild type, such that such a molecule has an altered affinity for an FcR, provided that such variant Fe Region does not have a substitution at positions that make direct contact with FcyR based on crystallographic and structural analysis of Fc-FcR interactions such as those described by Sondermann et al. (2000) Nature 406:267-73. Examples of positions within the Fe Region that make direct contact with FcyR are amino acid residues 234-239, amino acid residues

CbQb ίη/ΖΖΠΖ/Ε/ΥΙΛΙ

133

265-269 (loop B/C), amino acid residues 297-299 (loop C'/E), and amino acid residues 327-332 (loop F/G). In some embodiments, the molecules of the invention comprising variant Fe Regions comprise the modification of at least one residue that does not make direct contact with an FcyR based on structural and crystallographic analysis, for example, it is not located within the binding site. of Fc-FcyR.

[00198] Variant Fe Regions are well known in the art, and any known variant Fe Region can be used in the present invention to confer or modify the effector function exhibited by a molecule of the invention comprising an Fe Region (or portion thereof) as tested functionally, for example, in an NK-dependent or macrophage-dependent assay. For example, variant Fe Regions identified to alter effector function are described in PCT Publications Nos. WO 04/063351; WO 06/088494; WO 07/024249; WO 06/113665; WO 07/021841; WO 07/106707; and WO 2008/140603, and any suitable variant described herein can be used in the present molecules.

[00199] In certain embodiments, the PD-1 binding molecules of the present invention comprise a variant Fe Region, which has one or more amino acid modifications in one or more regions, which modifications alter (with cnan Ln / zznz / E/YiAi

134 relative to a wild type Fe Region) the Relationship of Affinities of the variant Fe Region with an activating FcyR (such as FcyRIIA or FcyRIIIA) relative to an inhibiting FcyR (such as FcyRIIB):

Change from Wild Type to Variant in Affinity to Relationship of Affinities^------------------------------------- ---Change from Natural Type to Variant in Affinity to

[00200] Particularly preferred are PD-1 binding molecules of the present invention that possess a variant Fe Region (relative to the wild type Fe Region) in which the variant Fe Region has an Affinity Ratio greater than 1. Such molecules have particular use for providing therapeutic or prophylactic treatment of a disease, disorder, or infection, or improvement of a symptom thereof, where improved efficiency of FcyR-mediated effector cell function (e.g., ADCC) is desired. , for example, cancer or infectious disease. In contrast, a variant Fe Region that has an Affinity Ratio less than 1 mediates decreased efficiency of effector cell function. Table 1 lists single, double, triple, quadruple and quintuple exemplary mutations in case their Affinity Ratio is greater or less than 1.

cnan Ln/zznz/E/YiAi

135 cnan ίη/ζζηζ/Ε/γίΛΐ

<td colspan="5">Table 1 Single and Multiple Exemplar Mutations Listed by Relationship of Affinities</td>

<td>Simple</td><td>Double</td><td>Triple</td><td>Quadruple</td><td>Quintuple</td>

<td></td><td></td><td colspan="3">Affinities Ratio &gt; 1</td>

<td>F243L</td><td>F243L &amp; R292P</td><td>F243L, P247L &amp; N421K</td><td>L234F, F243L, R292P &amp; Y300L</td><td>L235V, F243L, R292P, Y300L &amp; P396L</td>

<td>D270E</td><td>F243L &amp; Y300L</td><td>F243L, R292P &amp; Y300L</td><td>L235I, F243L, R292P &amp; Y300L</td><td>L235P, F243L, R292P, Y300L &amp; P396L</td>

<td>R292G</td><td>F243L &amp; P396L</td><td>F243L, R292P &amp; V305I</td><td>L235Q, F243L, R292P &amp; Y300L</td><td>F243L, R292P, V305I, Y300L &amp; P396L</td>

<td>R292P</td><td>D270E &amp; P396L</td><td>F243L, R292P &amp; P396L</td><td>F243L, P247L, D270E&amp;N421K</td><td></td>

<td></td><td>R292P &amp; Y3O0L</td><td>F243L, Y300L &amp; P396L</td><td>F243L, R255L, D270E &amp; P396L</td><td></td>

<td></td><td>R292P &amp; V3O5I</td><td>P247L, D270E &amp; N421K</td><td>F243L, D270E, G316D&amp;R416G</td><td></td>

<td></td><td>R292P &amp; P396L</td><td>R255L, D270E&amp; P396L</td><td>F243L, D270E, K392T &amp; P396L</td><td></td>

<td></td><td>Y300L&amp; P396L</td><td>D270E, G3 1 6D &amp; R416G</td><td>F243L, D270E, P396L&amp;Q419H</td><td></td>

<td></td><td>P396L &amp; Q419H</td><td>D270E, K392T &amp; P396L</td><td>F243L, R292P, Y300L, &amp; P396L</td><td></td>

<td></td><td></td><td>D270E, P396L &amp; Q419H</td><td>F243L, R292P, V305I &amp; P396L</td><td></td>

<td></td><td></td><td>V284M, R292L &amp; K370N</td><td>P247L, D270E, Y300L&amp;N421K</td><td></td>

<td></td><td></td><td>R292P, Y300L &amp; P396L</td><td>R255L, D270E, R292G &amp; P396L</td><td></td>

<td></td><td></td><td></td><td>R255L, D270E, Y300L &amp; P396L</td><td></td>

<td></td><td></td><td>Relation of</td><td>D270E, G316D, P396L &amp;R416G Affinities &lt; 1</td><td></td>

<td>Y300L</td><td>F243L &amp; P396L</td><td>F243L, R292P &amp; V305I</td><td></td><td></td>

<td>P396L</td><td>P247L &amp; N421K</td><td></td><td></td><td></td>

<td></td><td>R255L &amp; P396L</td><td></td><td></td><td></td>

<td></td><td>R292P &amp; V3051</td><td></td><td></td><td></td>

<td></td><td>K392T &amp; P396L</td><td></td><td></td><td></td>

<td></td><td>P396L &amp; Q419H</td><td></td><td></td><td></td>

136

[00201] In a specific embodiment, in variant Fe Regions, any amino acid modifications (e.g., substitutions) at any of positions 235, 240, 241, 243, 244, 247, 262, 263, 269, 298, 328 or 330 and preferably one or more of the following residues: A240, l240, L241, L243, H244, N298, 1328 or V330. In a different specific embodiment, in variant Fe Regions, any amino acid modifications (e.g., substitutions) at any of positions 268, 269, 270, 272, 276, 278, 283, 285, 286, 289, 292, 293 , 301, 303, 305, 307, 309, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438 or 439 and preferably one or more of the following residues: H280, Q280, Y280, G290, S290, T290, Y290, N294, K295, P296, D298, N298, P298, V298, 1300 or L300.

cnan Ln/zznz/E/YiAi

[00202] In a preferred embodiment, in Regions of Faith

<td colspan="2">variants</td><td>that</td><td>unite</td><td>a</td><td>FcyR</td><td>with</td><td>a</td><td colspan="2">affinity</td><td colspan="2">altered,</td>

<td colspan="9">any amino acid modifications</td><td>(by</td><td colspan="2">example,</td>

<td colspan="2">substitution</td><td>ones)</td><td colspan="3">in any of</td><td>the</td><td colspan="2">positions</td><td> 255,</td><td> 256,</td><td> 258,</td>

<td> 267,</td><td> 268,</td><td> 269,</td><td> 270,</td><td> 272,</td><td> 276,</td><td> 278,</td><td> 280,</td><td> 283,</td><td> 285,</td><td> 286,</td><td> 289,</td>

<td> 290,</td><td> 292,</td><td> 293,</td><td> 294,</td><td> 295,</td><td> 296,</td><td> 298,</td><td> 300,</td><td> 301,</td><td> 303,</td><td> 305,</td><td> 307,</td>

<td> 309,</td><td> 312,</td><td> 320,</td><td> 322,</td><td> 326,</td><td> 329,</td><td> 330,</td><td> 332,</td><td> 331,</td><td> 333,</td><td> 334,</td><td> 335,</td>

<td> 337,</td><td> 338,</td><td> 339,</td><td> 340,</td><td> 359,</td><td> 360,</td><td> 373,</td><td> 376,</td><td> 416,</td><td> 419,</td><td> 430,</td><td> 434,</td>

<td> 435,</td><td> 437,</td><td>438 or</td><td> 439 .</td><td colspan="3">Preferably,</td><td>the</td><td>Region</td><td>of faith</td><td colspan="2">i variant</td>

<td>has</td><td colspan="3">any of</td><td>the</td><td colspan="4">following waste</td><td colspan="2">: A256,</td><td>N268,</td>

137

Q272, D286, Q286, S286, Α290, S290, Α298, Μ301, Α312,Ε320,

Μ320, Q320, R320, Ε322, Α326, D326, Ε326, Ν326, S326,Κ330,

Τ339, Α333, Α334, Ε334, Η334, L334, Μ334, Q334, V334,Κ335,

Q335, Α359, Α360 or A430.

[00203] In a different embodiment, in variant Fe Regions that bind an FcyR (via its Fe Region) with a reduced affinity, any amino acid modifications (e.g., substitutions) at any of the positions

252, 254, 265, 268, 269, 270, 278, 289, 292, 293, 294,295,

296, 298, 300, 301, 303, 322, 324, 327, 329, 333, 335,338,

340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435,437,

438 or 439.

[00204] In a different embodiment, in variant Fe Regions that bind an FcyR (via its Fe Region) with improved affinity, any amino acid modifications (e.g., substitutions) at any of positions 280, 283, 285, 286, 290, 294, 295, 298, 300, 301, 305, 307, 309, 312, 315, 331, 333, 334, 337, 340, 360, 378, 398 or 430. In a different embodiment, in variant Fe Regions that bind FcyRIIA with improved affinity, any of the following residues: A255, A256, A258, A267, A268, N268,

A272, Q272, A276, A280, A283, A285, A286, D286, Q286, S286, A290, S290, M301, E320, M320, Q320, R320, E322, A326, D326, E326, S326, K330, A331, Q335 , A337 or A430.

[00205] Preferred variants include one or more cnan Ln/zznz/E/YiAi

138 modifications in any of the positions: 228, 230, 231, 232, 233, 234, 235, 239, 240, 241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 271, 273, 275 , 281, 284, 291, 296, 297, 298, 299, 302, 304, 305, 313, 323, 325, 326, 328, 330 or 332.

[00206] Particularly preferred variants include one or more modifications selected from groups A-AI:

[00207] Even more particularly preferred variants include one or more modifications selected from Groups 1-105:

CbQb ίη/77Ω7/Β/ΥΙΛΙ

139 cnan Ln/zznz/E/YiAi

<td>TO</td><td>228E, 228K, 228Y or 228G;</td>

<td>b</td><td>230A, 230E, 230Y or 230G;</td>

<td>c</td><td>231E, 231K, 231Y, 231P or 231G;</td>

<td>d</td><td>232E, 232K, 232Y, 232G;</td>

<td>AND</td><td>233D;</td>

<td>F</td><td>2341 or 234F;</td>

<td>g</td><td>235D, 235Q, 235P, 2351 or 235V;</td>

<td>h</td><td>239D, 239E, 239N or 239Q;</td>

<td>Yo</td><td>240A, 2401, 240M or 240T;</td>

<td>J.</td><td>243R, 243, 243Y, 243L, 243Q, 243W, 243H or 2431;</td>

<td>K</td><td>244H;</td>

<td>l</td><td>245A;</td>

<td>M</td><td>247G, 247V or 247L;</td>

<td>N</td><td>262A, 262E, 2621, 262T, 262E or 262F;</td>

<td> 0</td><td>263A, 2631, 263M or 263T;</td>

<td>Q</td><td>264F, 264E, 264R, 2641, 264A, 264T or 264W;</td>

<td>Q</td><td>265F, 265Y, 265H, 2651, 265L, 265T, 265V, 265N or 265Q;</td>

<td>R</td><td>266A, 2661, 266M or 266T;</td>

<td>yes</td><td>271D, 271E, 271N, 271Q, 271K, 271R, 271 S, 271T, 271H, 271A, 271V, 271L, 2711, 271F, 271M, 271Y, 271W or 271G;</td>

<td>T</td><td> 2731;</td>

<td>or</td><td>275Lo 275W;</td>

<td>V</td><td>281D, 281K, 281Y or 281P;</td>

<td>w</td><td>284E, 284N, 284T, 284L, 284Y or 284M;</td>

<td>x</td><td>291D, 291E, 291Q, 291T, 291H, 2911 or 291G;</td>

<td>AND</td><td>299A, 299D, 299E, 299F, 299G, 299H, 2991, 299K, 299L, 299M, 299N, 299P, 299Q, 299R, 299S, 299V, 299W or 299Y;</td>

<td>z</td><td> 3021;</td>

<td>AA</td><td>304D, 304N, 304T, 304H or 304L</td>

<td>AB</td><td> 3051;</td>

<td>A.C.</td><td>313F;</td>

<td>A.D.</td><td> 3231;</td>

<td>A.E.</td><td>325A, 325D, 325E, 325G, 325H, 3251, 325L, 325K, 325R, 325S, 325F, 325M, 325T, 325V, 325Y, 325W or 325P;</td>

<td>AF</td><td>328D, 328Q, 328K, 328R, 328S, 328T, 328V, 3281, 328Y, 328W, 328P, 328G, 328A, 328E, 328F, 328H, 328M or 328N;</td>

<td>AG</td><td>330L, 330Y, 3301 or 330V;</td>

<td>AH</td><td>332A, 332D, 332E, 332FI, 332N, 332Q, 332T, 332K, 332R, 332S, 332V, 332L, 332F, 332M, 332W, 332P, 332G or 332Y; and</td>

<td>AI</td><td>336E, 336K or 336Y</td>

[00208] In one embodiment a PD-1 binding molecule of

140 The invention will comprise a variant Fe Region having at least one modification to the Fe Region. In certain embodiments, the variant Fe Region comprises at least one substitution selected from the group consisting of L235V, F243L, R292P, Y300L, Y305I and P396L.

[00209] In a specific embodiment, the variant Faith Region comprises:

(A) at least one substitution selected from the group consisting of F243L, R292P, Y300L, V305I and P396L;

(B) at least two substitutions selected from the group consisting of:

(1) F243L and P396L;

(2) F243L and R292P; and (3) R292P and V305I;

(C) at least three substitutions selected from the group consisting of:

(1) F243L, R292P and Y300L;

(2) F243L, R292P and V305I;

(3) F243L, R292P and P396L; and (4) R292P, V305I and P396L;

(D) at least four substitutions selected from the group consisting of:

(1) F243L, R292P, Y300L and P396L; and (2) F243L, R292P, V305I and P396L; or (E) at least the five selected substitutions

CbQb ίη/ΖΖΩΖ/Ε/ΥΙΛΙ

141 of the group consisting of:

(1) F243L, R292P, Y300L, V305I and P396L; and (2) L235V, F243L, R292P, Y300L and P396L.

[00210] In another specific embodiment, the variant Faith Region comprises substitutions of:

(A) F243L, R292P, and Y300L;

(B) L235V, F243L, R292P, Y300L, and P396L; or (C) F243L, R292P, Y300L, V305I, and P396L.

[00211] In one embodiment, a PD-1 binding molecule of the invention comprises a variant Fe region that shows decreased (or substantially no) binding to FcyRIA (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD16a) or FcyRIIIB (CD16b) (relative to the binding shown by the Fe Region of wild-type IgGl (SEQ ID NO:1)). In one embodiment, a PD-1 binding molecule of the invention will comprise a variant Fe Region that exhibits reduced (or substantially no) binding to an FcyR (e.g., FcyRIIIA) and reduced (or substantially no) ADCC effector function. none). In certain embodiments, the variant Fe Region comprises at least one substitution selected from the group consisting of L234A, L235A, D265A, N297Q and N297G. In a specific embodiment, the variant Fe Region comprises the substitution of L234A; L235A; L234A and L235A; D265A; N297Q, or N297G.

[00212] A Preferred IgGl Sequence for Ln/zznz/E/YiAi cnan Domains

142

CH2 and CH3 of the PD-1 binding molecules of the invention will have the substitutions L234A/L235A (SEQ ID NO:5):

APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQ QG NVFSCSVMHE ALHNHYTQKS LSLSPGX where, X is a Power Plant (K) or is absent.

[00213] In a different embodiment, a PD-1 binding molecule of the invention comprises an Fe region that inherently exhibits decreased (or substantially no) binding to FcyRIIIA (CD16a) and/or reduced effector function ( relative to the binding shown by the Fe Region of wild-type IgGl (SEQ ID NO:1)). In a specific embodiment, a PD-1 binding molecule of the present invention comprises an IgG2 Fe Region (SEQ ID NO: 2) or an IgG4 Fe Region (SEQ ID NO: 4). When an IgG4 Fe Region is used, the present invention also encompasses the introduction of a stabilizing mutation, such as the IgG4 hinge region S228P substitution (see, for example, SEQ ID NO: 13: ESKYGPPCPPCP, (Lu et al., (2008) The Effect Of A Point Mutation On The Stability Of Igg4 As Monitored By Analytlcal Ultracentrlfugatlon J. Pharmaceutical Sciences 97:960-969) to reduce the incidence of a twisted exchange. Other stabilizing mutations known in the art can be introduced into a Fe cnan Ln/zznz/E/YiAi Region.

CbQb ίη/77Ω7/Β/ΥΙΛΙ

143 of IgG4 (Peters, P et al., (2012) Engineering an Improved IgG4 Molecule with Reduced Disulfide Bond Heterogeneity and Increased Fab Domain Thermal Stability, J. Biol. Chem., 287:24525-24533; PCT Patent Publication No.: WO 2008/145142).

[00214] In other embodiments, the invention encompasses the use of any variant Fe Region known in the art, such as that described in Jefferis, BJ et al (2002) Interaction Sites On Human IgG-Fc For FcgammaR: Current Models Immunol.

Lett. 82:57-65; Presta, LG et al (2002) Engineering

Therapeutic Antibodies For Improved Function, Biochem. Soc.

Trans. 30:487-90; Idusogie, EE et al (2001) Engineered Antibodies With Increased Activity To Recruit Complement, J.

Immunol. 166:2571-75; Shields, RL et al (2001) High Resolution Mapping Of The Binding Site On Human IgGl For Fe Gamma RI, Fe Gamma RII, Fe Gamma RUI, And FcRn And Design Of IgGl Variants With Improved Binding To The Fe gamma R, J.

Biol Chem 276:6591-6604; Idusogie, EE et al (2000) Mapping Of The Clq Binding Site On Rituxan, A Chimeric Antibody With A Human IgG Fe, J. Immunol. 164:4178-84;

Reddy, MP et al (2000) Elimination Of Fe ReceptorDependent Effector Functions Of A Modified IgG4 Monoclonal Antibody To Human CD4, J. Immunol. 164:1925-1933; Xu, D. et al (2000) In Vitro Characterization of Five Humanized OKT3 Effector Function Variant Antibodies, Cell. Immunol. 200:16144

26; Armour, KL et al (1999) Recombinant human IgG Molecules Lacking Fcgamma Receptor I Binding And Monocyte Triggering Activities, Eur. J. Immunol. 29:2613-24;

Jefferis, R. et al (1996) Modulation Of Fe (Gamma)R And Human Complement Activation By IgG3-Core Oligosaccharide

Interactions, Immunol. Lett. 54:101-04; Lund, J. et al (1996) Multiple Interactions Of IgG With Its Core Oligosaccharide Can Modulate Recognition By Complement And Human Fe Gamma Receptor I And Influence The Synthesis Of Its Oligosaccharide Chains, J. Immunol. 157:4963-4969; Hutchins et al (1995) Improved Biodistribution, Tumor Targeting, And Reduced Immunogenicity In Mice With A Gamma 4 Variant Of Campath-IH, Proc. Nati. Academic Sci. (USA) 92:11980-84;

Jefferis, R. et al (1995) Recognition Sites On Human IgG For Fe Gamma Receptors: The Role Of Glycosylation, Immunol. Lett. 44:111-17; Lund, J. et al (1995) OI igosaccharide Protein Interactions In IgG Can Modulate Recognition By Fe Gamma Receptors, FASEB J. 9:115- 19; Alegre, ML et al (1994) 'Ά Non-Activating Humanized Anti-CD3 Monoclonal Antibody Retains Immunosuppressive Properties In Vivo Transplantation 57:1537-1543; Lund et al. (1992) Multiple Binding Sites On The CH2 Domain Of IgG For Mouse Fe Gamma Rll, Mol. Immunol. 29:53-59; Lund et al (1991) Human Fe cnan Ln/zznz/E/YiAi

Gamma RI And Fe Gamma RII Interact With Distinct But

Overlapping Sites On Human IgG, J. Immunol. 147:2657-2662;

CbQb ίη/ΖΖΠΖ/Ε/ΥΙΛΙ

145

Duncan, AR et al (1988) Localization Of The Binding Site For The Human High-Affinity Fe Receptor On IgG Nature 332:563-564; United States Patent Nos. 5,624,821; 5,885,573; 6,194,551; 7,276,586; and 7,317,091; and PCT Publications WO 00/42072 and PCT WO 99/58572.

[00215] In some embodiments, the molecules of the invention further comprise one or more glycosylation sites, such that one or more carbohydrate moieties are covalently linked to the molecule. Preferably, molecules of the invention with one or more glycosylation sites and/or one or more modifications in the Fe Region confer or have an enhanced antibody-mediated effector function, for example, enhanced ADCC activity, compared to the antibody modified. In some embodiments, the invention further comprises molecules comprising one or more amino acid modifications that are known directly or indirectly to interact with a carbohydrate moiety of the Fe Region, including but not limited to amino acids at positions 241, 243, 244. , 245, 245, 249, 256, 258, 260, 262, 264, 265, 296, 299, and 301. Amino acids that interact directly or indirectly with a carbohydrate moiety of an Fe Region are known in the art, see, for example, Jefferis et al, 1995 Immunology Letters, 44: 111-7, which is incorporated herein for reference in its entirety.

146

[00216] In another embodiment, the invention encompasses molecules that have been modified by introducing one or more glycosylation sites at one or more sites on the molecules, preferably without altering the functionality of the molecules, for example, antigen binding activity. target or FcyR. The glycosylation sites can be introduced into the variable and/or constant region of the molecules of the invention. As used herein, glycosylation sites include any specific amino acid sequence in an antibody that will specifically and covalently bind to an oligosaccharide (i.e., carbohydrates containing two or more simple sugars linked together). Oligosaccharide side chains are typically linked into the backbone of an antibody via either N- or O-bonds. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid, e.g., serine, threonine. Molecules of the invention may comprise one or more glycosylation sites, including N-linked and 0-linked glycosylation sites. Any glycosylation site for N-linked or O-linked glycosylation known in the art can be used according to the instant invention.

A site of cnan Ln/zznz/E/YiAi cnan Ln/zznz/E/YiAi

147 Exemplary N-linked glycosylation that is useful according to the methods of the present invention is the amino acid sequence: Asn-X-Thr/Ser, where X can be any amino acid and Thr/Ser indicates a threonine or a serine. Such a site or sites may be introduced into a molecule of the invention using methods well known in the art to which this invention pertains (see for example, IN VITRO MUTAGENESIS, RECOMBINANT DNA: A SHORT COURSE, JD Watson, et al WH Freeman and Company, New York, 1983, chapter 8, pp. 106-116, which is incorporated herein by reference in its entirety. An exemplary method for introducing a glycosylation site into a molecule of the invention may comprise: modifying or mutating an amino acid sequence of the molecule such that the desired Asn-X-Thr/Ser sequence is obtained.

[00217] In some embodiments, the invention encompasses methods of modifying the carbohydrate content of a molecule of the invention by adding or deleting a glycosylation site. Methods for modifying the carbohydrate content of antibodies (and molecules comprising antibody domains, e.g. Fe region) are well known in the art and are encompassed within the invention, see, for example, United States Patent United No. 6,218,149; EP 0 359 096 Bl; United States Publication No. US 2002/0028486; WO 03/035835; Publication of the States

148

States No. 2003/0115614; United States Patent No. 6,218,149; United States Patent No. 6,472,511; all of which are incorporated herein by reference in their entirety. In other embodiments, the invention encompasses methods for modifying the carbohydrate content of a molecule of the invention by removing one or more endogenous carbohydrate moieties from the molecule. In a specific embodiment, the invention encompasses changing the glycosylation site of the Fe Region of an antibody, by modifying adjacent positions at 297. In a specific embodiment, the invention encompasses modifying position 296 so that position 296 and not position 297 is glycosylated. [00218] The effector function can also be modified by techniques such as by introducing one or more cysteine ​​residues into the Fe Region, thereby allowing the formation of disulfide bonds between chains in this region to occur, resulting in the generation of an antibody. homodimeric that may have an improved internalization capacity and/or cell killing mediated by increased complement and ADCC (Carón, PC et al. (1992) Engineered Humanized Dimeric Forms Of IgG Are More Effective Antibodies. J Exp Med 176:1191-1195; Shopes, B. (1992) A Genetically Engineered Human IgG Mutant with Enhanced Cytolytic Activity. J. Immunol. 148(9):2918-2922. cnan Ln/zznz/E/YiAi homodimeric antibodies with enhanced antitumor activity can also

149 prepare using heterobifunctional cross-linkers as described in Wolff, EA et al. (1993) Monoclonal Antibody Homodimers: Enhanced Antitumor Activity In Nude Mice Cancer Research 53:2560-2565. Alternatively, an antibody can be designed that has dual Fe regions and can thus enhance complement lysis and ADCC capabilities (Stevenson, GT et al. (1989) A Chimeric Antibody with Dual Fe Regions (bisFabFc) Prepared By Manipulations At The IgG Hinge Anti-Cancer Drug Design 3:219-230).

[00219] The mean serum pathway of molecules of the present invention comprising Fe Regions can be increased by increasing the binding affinity of the Fe Region for FcRn. The term half-life as used herein means a pharmacokinetic property of a molecule that is a measure of the average survival time of the molecules after administration. The half-life may be expressed as the time required to eliminate fifty percent (50%) of a known amount of the molecule from the body of a subject (e.g., human patient or other mammal) or a specific compartment thereof, e.g. when measured in serum, that is, circulating half-life, or in other tissues. In general, an increase in daily life results in an increase in the mean residence time (MRT) in cnan Ln/zznz/E/YiAi circulation for the administered molecule.

150

[00220] In some embodiments, the PD-1 binding molecules of the present invention comprise a variant Fe Region, wherein the variant Fe Region comprises at least one amino acid modification relative to a wild-type Fe Region , so that the molecule has an increased half-life (relative to a wild-type Fe Region).

[00221] In some embodiments, the PD-1 binding molecules of the present invention comprise a variant Fe Region, wherein the variant Fe Region comprises a half-life that extends the amino acid substitution at one or more selected positions of the group consisting of 238, 250, 252, 254, 256, 257, 256, 265, 272, 286, 288, 303, 305,

307, 308, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378,

380, 382, ​​413, 424, 428, 433, 434, 435, and 436. Numerous specific mutations capable of increasing the half-life of a molecule containing an Fe Region are known in the art and include, for example, M252Y, S254T , T256E, and combinations thereof. For example, see the mutations described in US Patent Nos. 6,277,375, 7,083,784; 7,217,797, 8,088,376;

United States Publications Nos. 2002/0147311; 2007/0148164; and International Publications Nos. WO 98/23289; WO 2009/058492; and WO 2010/033279, all of which are incorporated herein by reference in their entirety.

cnan Ln/zznz/E/YiAi

151

Molecules containing the Fe Region with enhanced half-life also include those with substitutions at two or more Fe Region residues 250, 252, 254, 256, 257, 288, 307, 308, 309, 311, 378, 428, 433, 434, 435 and 436. In particular, two or more substitutions selected from: T250Q, M252Y, S254T, T256E, K288D, T307Q, V308P, A378V, M428L, N434A, H435K, and Y436I.

[00222] In a specific embodiment, the variant Faith Region comprises substitutions of:

(A) M252Y, S254T and T256E;

(B) M252Y and S254T;

(C) M252Y and T256E;

(D) T250Q and M428L;

(E) T307Q and N434A;

(F) A378V and N434A;

(G) N434A and Y436I;

(H) V308P and N434A; or (I) K288D and FI435K.

[00223] Instant invention further encompasses variant Fe Regions comprising:

(A) one or more mutations that alter effector and/or FcyR function; and (B) one or more mutations that extend the mean serum cnan Ln/zznz/E/YiAi lifespan.

152

SAW. Bispecific anti-human PD-1 binding molecules

[00224] One embodiment of the present invention relates to bispecific binding molecules that are capable of binding to a first epitope and a second epitope, wherein the first epitope is a human PD-1 epitope and the second epitope is the same. epitope or different from PD-1, or is an epitope of another molecule that is present on the surface of an immune cell (such as a T cell) and is involved in regulating an immune checkpoint. In one embodiment, the second epitope is an epitope of B7-H3, B7-H4, BTLA, CD3, CD8, CD16, CD27, CD32, CD40, CD40L, CD47, CD64, CD70, CD80, CD86, CD94, CD137, CD137L , CD226, CTLA-4, Galectin-9, GITR, GITRL, HHLA2, ICOS, ICOSL, KIR, LAG-3, LIGHT, MHC class I or II, NKG2a, NKG2d, 0X40, OX40L, PD1H, PD1, PD-L1 , PD-L2, PVR, SIRPa, TCR, TIGIT, TIM-3 or VISTA. In one embodiment, the second epitope is not a PD-1 epitope. In a specific embodiment, the second epitope is CD137, CTLA-4, LAG-3, 0X40, TIGIT, or TIM-3. In certain embodiments, a bispecific molecule comprises more than two epitope binding sites. Such bispecific molecules may bind to two or more different LAG-3 epitopes and at least one epitope of a non-LAG-3 molecule.

[00225] The instant invention encompasses bispecific antibodies capable of simultaneously binding to PD-1 and the second epitope (e.g., B7-H3, B7-H4, BTLA, CD40, cnan Ln/zznz/E/YiAi

153

CD80, CD86, CD137, CTLA-4, ICOS, KIR, LAG-3, MHC class I or II, 0X40, PD-L1, TCR, TIM-3, etc.). In some embodiments, the bispecific antibody capable of simultaneously binding PD-1 and the second epitope is produced using any of the methods described in PCT Publications Nos. WO 1998/002463, WO 2005/070966, WO 2006/107786 WO 2007/024715, WO 2007/075270, WO 2006/107617, WO 2007/046893, WO 2007/146968, WO 20 08/003103, WO 2008/003116, WO 2008/027236, WO 2008/024188, WO 2009/132876, WO 2009/018386, WO 2010/028797, WO 2010028796, WO 2010/028795, WO 2010/108127, WO 2010 /136172, WO 2011/086091, WO 2011/ 133886, WO 2012/009544, WO 2013/003652, WO 2013/070565, WO 2012/162583, WO 2012/156430, WO 2013/174873, and WO 2014/022540, each of which is incorporated herein by reference in its entirety.

A. Fe regions lacking bispecific diabodies

[00226] One embodiment of the present invention relates to bispecific diabodies comprising, and most preferably consisting of, a first polypeptide chain and a second polypeptide chain, the sequences of which allow the polypeptide chains to link covalently. with each other to form covalently associated diabodies that are capable of cnan binding Ln/zznz/E/YiAi

154 simultaneous to a first epitope and a second epitope, such epitopes are not identical to each other. Such bispecific diabodies thus comprise VL1/VH1 Domains that are capable of binding to the first epitope and VL2/VH2 Domains that are capable of binding to the second epitope. The notation VL1 and VH1 denote, respectively, the Variable Light Chain Domain and the Variable Heavy Chain Domain that bind to the first epitope of such a bispecific diabody. Similarly, the notation VL2 and VH2 denote, respectively, the Variable Light Chain Domain and the Variable Heavy Chain Domain that bind to the second epitope of such a bispecific diabody. It is irrelevant whether a particular epitope is designated as the first versus the second epitope; Such notation only has relevance with respect to the presence and orientation of domains of the polypeptide chains of the binding molecules of the present invention. In one embodiment, one such epitope is a PD-1 epitope and the other such epitope is not a PD-1 epitope (e.g., an epitope of B7-H3, B7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, KIR, LAG-3, MHC class I or II, 0X40, PD-L1, TCR, TIM-3, etc.). [00227] The VL domain of the first polypeptide chain interacts with the VH domain of the second polypeptide chain to form a functional first antigen binding site that is specific for a first antigen (es cnan Ln / zznz / E / YiAi

155 i.e., either PD-1 or an antigen containing the second epitope). Likewise, the VL domain of the second polypeptide chain interacts with the VH domain of the first polypeptide chain to form a second functional antigen binding site that is specific for a second antigen (i.e., either an antigen that contain the second epitope or PD-1). In this way, the selection of the VL and VH Domains of the first and second polypeptide chains is coordinated, so that the two polypeptide chains of the diabody collectively comprise VL and VH Domains capable of binding to both a PD epitope -1 and the second epitope (i.e., they comprise VLpd-i/VHpd-i and VL2/VH2, where PD-1 is the first epitope, or VL1/VH1 and VLpd-i/VHpd-i, where PD- 1 is the second epitope).

[00228] The first polypeptide chain of one embodiment of such bispecific diabodies comprises, in the N-terminal to C-terminal direction, an N-terminus, the VL1 Domain of a monoclonal antibody capable of binding to any of the first or second epitope (i.e. , any of VL_P.S-io VL_Epitope 2), a first intervening spacer peptide (Linker 1), a VH2 Domain of a monoclonal antibody capable of binding to either the second epitope (if the first polypeptide chain contains VLpd-i) or the first epitope (if the cnan Ln/zznz/E/YiAi first polypeptide chain contains VLEpitopo 2), a second intervening spacer peptide (Linker 2) containing

CbQb ίη/ΖΖΩΖ/Ε/ΥΙΛΙ

156 optionally a cysteine ​​residue, a Heterodimer Promoter Domain and a C-terminus (Figure 1).

[00229] The second polypeptide chain of this embodiment of bispecitic diabodies comprises, in the N-terminal to C-terminal direction, an N-terminus, a VL2 Domain of a monoclonal antibody capable of binding to any PD-1 or to the second epitope (i.e. i.e., either VLpd-i or VLE (pitope 2, and the VL domain not selected for inclusion in the first polypeptide chain of the diabody), an intervening linker peptide (Linker 1), a VH1 Domain of a monoclonal antibody capable of binding to any of the second epitope (if the second polypeptide chain contains VLp_d-i) or to PD-1 (if the second polypeptide chain contains VL_Epitepe 2), a second intervening spacer peptide (Linker 2) that optionally contains a cysteine ​​residue, a Heterodimer Promoter Domain, and a C-terminus (Figure 1).

[00230] Most preferably, the length of the intervening linker peptide (e.g., Linker 1) separating such VL and VH Domains is selected to substantially or completely prevent the VL and VH Domains of the polypeptide chain from binding to each other. Thus, the VL and VH Domains of the first polypeptide chain are substantially or completely incapable of binding to each other. Similarly, the VL and VH Domains of the second chain of

157 polypeptides are substantially or completely incapable of binding to each other. A preferred intervening spacer peptide (Linker 1) has the sequence (SEQ ID NO: 14): GGGSGGGG.

[00231] The length and composition of the second intervening linker peptide (Linker 2) is selected based on the choice of heterodimer promoter domains. Typically, the second linker peptide of interest (Linker 2) will comprise 3-20 amino acid residues. In particular, where the Heterodimer Promoter Domains do not comprise a cysteine ​​residue, a second intervening linker peptide (Linker 2) containing cysteine ​​is used. A second intervening spacer peptide (Linker 2) containing cysteine ​​will contain 1, 2, 3 or more cysteines. A spacer peptide (Linker 2) containing cysteine ​​has the sequence that is SEQ ID NO: 15: GGCGGG. Alternatively, Linker 2 does not comprise a cysteine ​​(e.g., GGG, GGGS (SEQ ID NO:29), LGGGSG (SEQ ID NO:261), GGGSGGGSGGG (SEQ ID NO:262), ASTKG (SEQ ID NO:30) , LEPKSS (SEQ ID NO:33), APSSS (SEQ ID NO:34), etc.) and a Cysteine-Containing Heterodimer Promoter Domain is used, as described below. Optionally, both a cysteine-containing Linker 2 and a Cysteine-Containing Heterodimer Promoter Domain are used.

CbQb ίη/ΖΖΠΖ/Ε/ΥΙΛΙ

158

[00232] Heterodimer Promoter Domains may be GVEPKSC (SEQ ID NO:16) or VEPKSC (SEQ ID NO:17) or AEPKSC (SEQ ID NO:18) in a polypeptide chain and GFNRGEC (SEQ ID NO: 19) or FNRGEC (SEQ ID NO:20) on the other polypeptide chain (US2007/0004909).

[00233] However, more preferably, the heterodimer Promoter Domains of such diabodies are formed from one, two, three or four tandemly repeated helical Domains of opposite charge comprising a sequence of at least six, at least seven or at least eight amino acid residues so that the Heterodimer Promoter Domain has a net charge (Apostolovic, B. et al. (2008) pH-Sensiti vity of the E3/K3 Heterodimeric Coiled Coil of Biomacromolecules 9:3173-3180; Arndt, K.M. et al. (2001) Helix-stabilized Fv (hsFv) Antibody Fragments: Substituting the Constant Domains of a Fab Fragment for a Heterodimeric Coiled-coil Domain, J. Molec. Biol. 312:221-228; Arndt, K.M. et al. (2002) Comparison of In Vivo Selection and Rational Design of Heterodimeric Coiled Coils of Structure 10:12351248; Boucher, C. et al. (2010) Protein Detection By Western Blot Via Coiled-Coil Interactions of Analytical Biochemistry 399:138-140; Cachúa, PJ et al. (2004) Synthetic Peptide Vaccine Development: Measurement Of Polyclonal Antibody Affinity and Cross-Reactivity Using A New Peptide Capture and Pelease System For Sur face Plasmon Resonance Spectroscopy J.

cnan Ln/zznz/E/YiAi cnan Ln/zznz/E/YiAi

159

Mol. Recognit. 17:540-557; De Crescenzo, GD et al. (2003) Real-Time Monitoring of the Interactions of Two-Stranded de novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding, Biochemistry 42:1754-1763; Fernandez-Rodríguez, J. et al. (2012) Induced Heterodimerization and Purification Of Two Target Proteins By A Synthetic Coiled-Coil Tag, Protein Science 21:511-519; Ghosh, T. S. et al. (2009) End-To-End and End-To-Middle Interhelical Interactions: New Classes Of Interacting Helix Pairs In Protein Structures, Acta Crystallographica D65:1032-1041; Grigoryan, G. et al. (2008) Structural Specificity In Coiled-Coil Interactions, Curr. Opinion. Struc. Biol. 18:477-483; Litowski, JR et al. (2002) Designing Heterodimeric Two-Stranded a-Helical Coiled-Coils: The Effects Of Hydrophobicity and a-Helical Propensity On Protein Folding, Stability, and Specificity, J. Biol. Chem. 277:37272-37279; Steinkruger, J.D. et al. (2012) The d'—d— d' Vertical Triad is Less Discriminating Than the a'—a—a' Vertical Triad in the Antiparallel Coiled-coil Dimer Motif J. Amer. Chem Soc 134 (5):2626-2633; Straussman, R. et al. (2007) Kinking the Coiled Coil - Negatively Charged Residues at the Coiled-coil Interface, J. Molec. Biol. 366:1232-1242; Tripet, B. et al. (2002) Kinetic Analysis of the Interactions between Troponin C and the C-terminal Troponin I Regulatory Region and Validation of a New Peptide

160

Delivery/Capture System used for Surface Plasmon Resonance, J. Molec. Biol. 323:345-362; Woolfson, D.N. (2005) The Design Of Coiled-Coil Structures and Assemblies, Adv. Prot Chem 70:79-112; Zeng, Y. et al. (2008) A LigandPseudoreceptor System Based On de novo Designed Peptides For The Generation Of Adenoviral Vectors with Altered Tropism, J. Gene Med. 10:355-367).

[00234] Such repeated helical domains may be exact repeats or may have substitutions. For example, the helical domain of the Heterodimer Promoting Domain of the first polypeptide chain may comprise a sequence of eight amino acid residues selected to confer a negative charge to such Heterodimer Promoting Domain, and the helical domain of the Heterodimer Promoting Domain of the second polypeptide chain may comprise a sequence of eight amino acid residues selected to confer a positive charge to such Heterodimer Promoting Domain. It is irrelevant whether the helix is ​​provided in the first or second polypeptide chain, as long as a helix of opposite charge is used for the other polypeptide chain. The positively charged amino acid can be Usine, arginine, histidine, etc. and/or the negatively charged amino acid may be glutamic acid, aspartic acid, etc. The positively charged amino acid is preferably lysine

CbQb ίη/77Π7/Ε/ΥΙΛΙ

161 and/or the negatively charged amino acid is preferably glutamic acid. It is possible that only a single Heterodimer Promoting Domain may be employed (since such a domain will inhibit homodimerization and thus promote heterodimerization), however, it is preferred for the first and second polypeptide chains of the diabodies of the present invention that contain heterodimer promoter domains.

[00235] In a preferred embodiment, one of the Heterodimer Promoter Domains will comprise four tandem E-Helix Helical Domains (SEQ ID NO:21: EVAALEKEVAALEK-EVAALEK-EVAALEK), the glutamate residues of which will form a negative charge at pH 7, while the other Heterodimer Promoter Domains will comprise four tandem K-Helix Domains (SEQ ID NO:22: KVAALKE-KVAALKEKVAALKE-KVAALKE), whose lysine residues will form a positive charge at pH 7. The presence of such charged domains promotes the association between the first and second polypeptides, and thus enhances the formation of heterodimers. Especially preferred is a Heterodimer Promoter Domain in which one of the four tandem E-Helix helical Domains of SEQ ID NO: 21 has been modified to contain a cysteine ​​residue: EVAACEKEVAALEK-EVAALEK-EVAALEK (SEQ ID NO:23). Similarly, a Heterodimer Promoter Domain cnan Ln/zznz/E/YiAi is especially preferred.

162 wherein one of the four K-Helix Helical Domains of SEQ ID NO:22 has been modified to contain a cysteine ​​residue: KVAACKE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO:24).

[00236] As described in WO 2012/018687, to improve the in vivo pharmacokinetic properties of diabodies, a diabody can be modified to contain a polypeptide portion of a serum binding protein at one or more of the diabody termini. More preferably, such a polypeptide portion of a serum binding protein will be installed at the C-terminus of the diabody. Albumin is the most abundant protein in plasma and has a half-life of 19 days in humans. Albumin has various small molecule binding sites that allow it to bind non-covalently to other proteins and thus extend their half-lives in serum. Albumin Binding Domain 3 (ABD3) of the G protein of Streptococcus strain G148 consists of 46 amino acid residues that form a stable three-helix bundle and has a high and broad albumin binding specificity (Johansson, MU et al. (2002) Structure, Specificity, and Mode Of Interaction For Bacterial Albumin-Binding Modules, J. Biol. Chem. 277(10):8114-8120. Thus, a particularly preferred polypeptide portion of a serum binding protein for improving the in vivo pharmacokinetic properties of a diabody is the Albumin Binding Domain (ABD) of cnan Ln/zznz/E/YiAi cnan Ln/ zznz/E/YiAi

163 streptococcal G protein, and more preferably, the Albumin Binding Domain 3 (ABD3) of the G protein of Streptococcus dysgalactiae strain G148 (SEQ ID NO:25): LAEAKVLANR ELDKYGVSDY YKNLIDNAKS AEGVKALIDE ILAALP.

[00237] As described in WO 2012/162068 (incorporated herein by reference), deimmunized variants of SEQ ID NO:25 have the ability to attenuate or eliminate MHC class II binding. Based on the combination mutation results, the following combinations of substitutions are considered to be preferred substitutions to form such deimmunized ABD: 66D/70S +71 A; 66S/70S +71 A; 66S/70S +79A; 64A/65A/71A; 64A/65A/71A+66S; 64A/65A/71A+66D; 64A/65A/71A+66E; 64A/65A/79A+66S; 64A/65A/79A+66D; 64A/65A/79A+66E. ABD Variants have modifications L64A, I65A and D79A or modifications N66S, T70S and D79A. The ABD Deimmunized Variants have the amino acid sequence:

LAEAKVLANR ELDKYGVSDY YKNLID_G6NAKS₇₀ A71EGVKALIDE ILAALP (SEQ ID NO:26), or the amino acid sequence:

LAEAKVLANR ELDKYGVSDY YKNA₆₄TO₆₅NNAKT VEGVKALIA79E ILAALP (SEQ ID NO:27), or the amino acid sequence:

LAEAKVLANR ELDKYGVSDY YKNLIS₆₆NAKS7₀ VEGVKALIA79E ILAALP (SEQ ID NO:28), cnan Ln/zznz/E/YiAi

164 Particularly preferred as such are deimmunized ABD that shows substantially wild-type binding providing attenuated MHC class II binding. Thus, the first polypeptide chain of such a diabody having an ABD contains a peptide linker preferably placed C-terminally in the Helix E Domain (or helix K) of such polypeptide chain so as to intervene between the Helix Domain. Helix E (or helix K) and the ABD (which is preferably a deimmunized ABD). A preferred sequence for such a peptide linker is SEQ ID NO:29:GGGS.

B. Bispecific Diabodies Containing Fe Regions

[00238] One embodiment of the present invention relates to bispecific diabodies that comprise an Fe Region capable of simultaneously binding PD-1 and a second epitope (for example B7-H3, B7-H4, BTLA, CD40, CD80 , CD86, CD137, CTLA-4, ICOS, KIR, LAG-3, MHC class I or II, 0X40, PD-1, PD-L1, TCR, TIM-3, etc.). The addition of a CH2-CH3 Domain of an IgG to one or both polypeptide chains of the diabody, so that the complexed diabody chains result in the formation of an Fe Region, increases the biological half-life and/or alters the valence of the diabody. Incorporating one of the CH2-CH3 Domains of IgG onto the diabody polypeptides will allow a diabody to form that contains an Fe Region.

165 two-chain bispecific (Figure 2).

[00239] Alternatively, incorporating an IgG CH2-CH3 Domain solely onto one of the diabody polypeptides will allow a diabody containing a more complex four-chain bispecific Fe Region to form (Figures 3A-3C). Figure 3C shows a representative four-chain diabody possessing the Constant Light Domain (CL) and Constant Heavy CH1 Domain; however, fragments of such Domains as well as other polypeptides may alternatively be used (see, for example, Figures 3A and 3B, United States Patent Publication No. 20130295121; 2010-0174053 and 2009-0060910; European Patent Publication No. EP 2714079; EP 2601216; EP 2376109; EP 2158221 and PCT Publications No. WO 2012/162068; WO 2012/018687; WO 2010/080538). Thus, for example, in view of the CH1 Domain, one can employ a peptide having the amino acid sequence GVEPKSC (SEQ ID NO:16), VEPKSC (SEQ ID NO:17), or AEPKSC (SEQ ID NO:18). , derived from the hinge domain of a human IgG, and in view of the CL Domain, one can employ 6 C-terminal amino acids of the human kappa light chain, GFNRGEC (SEQ ID NO: 19) or FNRGEC (SEQ ID NO: 20) . A representative peptide containing a four-chain diabody is shown in Figure 3A. Alternatively, or in addition, one may employ a peptide comprising tandem helical domains comprised of opposite charge such as the Ln/zznz/E/YiAi cnan Domains.

6 Helix E helicals (SEQ ID NO:21: EVAALEK-EVAALEKEVAALEK-EVAALEK or SEQ ID NO:23: EVAACEK-EVAALEK-EVAALEKEVAALEK); and the K helix domains (SEQ ID NO:22: KVAALKEKVAALKE-KVAALKE-KVAALKE or SEQ ID NO:24: KVAACKE-KVAALKEKVAALKE-KVAALKE). A representative helical domain containing a four-stranded diabody is shown in Figure 3B.

[00240] The Fe Region-containing diabody molecules of the present invention generally include additional intervening linker peptides (Linkers). Typically, additional Linkers will comprise 3-20 amino acid residues. Additional or alternative linkers that may be employed in the Fe Region-containing diabody molecules of the present invention include: GGGS (SEQ ID NO:29), LGGGSG (SEQ ID NO:261), GGGSGGGSGGG (SEQ ID NO:262 ), ASTKG (SEQ ID NO:30), DKTHTCPPCP (SEQ ID NO:31), EPKSCDKTHTCPPCP (SEQ ID NO:32), LEPKSS (SEQ ID NO:33), APSSS (SEQ ID NO:34), and APSSSPME ( SEQ ID NO:35), LEPKSADKTHTCPPC (SEQ ID NO:36), GGC, and GGG. SEQ ID NO:33 can be used in view of GGG or GGC for ease of cloning. Additionally, the amino acids GGG, or SEQ ID NO:33 can be immediately followed by SEQ ID NO:31 to form the alternate linkers: GGGDKTHTCPPCP (SEQ ID NO:263); and LEPKSSDKTHTCPPCP (SEQ ID NO:37). The diabody molecule containing the Fe Region of cnan Ln/zznz/E/YiAi cnan Ln/zznz/E/YiAi

167 The present invention may incorporate an IgG hinge region in addition to or instead of a linker. Exemplary hinge regions include: EPKSCDKTHTCPPCP (SEQ ID NO: 32) of IgGl, ERKCCVECPPCP (SEQ ID NO: 11) of IgG2, ESKYGPPCPSCP (SEQ ID NO: 12) of IgG4, and an IgG4 hinge variant ESKYGPPCPPCP (SEQ ID NO: 13) comprising a stabilization substitute to reduce strand exchange.

[00241] As provided in Figure 3A-3C, the diabodies of the invention may comprise four different chains. The first and third polypeptide chains of such a diabody contain three Domains: (i) a VL1-containing Domain, (ii) a VH2-containing Domain, (iii) Heterodimer Promoter Domain and (iv) a Domain containing a CH2 sequence. -CH3. The second and fourth polypeptide chains contain: (i) a VL2-containing Domain, (ii) a VH1-containing Domain and (iii) a Heterodimer Promoter Domain, where the heterodimer Promoter Domains promote dimerization of the first/third polypeptide chain with the second/fourth polypeptide chain. The VL and/or VH Domains of the third and fourth polypeptide chains, and VL and/or VH Domains of the first and second polypeptide chains may be the same or different so as to allow tetravalent binding that is either monospecific, bispecific or tetraspecific. The notation VL3 and VH3 denote respectively, the Chain Domain cnan Ln/zznz/E/YiAi

168

Variable Light and the Variable Heavy Chain Domain that bind to the third epitope of such a diabody. Similarly, the notation VL4 and VH4 denote, respectively, the Variable Light Chain Domain and the Variable Heavy Chain Domain 5 that bind to the fourth epitope of such a diabody. The general structure of the polypeptide chains of a representative four-chain Fe Region containing the diabodies of the invention is provided in Table 2:

<td colspan="3">Table 2</td>

<td rowspan="4">Bispecific</td><td> 2⁵ Chain</td><td>N.H.₂-VL2-VH1-HPD-COOH</td>

<td>1¿ Chain</td><td>N.H.₂-VL1-VH2-HPD-CH2-CH3-COOH</td>

<td>1¿ Chain</td><td>NH2-VL1-VH2-HPD-CH2-CH3-COOH</td>

<td>21 Chain</td><td>N.H.₂-VL2-VH1-HPD-COOH</td>

<td rowspan="4">Tetraspecific</td><td>21 Chain</td><td>N.H.₂-VL2-VH1-HPD-COOH</td>

<td>1* Chain</td><td>NH2-VL1-VH2-HPD-CH2-CH3-COOH</td>

<td>3rd Chain</td><td>N.H.₂-VL3-VH4-HPD-CH2-CH3-COOH</td>

<td>4th Chain</td><td>N.H.₂-VL4-VH3-HPD-COOH</td>

HPD = Heterodimer Promoter Domain

[00242] In a specific embodiment, the diabodies of the present invention are bispecific, tetravalent (i.e., possess four epitope binding sites), Fe-containing diabodies (Figures 3A-3C) that are composed of four total chains. of polypeptides. The bispecific, tetravalent, Fe-containing diabodies of the invention comprise two epitope binding sites.

169 immunospecific for PD-1 (which may be able to bind to the same epitope of PD-1 or to different epitopes of PD-1), and two epitope binding sites specific for a second epitope (e.g., B7-H3, B7- H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, KIR, LAG-3 of MHC class I or II, 0X40, PD-L1, TCR, TIM-3, etc.).

[00243] In a further embodiment, diabodies containing the bispecific Fe Region may comprise three polypeptide chains. The first polypeptide of such a diabody contains three Domains: (i) a Domain containing VL1, (ii) a Domain containing VH2 and (iii) a Domain containing a CH2-CH3 sequence. The second polypeptide of such diabodies contains: (i) a VL2-containing Domain, (ii) a VH1-containing Domain, and (iii) a Domain that promotes heterodimerization and covalently links to the first polypeptide chain of the diabody. The third polypeptide of such diabodies comprises a CH2-CH3 sequence. In this way, the first and second polypeptide chains of such diabodies associate together to form a VL1/VH1 binding site that is capable of binding to the first epitope, as well as a VL2/VH2 binding site that is capable of binding to the first epitope. second epitope. The first and second polypeptides are linked to each other through a disulfide bond involving cysteine ​​residues in their respective Third Domains. Notably, the complex of the first and

CbQb ίη/ΖΖΩΖ/Ε/ΥΙΛΙ cnan Ln/zznz/E/YiAi

170 third polypeptide chains with each other to form an Fe Region that is stabilized by a disulfide bond. Such diabodies have improved potency. Figures 4A and 4B illustrate the structures of such diabodies.

Such bispecific diabodies containing the Fe Region can have either of two orientations (Table 3):

<td colspan="3">Table 3</td>

<td rowspan="3">First Orientation</td><td> 3^to chain</td><td>NH2-CH2-CH3-COOH</td>

<td>Yo^to chain</td><td>N.H.₂-VL1-VH2-HPD-CH2-CH3-COOH</td>

<td> 2^to chain</td><td>N.H.₂-VL2-VH1-HPD-COOH</td>

<td rowspan="3">Second Orientation</td><td> 3^to chain</td><td>NH2-CH2-CH3-COOH</td>

<td>Yo^to chain</td><td>N.H.₂-CH2-CH3-VL1-VH2-HPD-COOH</td>

<td> 2^to chain</td><td>N.H.₂-VL2-VH1-HPD-COOH</td>

HPD = Heterodimer Promoter Domain

[00244] In a specific embodiment, the diabodies of the present invention are bispecific, bivalent (i.e., possess two epitope binding sites), Fe-containing diabodies (Figures 4A-4B) that are composed of three total chains of polypeptides. The bispecific, bivalent Fe-containing diabodies of the invention comprise an immunospecific epitope binding site for PD-1, and a specific epitope binding site for a second epitope (e.g., B7-H3, B7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, KIR, LAG-3 OF MHC class I or II, 0X40, PD-L1, TCR, TIM171

3, etc.).

[00245] In a further embodiment, diabodies containing the bispecific Fe Region may comprise a total of five polypeptide chains. In a particular embodiment, two of the five polypeptide chains have the same amino acid sequence. The first polypeptide chain of such diabodies contains: (i) a Domain containing VH1, (ii) a Domain containing CH1, and (iii) a Domain containing a CH2-CH3 sequence. The first polypeptide chain may be the heavy chain of an antibody containing a heavy chain constant region and VH1. The second and fifth polypeptide chains of such diabodies contain: (i) a VL1-containing domain, and (ii) a CL-containing domain. The second and/or fifth polypeptide chains of such diabodies may be the light chains of an antibody containing a VL1 complementary to the VH1 of the first/third polypeptide chain. The first, second and/or fifth polypeptide chains can be isolated from naturally occurring antibodies. Alternatively, they can be constructed recombinantly. The third polypeptide chain of such diabodies contains: (i) a Domain containing VH1, (ii) a Domain containing CH1, (iii) a Domain containing a CH2-CH3 sequence, (iv) a Domain containing VL2, (v) a VH3-containing Domain and (vi) a Heterodimer Promoting Domain, where the Domains

CbQb ίη/77Ω7/Β/ΥΙΛΙ

172 Heterodimer promoters promote dimerization of the third strand with the fourth strand. The fourth polypeptide of such diabodies contains: (i) a VL3-containing Domain, (ii) a VH2-containing Domain, and (iii) a Domain that promotes heterodimerization and covalently links to the third polypeptide chain of the diabody.

[00246] Thus, the first and second, and the third and fifth polypeptide chains of such diabodies associate together to form two VL1/VH1 binding sites capable of binding to a first epitope. The third and fourth polypeptide chains of such diabodies associate together to form a VL2/VH2 binding site that is capable of binding to a second epitope, as well as a VL3/VH3 binding site that is capable of binding to a third epitope. . The first and third polypeptides are linked to each other through a disulfide bond involving cysteine ​​residues in their respective constant regions. Notably, the complex of the first and third polypeptide chains with each other forms an Fe Region. Such diabodies have enhanced potency. Figure 5 illustrates the structure of such diabodies. It will be understood that Domains VL1/VH1, VL2/VH2, and VL3/VH3 may be the same or different so as to allow binding that is monospecific, bispecific, or trispecific. However, as provided herein, these Preference Domains are selected by way of binding PD-1 and a second

CbQb ίη/ΖΖΠΖ/Ε/ΥΙΛΙ cnan Ln/zznz/E/YiAi

173 epitope (e.g., B7-H3, B7-H4, BTLA, CD40, CD80, CD86, CD 137, CTLA- 4, ICOS, KIR, LAG-3 of MHC class I or II, 0X40, PD-L1, TCR, TIM-3, etc.).

[00247] The VL and VH Domains of the polypeptide chains are selected so as to form VL/VH binding sites specific for a desired epitope. The VL/VH binding sites formed by the association of the polypeptide chains may be the same or different so as to allow tetravalent binding that is monospecific, bispecific, trispecific or tetraspecific. In particular, the VL and VH Domains can be selected so that a bispecific diabody can comprise two binding sites for a first epitope and two binding sites for a second epitope, or three binding sites for a first epitope and one binding site. for a second epitope, or two binding sites for a first epitope, one binding site for a second epitope and one binding site for a third epitope (as depicted in Figure 5). The polypeptide chain structure of representative five-chain Fe Region-containing diabodies is given in Table

4:

174 cnan Ln/zznz/E/YiAi

Table 4

<td rowspan="5">Bispecific (2 x 2)</td><td> 2^to Chain</td><td>NH2-VLI-CL-COOH</td>

<td>Yo^to Chain</td><td>N.H.₂-VH1-CH1-CH2-CH3-COOH</td>

<td> 3^to Chain</td><td>N.H.₂-VH1-CH1-CH2-CH3-VL2-VH2-HPD-COOH</td>

<td> 5^to Chain</td><td>NH2-VLI-CL-COOH</td>

<td> 4^to Chain</td><td>NH2-VL2-VH2-HPD-COOH</td>

<td rowspan="5">Bispecific (3x1)</td><td> 2^to Chain</td><td>NH2-VLI-CL-COOH</td>

<td>Yo^to Chain</td><td>N.H.₂-VH1-CH1-CH2-CH3-COOH</td>

<td> 3^to Chain</td><td>N.H.₂-VH1-CH1-CH2-CH3-VL1-VH2-HPD-COOH</td>

<td> 5^to Chain</td><td>NH2-VLI-CL-COOH</td>

<td> 4^to Chain</td><td>NH2-VL2-VH1-HPD-COOH</td>

<td rowspan="5">Trispecific (2x1x1)</td><td> 2^to Chain</td><td>NH2-VLI-CL-COOH</td>

<td>Yo^to Chain</td><td>N.H.₂-VH1-CH1-CH2-CH3-COOH</td>

<td> 3^to Chain</td><td>N.H.₂-VH1-CH1-CH2-CH3-VL2-VH3-HPD-COOH</td>

<td> 5^to Chain</td><td>NH2-VLI-CL-COOH</td>

<td> 4^to Chain</td><td>N.H.₂-VL3-VH2-HPD-COOH</td>

HPD = Heterodimer Promoter Domain

[00248] In a specific embodiment, the diabodies of the present invention are bispecific, tetravalent (i.e., they possess four epitope binding sites), Fe-containing diabodies that are composed of five total polypeptide chains have two binding sites. binding for a first epitope and two binding sites for a second epitope.

cnan Ln/zznz/E/YiAi

175

In one embodiment, the bispecific, tetravalent Fe-containing diabodies of the invention comprise two immunospecific epitope binding sites for PD-1 (which may be capable of binding to the same PD-1 epitope or to different PD-1 epitopes). ), and two epitope binding sites specific for a second epitope (e.g., B7H3, B7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, KIR, LAG-3 MHC class I or II, 0X40, PD-L1, TCR, TIM-3, etc.). In another embodiment, the bispecific, tetravalent Fe-containing diabodies of the invention comprise three immunospecific epitope binding sites for PD-1 that may be capable of binding to the same epitope of PD-1 or to different epitopes of PD-1). , and an epitope binding site specific for a second epitope (e.g., B7-H3, B7H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, KIR, LAG-3 MHC class I or II , 0X40, PD-L1, TCR, TIM-3, etc.). In another embodiment, the bispecific, tetravalent Fe-containing diabodies of the invention comprise one epitope binding site immunospecific to PD-1, and three epitope binding sites specific to a second epitope (e.g., B7-H3, B7- H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, KIR, MHC class I or II LAG3, 0X40, PD-L1, TCR, TIM-3, etc.).

C. Bispecific Trivalent Bonding Molecules Containing Fe Regions

176

[00249] An additional embodiment of the present invention relates to trivalent bispecific binding molecules, comprising an Fe Region, and being capable of simultaneously binding to a first epitope, a second epitope and a third epitope, where at At least one of such epitopes is not identical to the others. Such bispecific diabodies thus comprise VL1/VH1 domains that are capable of binding to the first epitope, VL2/VH2 domains that are capable of binding to the second epitope and VL3/VH3 domains that are capable of binding to the third epitope. In one embodiment, one or two of such epitopes is a PD-1 epitope and the other (or the other) of such epitopes is not a PD-1 epitope (e.g., a B7-H3, B7-H4 epitope). , BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, KIR, LAG-3, MHC class I or II, 0X40, PD1, PD-L1, TCR, TIM-3, etc.). Such bispecific trivalent binding molecules comprise three epitope binding sites, two of which are diabody binding domains, providing a binding Site A and a binding Site B, and one of which is a non-diabody binding domain. diabody type binding, which provides binding Site C (see, for example, Figures 6A-6F, and PCT Application No: PCT/US15/33081; and PCT/US15/33076).

[00250] Typically, the trivalent linker molecules of the present invention will comprise four different polypeptide chains (see Figures 6A-6B); however,

CbQb ίη/77Π7/Ε/ΥΙΛΙ

177 The molecules may comprise smaller or larger numbers of polypeptide chains, for example, by fusing such polypeptide chains together (for example, by a peptide bond) or by cleaving such polypeptide chains to form additional polypeptide chains, or by associating few or additional polypeptide chains through disulfide bonds. Figures 6B-6F illustrate this aspect of the present invention by schematically representing such molecules having three polypeptide chains. As provided in Figures 6A-6F, the trivalent binding molecules of the present invention can have alternative orientations in which the diabody-like binding domains are N-terminal (Figures 6A, 6C and 6D) or C-terminal (Figures 6B, 6E and 6F) to a Region of Faith.

[00251] In certain embodiments, the first polypeptide chain of such trivalent linker molecules of the present invention contains: (i) a VL1-containing Domain, (ii) a VH2-containing Domain, (iii) a Heterodimer Promoter Domain , and (iv) a Domain containing a CH2-CH3 sequence. Domains VL1 and VL2 are located N-terminal or C-terminal to the CH2-CH3-containing domain as presented in Table 5 (Figures 6A and 6B). The second polypeptide chain of such embodiments contains: (i) a VL2-containing Domain, (ii) a VH1-containing Domain, and (iii) a Heterodimer Promoter Domain. The third cnan chain Ln/zznz/E/YiAi

178 Polypeptides of such embodiments contain: (i) a Domain containing VH3, (ii) a Domain containing CH1 and (iii) a Domain containing a CH2-CH3 sequence. The third polypeptide chain may be the heavy chain of an antibody containing a heavy chain constant region and a VH3. The fourth polypeptide of such embodiments contains: (i) a VL3-containing domain and (ii) a CL-containing domain. The fourth polypeptide chains may be an antibody light chain containing a VL3 complementary to the VH3 of the third polypeptide chain. The third or fourth polypeptide chains can be isolated from naturally occurring antibodies. Alternatively, they can be constructed recombinantly, synthetically or by other means.

[00252] The Variable Light Chain Domain of the first and second polypeptide chains are separated from the Variable Heavy Chain Domains of such polypeptide chains by an intervening spacer linker that has a length that is very short to allow its domains VL1/VH2 (or their VL2/VH1) associate together to form epitope binding sites capable of binding to either the first or second epitope. A preferred intervening spacer peptide (Linker 1) for this purpose has the sequence (SEQ ID NO: 14): GGGSGGGG. Other domains of the trivalent binding molecules can be separated by one or more intervening spacer peptides, comprising cnan Ln/zznz/E/YiAi

181

Table 5:

cnan Ln/zznz/E/YiAi

<td colspan="3">Table 5</td>

<td rowspan="4">Orientation of four Yo^to chains</td><td> 2^to Chain</td><td>NH2-VL2-VH1-HPD-COOH</td>

<td>Yo^to Chain</td><td>N.H.₂-VL1-VH2-HPD-CH2-CH3-COOH</td>

<td> 3^to Chain</td><td>N.H.₂-VH3-CH1-CH2-CH3-COOH</td>

<td> 4^to Chain</td><td>N.H.₂-VL3-CL-COOH</td>

<td rowspan="4">Four orientation 2^to chains</td><td> 2^to Chain</td><td>NH2-VL2-VH1-HPD-COOH</td>

<td>Yo^to Chain</td><td>N.H.₂-CH2-CH3-VL1-VH2-HPD COOH</td>

<td> 3^to Chain</td><td>NH2-VH3-CH1-CH2-CH3-COOH</td>

<td> 4^to Chain</td><td>NH2-VL3-CL-COOH</td>

<td rowspan="3">Three I orientation^tochains</td><td> 2^to Chain</td><td>N.H.₂-VL2-VH1-HPD-COOH</td>

<td>Yo^to Chain</td><td>N.H.₂-VL1-VH2-HPD-CH2-CH3-COOH</td>

<td> 3^to Chain</td><td>N.H.₂-VL3-VH3-HPD-CH2-CH3-COOH</td>

<td rowspan="3">orientation of three 2^tochains</td><td> 2^to Chain</td><td>NH2-VL2-VH1-HPD-COOH</td>

<td>Yo^to Chain</td><td>N.H.₂-CH2-CH3-VL1-VH2-HPD-COOH</td>

<td> 3^to Chain</td><td>N.H.₂-VL3-VH3-HPD-CH2-CH3-COOH</td>

HPD = Heterodimer Promoter Domain

[00256] One embodiment of the present invention relates to bispecific trivalent binding molecules comprising two epitope binding sites for PD-1 and one epitope binding site for the second epitope present on a molecule other than PD-1 ( for example B7-H3, B7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, KIR, LAG-3, MHC class I or II, 0X40, PD-L1, TCR, TIM-3, etc.). The two sites

182 Epitope binding for PD-1 can bind the same epitope or different epitopes. Another embodiment of the present invention relates to bispecific trivalent binding molecules that comprise one epitope binding site for PD-1 and two epitope binding sites that bind to a second antigen present on a molecule other than PD-1. (e.g. B7-H3, B7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, KIR, LAG-3, MHC class I or II, 0X40, PD-L1, TCR, TIM-3 , etc.). The two epitope binding sites for the second antigen may bind the same epitope or different epitopes of the antigen (for example, the same or different epitopes of LAG-3). As provided above, such bispecific trivalent binding molecules may comprise three or four polypeptide chains.

VII. Constant Domains and Faith Regions

[00257] Provided herein are Antibody Constant Domains useful in generating the PD-1 binding molecules (e.g., antibodies, diabodies, trivalent binding molecules, etc.) of the invention.

[00258] A preferred CL Domain is a human IgG Kappa CL Domain. The amino acid sequence of an exemplary human Kappa CL Domain is (SEQ ID NO:8):

RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC cnan Ln/zznz/E/YiAi

183

[00259] Alternatively, an exemplary CL Domain is a human IgG Lambda CL Domain. The amino acid sequence of an exemplary human Kappa CL Domain is (SEQ ID NO:9):

QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA GVETTPSKQS NNKYAASSYL SLTPEQWKSH RSYSCQVTHE GSTVEKTVAP TECS

[00260] As provided herein, PD-1 binding molecules of the invention may comprise an Fe Region. The Fe Region of such molecules of the invention may be any isotype (e.g., IgGl, IgG2, IgG3, or IgG4). PD-1 binding molecules of the invention may further comprise a CHI Domain and/or a hinge region. When present, the CHI Domain and/or hinge region may be any isotype (e.g., IgGl, IgG2, IgG3, or IgG4), and is preferably of the same isotype as the desired Fe Region.

[00261] An exemplary CHI Domain is a human IgGl CHI Domain. The amino acid sequence of an exemplary human IgGl CHI Domain is (SEQ ID NO.10):

ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRV

[00262] An exemplary CHI Domain is a human IgG2 CHI Domain. The amino acid sequence of an exemplary human IgG2 CHI Domain is (SEQ ID NO:257):

cnan Ln/zznz/E/YiAi

184 cnan Ln/zznz/E/YiAi

ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTV

[00263] An exemplary CHI Domain is a human IgG4 CHI Domain. The amino acid sequence of an exemplary human IgG4 CHI Domain is (SEQ ID NO:254):

ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRV

[00264] An exemplary hinge region is a human IgGl hinge region. The amino acid sequence of an exemplary human IgGl hinge region is (SEQ ID NO:32): EPKSCDKTHTCPPCP.

[00265] Another exemplary hinge region is a human IgG2 hinge region. The amino acid sequence of an exemplary human lgG2 hinge region is (SEQ ID NO:11): ERKCCVECPPCP.

[00266] Another exemplary hinge region is a human IgG4 hinge region. The amino acid sequence of an exemplary human IgG4 hinge region is (SEQ ID NO:12): ESKYGPPCPSCP. As described herein, an IgG4 hinge region may comprise a stabilizing mutation such as the S228P substitution. The amino acid sequence of an exemplary stabilized IgG4 hinge region is (SEQ ID NO:13): ESKYGPPCPPCP.

[00267] The Fe Region of molecules (e.g., antibodies, diabodies, and trivalent molecules) that

185 containing the Fe Region of the present invention may be a complete Fe Region (for example, a complete IgG Fe Region) or only a fragment of an Fe Region. Optionally, the Fe Region of molecules containing the Fe Region of The present invention lacks the C-terminal Usine amino acid residue. In particular, the Fe Region of the Fe Region-containing molecules of the present invention may be an engineered variant Fe Region. Although the Fe Region of the bispecific Fe Region-containing molecules of the present invention may possess the ability to bind to one or more Fe receptors (e.g., FcyR(s)), more preferably such variant Fe Region has altered binding to FcyRIA (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (GD16a) or FcyRIIIB (CD16b) (relative to the binding shown by a wild-type Fe Region) or will or will not have substantially reduced capacity to bind to receptors inhibitors. Thus, the Fe Region of the Fe Region-containing molecules of the present invention may include some or all of the CH2 Domains and/or some or all of the CH3 Domains of a complete Fe Region, or may comprise a variant CH2 sequence. and/or a variant CH3 sequence (this may include, for example, one or more insertions and/or one or more deletions with respect to the CH2 or CH3 domains of a complete Fe Region). Such Fe Regions may comprise portions of non-Fe polypeptides, or cnan Ln/zznz/E/YiAi

CbQb ίη/77Ω7/Β/ΥΙΛΙ

186 they may comprise portions of non-naturally complete Fe Regions, or they may comprise orientations of CH2 and/or CH3 Domains that do not occur naturally (such as, for example, two CH2 domains or two CH3 domains, or in an N-terminal direction to C-terminal, a CH3 Domain linked to a CH2 Domain, etc.).

[00268] Fe Region modifications identified as altering effector function are known in the art, including modifications that increase binding to activating receptors (eg, FcyRIIA (CD16A) and reduce binding to inhibitory receptors (eg , FcyRIIB (CD32B) (see, for example, Stavenhagen, JB et al. (2007) Fe Optimization Of Therapeutic Antibodies Enhances Their Ability To Kill Tumor Cells In Vitro And Controls Tumor Expansion In Vivo Via Low-Affinity Activating Fcgamma Receptora Cancer Res. 57 (18):8882-8890). Exemplary variants of the Fe Regions of human IgGl with reduced binding to CD32B and/or increased binding to CD16A contain substitutions F243L, R292P, Y300L, V305I or P296L. These amino acid substitutions can occur in an Fe Region of human IgGl in any combination or subcombination. In one embodiment, the human IgGl Fe Region variant contains an F243L, R292P and Y300L substitution. In another embodiment, the Fe Region variant of human IgGl contains substitutions F243L, R292P, Y300L, V305I and P296L.

187

[00269] In particular, it is preferred that the Fe Regions of the polypeptide chains of the Fe Region-containing molecules of the present invention show decreased binding (or substantially no binding) to FcyRIA (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD16a) or FcyRIIIB (CD16b) (relative to the binding shown by the Fe Region of wild-type IgGl (SEQ ID NO:1). Variant Fe Regions and mutant forms capable of mediating such altered binding are described above. In a specific embodiment, the Fe Region-containing molecules of the present invention comprise an IgG Fe Region that exhibits reduced ADCC effector function. In a preferred embodiment, the CHICHI Domain of the first and/or third polypeptide chains of such Fe Region-containing molecules includes any of 1, 2, or 3, of the substitutions: L234A, L235A, N297Q, and N297G. In another embodiment, the human IgG Fe Region variant contains an N297Q substitution, an N297G substitution, L234A and L235A substitutions, or a D265A substitution, as these mutations abrogate FcR binding. Alternatively, a CH2-CH3 Domain is used from an Fe region that inherently shows decreased (or substantially no) binding to FcyRIIIA (CD16a) and/or reduced effector function (relative to the binding shown by the Fe region). Fe of IgGl in its natural state (SEQ ID NO: 1)). In a specific embodiment, the molecules containing the Region

CbQb ίη/ΖΖΠΖ/Ε/ΥΙΛΙ

188

Fe of the present invention comprise an IgG Fe Region (SEQ ID NO: 2) or an IgG2 Fe Region (SEQ ID NO: 4). When an IgG4 Fe Region is used, the present invention also encompasses the presentation of a stabilizing mutation, such as the hinge region S228P substitution described above (see, for example, SEQ ID NO:13). Because the N297G, N297Q, L234A, L235A, and D265A substitutions abrogate effector function, in circumstances where effector function is desired, these preference substitutions cannot be employed.

[00270] In particular, it is preferred that the Fe Regions of the polypeptide chains of the molecules containing the Fe Region of the present invention show increased serum half-life (relative to the half-life shown by the corresponding wild-type Fe ). Variant Fe Regions and mutant forms showing extended serum half-life are described above. In a preferred embodiment, the CH2-CH3 Domain of the first and/or third polypeptide chains of such molecules containing the Fe Region includes any of 1, 2, or 3, of the substitutions: M252Y, S254T and T256E. The invention further encompasses molecules containing the Fe Region of the present invention comprising variant Fe Regions comprising:

CbQb ίη/ΖΖΩΖ/Ε/ΥΙΛΙ (A) one or more mutations that alter function

189 effector and/or FcyR; and (B) one or more mutations that extend serum half-life.

[00271] A preferred IgGl sequence for Domains

CH2 and CH3 of the molecules containing the Fe Region of the present invention will comprise the substitutions

L234A/L235A/M252Y/S254T/T256E (SEQ ID NO:258):

APEAAGGPSV FLFPPKPKDT LYiTREPEVT CVVVDVSHED PEVKFNWYVD

GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA

PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE

WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE

ALHNHYTQKS LSLSPGX where, X is a Power Plant (K) or is absent.

[00272] A preferred IgG4 sequence for Domains

CH2 and CH3 of the molecules containing the Fe Region of the present invention will comprise the substitutions

M252Y/S254T/T256E (SEQ ID NO:259):

cnan Ln/zznz/E/YiAi

APEFLGGPSV FLFPPKPKDT LYITREPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSR WQEG NVFSCSVMHE ALHNHYTQKS LSLSLGX where, X is a lysine (K) or is absent.

[00273] For diabodies and trivalent linker molecules whose first and third polypeptide chains

190 are not identical), it is desirable to reduce or prevent heterodimerization from occurring between the CH2-CH3 Domains of the first two polypeptide chains or between the CH2-CH3 Domains of two third polypeptide chains. The CH2 and/or CH3 Domains of such polypeptide chains need not be identical in sequence, and are advantageously modified to promote complexing between the two polypeptide chains. For example, an amino acid substitution (preferably a substitution with an amino acid comprising a bulky side group that forms a bulge, e.g., tryptophan) can be introduced into the CH2 or CH3 domain such that spherical interference will prevent the interaction. with a similarly mutated domain and will force the mutated domain to be paired with a domain into which a complementary or accommodative mutation has been designed, i.e. the hole (for example, a substitution with wisteria). Such sets of mutations can be designed into any pair of polypeptides comprising CH2-CH3 Domains that form an Fe Region. Protein design methods to favor heterodimerization over homodimerization are well known in the art, particularly with respect to the design of immunoglobulin-like molecules, and are covered herein (see for example, Ridgway et al. (1996) 'Knobscnan Ln/zznz/E/YiAi

Into-Holes' Engineering Of Antibody CH3 Domains For Heavy cnan Ln/zznz/E/YiAi

191

Chain Heterodimerization, Protein Engr. 9:617-621, Atwell et al. (1997) Stable Heterodimers From Remodeling The Domain Interface Of A Homodimer Using A Phage Display Library, J. Mol. Biol. 270: 26-35, and Xie et al. (2005) A New Format Of Bispecific Antibody: Highly Efficient Heterodimerization, Expression And Tumor Cell Lysis, J. Immunol. Methods 296:95-101; each of which is incorporated herein by reference in its entirety. Preferably, the button is designed in the CH2-CH3 Domains of the first polypeptide chain and the hole is designed in the CH2-CH3 Domains of the third polypeptide chain of the diabodies comprising three polypeptide chains. In this way, the protrusion will help prevent the first polypeptide chain from homodimerizing through its CH2 and/or CH3 Domains. As the third polypeptide chain preferably contains the hole substitution it will heterodimerize with the first polypeptide chain, just as it homodimerizes with itself. This strategy can be used for diabodies and trivalent linker molecules comprising three, four or five chains as detailed above, where the button is designed in the CH2-CH3 Domains of the first polypeptide chain and the hole is designed in the CH2-CH3 domains of the third polypeptide chain.

[00274] A preferred bump is created by modifying cnan Ln/zznz/E/YiAi

192 an IgG Fe Region to contain T366W modification. A preferred hole is created by modifying an Fe Region of IgG to contain the T366S, L368A and Y407V modification. To assist in the purification of the third polypeptide chain homodimer from the molecule containing the final bispecific heterodimeric Fe Region, the protein A binding site of the CH2 and CH3 Domains that carry the hole of the third polypeptide chain is mutates preferentially by the amino acid substitution at position 435 (H435R). Therefore, the third polypeptide chain homodimer carrying the hole will not bind to protein A, while the bispecific heterodimer will retain its ability to bind to protein A through the protein A binding site in the first polypeptide chain. In an alternative embodiment, the third polypeptide chain carrying the hole can be incorporated into amino acid substitutions at positions 434 and 435 (N434A/N435K).

[00275] A preferred IgGl amino acid sequence for the CH2 and CH3 Domains of the first polypeptide chain of a molecule containing the Fe Region of the present invention will have the button-bearing sequence (SEQ ID NO: 6):

193

APEAAGGPSV GVEVHNAKTK

PIEKTISKAK WESNGQPENN ALHNHYTQKS

FLFPPKPKDT PREEQYNSTY

GQPREPQVYT YKTTPPVLDS LSLSPGX

LMISRTPEVT

RVVSVLTVLH

LPPSREEMTK DGSFFLYSKL

CVVVDVSHED

QDWLNGKEYK

NQVSLWCLVK

TVDKSRWQQG

PEVKFNWYVD

CKVSNKALPA

GFYPSDTAVE

NVFSCSVMHE cnan Ln/zznz/E/YiAi where, X is a Power Plant (K) or is absent.

[00276] A preferred IgGl amino acid sequence for the CH2 and CH3 Domains of the second polypeptide chain of a molecule containing the Fe Region of the present invention that has two polypeptide chains (or the third polypeptide chain of a molecule that contains the Fe Region that has three, four, or five polypeptide chains) will have the sequence that carries the hole (SEQ ID NO: 7):

APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQ QG NVFSCSVMHE ALHNRYTQKS LSLSPGX where, X is a Power Plant (K) or is absent.

[00277] As will be seen, the CH2-CH3 domains of SEQ ID NO: 6, and SEQ ID NO: 7 include a substitution at position 234 with alanine and 235 with alanine, and therefore form an exposition of the Region Fe decreased (or substantially no) binding to FcyRIA (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), cnan Ln/zznz/E/YiAi

194

FcyRIIIA (CD 16a) or FcyRIIIB (CD16b) (relative to the binding shown by the wild type Fe Region (SEQ ID NO: 1). The invention also encompasses such CH2-CH3 Domains, which comprise alternative and/or additional substitutions that They modify the effector function and/or the FyR binding activity of the Fe region. The invention also encompasses such CH2-CH3 Domains, which further comprise one or more amino acid substitutions that extend the half-life. In particular, the invention encompasses such hole-bearing and button-bearing CH2-CH3 Domains further comprising M252Y/S254T/T256E.

[00278] It is preferred that the first polypeptide chain will have a button-bearing CH2-CH3 sequence, such as that of SEQ ID NO: 6. However, as will be recognized, a hole-bearing CH2-CH3 Domain (e.g., SEQ ID NO:7) could be employed in the first polypeptide chain, in which case, a knob-bearing CH2-CH3 Domain (e.g. example, SEQ ID NO: 6) is used in the second polypeptide chain of a molecule containing the Fe Region of the present invention that has two polypeptide chains (or in the third polypeptide chain of a molecule containing the Fe Region that has three, four, or five polypeptide chains).

[00279] As detailed above, the invention encompasses molecules containing the Fe Region (e.g.

195 antibodies and diabodies containing the Fe Region) that have the wild-type CH2 and CH3 Domains, or have the CH2 and CH3 Domains that comprise the combinations of the substitutions described above. An exemplary amino acid sequence of an IgGl CH2-CH3 Domain encompassing such variations is (SEQ ID NO: 260):

APEXiX₂GGPSV FLFPPKPKDT LX₃IX₄RX₅PEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLX₆C.X.₇VK GFYPSDIAVE WESNGQPENN YKTTPPV1DS DGSFFLX₈SKL TVDKSRWQQG NVFSCSVMHE ALHX₉x₁₀YTQKS LSLSPGXn cnan Ln/zznz/E/YiAi where:

(a) Xi and X2 are either L (wild type), or are A (decreased FcyR binding);

(b) X3, X4, and X5, respectively, are H, S, and T (wild type), or are Y, T, and E (extended half-life), (c) L and Y (wild type), or are W, L and Y (button), or S, A and V (hole) (d) X9 and X10, respectively, are N and H (wild type), or are N and R (non-binding protein A), or A and K (non-binding protein A); and (e) X11 is K or absent.

[00280] In other embodiments, the invention encompasses PD-1 binding molecules that comprise CH2 and/or CH3 Domains that have been designed to favor heterodimerization over

196 homodimerization using mutations known in the art, such as those described in PCT Publication No. WO 2007/110205; WO 2011/143545; WO 2012/058768; WO 2013/06867, all of which are incorporated herein by reference in their entirety.

cnan Ln/zznz/E/YiAi

VIII. PD-1 x LAG-3 Bispecific Binding Molecules

[00281] The present invention particularly relates to bispecific PD-1 X LAG-3 binding molecules (e.g., bispecific antibodies, bispecific diabodies, etc.) comprising an epitope binding fragment of an anti-PD- 1, and preferably one of the novel anti-human PD-1 antibodies provided herein, and an epitope binding fragment of an anti-human LAG-3 antibody, preferably one of the novel anti-human LAG 3 antibodies provided herein.

The preferred PD-1 x LAG-3 bispecific binding molecules of the present invention possess antibody epitope binding fragments that allow them to be able to coordinately bind to two different epitopes: a PD-1 epitope and a PD-1 epitope. of LAG-3, in order to attenuate the inhibitory activities of such molecules. As used herein, such attenuation refers to a decrease of at least 20%, a decrease of at least 50%, a decrease of at least 80%, or a decrease of at least 80%.

197 less than 90% in detectable PD-1 and/or LAG3 inhibitory activity, or complete elimination of detectable PD-1 and/or LAG-3 inhibitory activity. The selection of the epitope binding fragments (e.g., the VL and VH Domains) of the anti-human PD-1 antibody and the anti-LAG-3 antibody is coordinated such that the polypeptide chains that compose such bispecific binding PD-1 x LAG-3 assembled to form at least one functional antigen binding site that is specific for the first antigen (i.e. either PD-1 or LAG-3) and at least one functional antigen binding site that is specific for the second antigen (i.e., either PD-1 or LAG-3, depending on the identity of the first antigen)

[00282] In a particular embodiment, a PD-1 x LAG-3 bispecific binding molecule of the current invention is a bispecific diabody, preferably comprising two, three, four, or five polypeptide chains as described herein. In another particular embodiment, a PD-1 x LAG-3 bispecific binding molecule of the current invention is a bispecific antibody, preferably comprising two, three, or four polypeptide chains as described herein (see also, for example, document WO 2007/024715; W02007/110205; WO 2009/080251; WO 2009/080254; WO 2009/089004; WO 2011/069104; WO 2011/117329; WO 2011/131746; WO 2011/133886; WO 2011/143545 ; WO 2012/023053;

CbQb ίη/77Π7/Ε/ΥΙΛΙ

198

WO 2013/060867, all of which disclosures are incorporated herein by reference in their entirety) cnan Ln/zznz/E/YiAi

A. Anti-Human LAG-3 Antibodies

[00283] Exemplary antibodies that are immunospecific for human LAG3 are provided in the following. Additional desired antibodies can be made by isolating antibody-secreting hybridomas raised using LAG-3 or a peptide fragment thereof, or by screening recombinant antibody libraries for binding to LAG-3 or a peptide fragment thereof. peptide thereof. Human LAG-3 (including a 28 amino acid residue signal sequence (shown underlined) and the 497 amino acid residue mature protein) have the amino acid sequence (SEQ ID NO: 38):

MWEAQFLGLL FLQPLWVAPV KPLQPGAEVP VVWAQEGAPA QLPCSPTIPL QDLSLLRRAG VTWQHQPDSG PPAAAPGHPL APGPHPAAPS SWGPRPRRYT VLSVGPGGLR SGRLPLQPRV QLDERGRQRG DFSLWLRPAR RADAGEYRAA VHLRDRALSC RLRLRLGQAS MTASPPGSLR ASDWVILNCS FSRPDRPASV HWFRNRG QGR VPVRESPHHH LAESFLFLPQ VSPMDSGPWG CILTYRDGFN VSIMYNLTVL GLEPPTPLTV YAGAGSRVGL PCRLPAGVGT RSFLTAKWTP PGGGPDLLVT GDNGDFTLRL EDVSQAQAGT YTCHIHLQEQ QLNATVTLAI ITVTPKSFGS PGSLGKLLCE VTPVSGQERF VWSSLDTPSQ RSFSGPWLEA QEAQLLSQ PW QCQLYQGERL LGAAVYFTEL SSPGAQRSGR APGALPAGHL LLFLILGVLS LLLLVTGAFG FHLWRRQWRP RRFSALEQGI HPPQAQSKIE ELEQEPEPEP EPEPEPEPEP EPEQL

1. LAG-3 mAb A

[00284] The anti-human BMS-986016 LAG-3 antibody (25F7;

199

Medarex/BMS), designated herein as LAG-3 mAb A, and variants thereof have been described (see, for example, WO 2014/008218). The amino acid sequence of the LAG-3 mAb A Heavy Chain Variable Domain has the amino acid sequence (SEQ ID NO: 39) (CDRs are shown underlined):

QVQLQQWGAG LLKPSETLSL TCAVYGGSFS DYYWNWIRQP PGKGLEWIGE INHNGNTNSN PSLKSRVTLS LDTSKNQFSL KLRSVTAADT AVYYCAFGYS DYEYNWFDPW GQGTLVTVSS

[00285] The amino acid sequence of the Light Chain Variable Domain of mAb A from LAG-3 has the amino acid sequence (SEQ ID NO: 40) (CDRs are shown underlined):

EIVLTQSPAT LSLSPGERAT LSCRASQSIS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPLTFGQ GTNLEIK

[00286] Additional murine anti-human LAG-3 antibodies have recently been identified that possess unique binding characteristics (see, United States Patent Application No. 62/172277). Preferred PD-1 a novel epitope and do not compete with BMS-986016 binding for LAG-3. Particularly preferred are the bispecific binding molecules PD-1 X cnan Ln/zznz/E/YiAi

200

LAG3 of the present invention that have VH Domains and/or

Humanized VL of LAG-3 mAb 1 or LAG-3 mAb 6.

cnan Ln/zznz/E/YiAi

2. LAG-1 mAb 1

[00287] The amino acid sequence of the VH domain of LAG-3 mAb (SEQ ID NO: 41) is shown below (hCDR residues are shown underlined).

QIQLVQSGPE LKKPGETVKI SCKASGYTFR NYGMNWVKQA PGKVLKWMGW INTYTGESTY ADDFEGRFAF SLGTSASTAY LQINILKNED TATYFCARES LYDYYSMDYW GQGTSVTVSS

LAG-3 mAb 1 CDRhI (SEQ ID NO:42): RNYGMN

CDRh2 of LAG-3 mAb 1 (SEQ ID N043): WINTYTGESTYADDFEG

CDRh3 of LAG-3 mAb 1 (SEQ ID NO:44): ESLYDYYSMDY

[00288] The amino acid sequence of the VL domain of LAG-3 mAb (SEQ ID NO: 45) is shown below (CDRL residues are shown underlined):

DVVVTQTPLT LSVTIGQPAS ISCKSSQSLL HSDGKTYLNW LLQRPGQSPE

RLIYLVSELD SGVPDRFTGS GSGTDFTLKI SRVEAEDLGV YYCWQGTHFP YTFGGGTKLE IK

LAG-3 mAb 1 CDRlI(SEQ ID NO:46): KSSQSLLHSDGKTYLN

CDRl2 of LAG-3 mAb 1(SEQ ID NO:47): LVSELDS

LAG-3 mAb 1 CDRl3(SEQ ID NO:48): WQGTHFPYT

[00289] Two humanized exemplars of the VH Domains of mAb 1 of LAG-3 designated here as mAb 1 VH1 of hLAG-3, and mAb 1 VH2 of hLAG-3, and four exemplars of the VL Domains

201 humanized mAb 1 of LAG-3 mAb 1 VL1 of hLAG-3, mAb 1 VL2 of hLAG-3, mAb 1 VL3 of hLAG-3, and mAb 1 VL4 of hLAG3, are provided in the following. Any of the humanized VL Domains can be paired with any of the humanized VH Domains to generate a LAG-3 binding domain. Accordingly, any antibody comprising one of the humanized VL Domains in pairs with the humanized VH Domain is generically referred to as hLAG-3 mAb 1, and particular combinations of humanized VH/VL Domains are referred to by reference to the VH/VL Domains. specific, for example a humanized antibody comprising hLAG-3 VH1 mAb 1 and hLAG-3 VL2 mAb 1 is specifically known as hLAG-3 mAb 1 (1,2)

[00290] The amino acid sequence of the hLAG-3 VH1 mAb 1 domain (SEQ ID NO: 49) is shown below (hCDR residues are shown underlined).

QVQLVQSGAE VKKPGASVKV SCKASGYTFT NYGMNWVRQA PGQGLEWMGW INTYTGESTY ADDFEGRFVF SMDTSASTAY LQISSLKAED TAVYYCARES LYDYYSMDYW GQGTTVTVSS

[00291] The amino acid sequence of the mAb 1 VH2 domain of hLAG-3 (SEQ ID NO: 50) is shown below (hCDR residues are shown underlined).

QVQLVQSGAE VKKPGASVKV SCKASGYTFT NYGMNWVRQA PGQGLEWMGW INTYTGESTY ADDFEGRFVF SMDTSASTAY LQISSLKAED TAVYFCARES cnan Ln/zznz/E/YiAi

LYDYYSMDYW GQGTTVTVSS

202

[00292] The amino acid sequence of the mAb 1 domain of hLAG-3 VL1 (SEQ ID NO: 51) is shown below (hCDR residues are shown underlined).

DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL HSDGKTYLNW LLQKPGQSPE

RLIYLVSELD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP YTFGGGTKVE IK

[00293] The amino acid sequence of the VL Domain of hLAG-3 VL2 mAb 1 (SEQ ID NO: 52) is shown below (hCDR residues are shown underlined).

DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL HSDGKTYLNW LLQRPGQSPE RLIYLVSELD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP YTFGGGTKVE IK

[00294] The amino acid sequence of the VL Domain of mAb 1

VL3 of hLAG-3 (SEQ ID NO: 53) is shown below (hCDR residues are shown underlined).

DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL HSDGKTYLNW LLQKPGQPPE RLIYLVSELD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP YTFGGGTKVE IK

[00295] The amino acid sequence of the VL Domain of mAb 1

VL4 of hLAG-3 (SEQ ID NO: 54) is shown below (hCDR residues are shown underlined).

DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL HSDAKTYLNW LLQKPGQPPE RLIYLVSELD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP YTFGGGTKVE IK

CbQb ίη/ΖΖΠΖ/Ε/ΥΙΛΙ

[00296] The CDRlI of the VL Domain of mAb 1 VL4 of hLAG-3

CbQb ίη/77Ω7/Β/ΥΙΛΙ

203 comprises a glycine to alanine amino acid substitution and has the amino acid sequence: KSSQSLLHSDAKTYLN (SEQ ID NO: 55, 1st substituted alanine is shown underlined). It is contemplated that a similar substitution can be incorporated into any of the LAG-3 mAb 1 CDRlI Domains described above.

3. LAG-3 mAb 6

[00297] The amino acid sequence of the VH Domain of LAG-3 mAb 6 (SEQ ID NO: 56) is shown below (hCDR residues are shown underlined):

EVLLQQSGPE LVKPGASVKI PCKASGYTFT DYNMDWVKQS HGESLEWIGD INPDNGVTIY NQKFEGKATL TVDKSSSTAY MELRSLTSED TAVYYCAREA DYFYFDYWGQ GTTLTVSS

LAG-3 mAb 6 CDRhI (SEQ ID NO:57): DYNMD

CDRh2 of mAb 6 from LAG-3 (SEQ ID NO:58): DINPDNGVTIYNQKFEG

CDRh3 of LAG-3 mAb 6 (SEQ ID NO:59): EADYFYFDY

[00298] The amino acid sequence of the VL domain of LAG-3 mAb 6 (SEQ ID NO: 60) is shown below (CDRL residues are shown underlined):

DIVMTQSHRF MSTSVGDRVS ITCKASQDVS SWAWYQQKP GQSPKLLIFS ASYRYTGVPD RFTGSGSGTD FTFTISSVQA ADLAVYYCQQ HYSTPWTFGG GTKLEIK

LAG-3 mAb 6 CDRlI (SEQ ID NO:61): KASQDVSSWA

CDRl2 of mAb 6 of LAG-3 (SEQ ID NO:62): SASYRYT

204

CDRl3 of mAb 6 of LAG-3 (SEQ ID NO:63): HYSTPWT

[00299] Two humanized exemplars of the VH Domains of mAb 6 of LAG-3 designated herein as mAb 6 VH1 of hLAG-3, and mAb 6 VH2 of hLAG-3, and two exemplars of the humanized VL Domains of mAb 6 of LAG-3 mAb 1 VL1 of hLAG-3, and mAb 1 VL2 of hLAG-3, are provided below. Any of the humanized VL Domains can be paired with any of the humanized VH Domains to generate a LAG-3 binding domain. Accordingly, any antibody comprising one of the humanized VL Domains in pairs with the humanized VH Domain is generically referred to as LAG-3 hmAb 6, and particular combinations of humanized VH/VL Domains are referred to by reference to the VH/VL Domains. specific, for example a humanized antibody comprising hLAG-3 mAb 6 VH1 and LAG-3 VL2 hmAb 6 is specifically known as hLAG-3 mAb 6 (1.2).

[00300] The amino acid sequence of the mAb 6 VH1 domain of hLAG-3 (SEQ ID NO: 294) is shown below (hCDR residues are shown underlined):

QVQLVQSGAE VKKPGASVKV SCKASGYTFT DYNMDWVRQA PGQGLEWMGD INPDNGVTIY NQKFEGRVTM TTDTSTSTAY MELRSLRSDD TAVYYCAREA DYFYFDYWGQ GTTLTVSS

[00301] An amino acid sequence of the VH Domain of mAb 6 cnan Ln/zznz/E/YiAi

VH2 of hLAG-3 (SEQ ID NO: 295) is shown below

205 (CDRh residues are shown underlined):

EVQLVESGGG LVKPGGSLRL SCAASGFTFS DYNMDWVRQA PGKGLEWVSD INPDNGVTIY NQKFEGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREA DYFYFDYWGQ GTTLTVSS

[00302] The amino acid sequence of the VL Domain of hLAG-3 mAb 6 VL1 (SEQ ID NO: 296) is shown below (CDRl residues are shown underlined).

DIQMTQSPSS LSASVGDRVT ITCRASQDVS SWAWYQQKP GKAPKLLIYS ASYRYTGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ HYSTPWTFGG GTKLEIK

[00303] The amino acid sequence of the VL Domain of hLAG-3 mAb 6 VL2 (SEQ ID NO: 297) is shown below (CDRl residues are shown underlined).

DIVMTQSPSS LSASVGDRVT ITCRASQDVS SWAWYQQKP GKAPKLLIYS ASYRYTGVPD RFSGSGSGTD FTFTISSLQP EDIAVYYCQQ HYSTPWTFGG GTKLEIK

[00304] The CDRlI of the VL Domain of mAb 6 VL1 of hLAG-3 and VL2 comprises an amino acid substitution from Usine to arginine and has the amino acid sequence: RASQDVSSWA (SEQ ID NO:298), the substituted arginine is shown underlined) . It is contemplated that a similar substitution can be incorporated into any of the LAG-3 CDRlI mAb 6 Domains described above.

cnan Ln/zznz/E/YiAi

B. Diabodies Containing the Faith Region of Four

206

Exemplary Chains Having E/K-helices.

[00305] Four exemplars of diabodies containing the Fe Region of four bispecific PD-1 designated). The structure of these diabodies containing the Fe Region is detailed below. These exemplary PD-1 x LAG3 diabodies are intended to illustrate, but in no way limit, the scope of the invention.

1. DART A

[00306] DART A is a diabody containing the four-chain, bispecific Fe Region, which has two specific binding sites for PD-1, two specific binding sites for LAG-3, a variant IgG4 Fe Region designed for Domains Promoting Heterodimer of E/K-helices containing cysteine ​​and extended half-life. The first and third polypeptide chains of DART A comprise, in the N-terminal to C-terminal direction: an N-terminal, a VL Domain of a monoclonal antibody capable of binding to LAG-3 (VLlag-3 mAb 1 VL4 hLAG-3) (SEQ ID NO:54); an intervening linker peptide (Linker 1: GGGSGGGG (SEQ ID NO: 14)); a VH domain of a monoclonal antibody capable of binding PD-1 (VHpd-i hPD-1 mAb 7 VH1) (SEQ ID NO: 147); a cysteine-containing intervening linker peptide (Linker 2:

cnan Ln/zznz/E/YiAi

207

GGCGGG (SEQ ID NO: 15)); a Heterodimer Promoter Domain (E-helix) containing cistern (EVAACEK-EVAALEK-EVAALEKEVAALEK (SEQ ID NO: 23)) a stabilized IgG4 hinge region (SEQ ID NO: 13); a variant IgG4 CH2-CH3 Domain comprising M252Y/S254T/T256E substitutions and lacking the C-terminal residue (SEQ ID NO: 259); and a Cterminal

[00307] The amino acid sequence of the first and third polypeptide chains of DART A is a variant of SEQ ID NO: 267:

cnan Ln/zznz/E/YiAi

DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL HSDXiKTYLNW LLQKPGQPPE

RLIYLVSELD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP

YTFGGGTKVE IKGGGSGGGG QVQLVQSGAE VKKPGASVKV SCKASGYSFT

SYWMNWVRQA PGQGLEWIGV IHPSDSETWL DQKFKDRVTI TVDKSTSTAY

MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSSG GCGGGEVAAC

EKEVAALEKE VAALEKEVAA LEKESKYGPP CPPCPAPEFL GGPSVFLFPP

KPKDTLX₂IX₃R X4PEVTCVVVD VSQEDPEVQF NWYVDGVEVH NAKTKPREEQ

FNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KGLPSSIEKT ISKAKGQPRE

PQVYTLPPSQ EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP

PVLDSDGSFF LYSRLTVDKS RWQEGNVFSC SVMHEALHNH YTQKSLSLSL eri^ where Xi, X2 is Y or Μ; X3 is T or S; and X4 is E or

T.

[00308] The amino acid sequences of the first and third polypeptide chains of DART A is SEQ ID NO: 267, where

XI is A; X2 is Y; X3 is T; and X4 is E.

[00309] The second and fourth polypeptide chains of DART A comprise, in the N-terminal to C-terminal direction: a

CbQb ίη/77Ω7/Β/ΥΙΛΙ

208

VL domain, N-terminal, of a monoclonal antibody capable of binding PD-1 (VLpd-i hPD-1 mAb 7 VL2) (SEQ ID NO: 153); an intervening linker peptide (Linker 1: GGGSGGGG (SEQ ID NO:14)); a VH Domain of a monoclonal antibody capable of binding LAG-3 (VHlag-3 hLAG-3 mAb 1 VH1) (SEQ ID NO:49); a cysteine-containing intervening linker peptide (Linker 2: GGCGGG (SEQ ID NO: 15)); a Cysteine-containing Heterodimer Promoter Domain (K-helix) (KVAACKEKVAALKE-KVAALKE-KVAALKE (SEQ ID NO:24); and a C-terminus. [00310] The amino acid sequence of the second and fourth polypeptide chains of DART A is (SEQ ID NO:268):

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVQSGAEV KKPGASVKVS CKASGYTFTN YGMNWVRQAP GQGLEWMGWI NTYTGESTYA DDFEGRFVFS MDTS ASTAYL QISSLKAEDT AVYYCARESL YDYYSMDYWG QGTTVTVSSG GCGGGKVAAC KEKVAALKEK VAALKEKVAA LKE

2. DART B

[00311] DART B is identical to DART A, except that the first and third polypeptide chains of DART B comprise the VL Domain of hLAG-3 mAb 1 VL3 (SEQ ID NO:53), which comprises an amino acid substitution in CDRlI. Thus, the first and third DART B polypeptide chains comprise, in the N-terminal to C-terminal direction: a to N-terminal; a VL Domain of a monoclonal antibody capable of binding LAG-3 (VLlag-3 hLAG-3 mAb 1 VL3) (SEQ ID NO: 53); a cnan Ln/zznz/E/YiAi

209 intervention linker peptide (Linker 1: GGGSGGGG (SEQ ID NO:14)); a VH Domain of a monoclonal antibody capable of binding to PD-1 (VHpd-i hPD-1 mAb 7 VH1) (SEQ ID NO: 147); an intervening linker peptide (Linker 2: GGGGGG (SEQ ID NO: 15)); a Heterodimer Promoter (E-helix) domain (EVAACEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO: 23)) containing cysteine; a stabilized IgG4 hinge region (SEQ ID NO: 13); an IgG4 CH2-CH3 Domain variant comprising the M252Y/S254T/T256E substitutions and lacking the C-terminal residue (SEQ ID NO: 259); and a C-terminal.

[00312] The amino acid sequence of the first and third polypeptide chains of DART B is SEQ ID NO: 267, where Xi is G; X2 is Y; X3 is T; and₄ that.

[00313] The amino acid sequence of the second and fourth polypeptide chains of DART B is SEQ ID NO: 268.

3. DART C

[00314] DART C is identical to DART B, except that the first and third polypeptide chains of DART B comprise a wild-type IgG4 CH2-CH3 domain that lacks the C-terminal residue (SEQ ID NO: 4). Thus, the first and third DART C polypeptide chains comprise, in the N-terminal to C-terminal direction: a to N-terminal; a VL Domain of a monoclonal antibody capable of binding LAG-3 (VLlag-3 hLAG-3 mAb 1 VL3) (SEQ ID NO: 53); a cnan peptide Ln/zznz/E/YiAi

210 intervention linker (Linker 1: GGGSGGGG (SEQ ID NO:14)); a VH Domain of a monoclonal antibody capable of binding to PD-1 (VHpd-i hPD-1 mAb 7 VH1) (SEQ ID NO: 147); an intervening linker peptide (Linker 2: GGGGGG (SEQ ID NO: 15)); a cysteine-containing Heterodimer Promoter (E-helix) domain (EVAACEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO: 23)) a stabilized IgG4 hinge region (SEQ ID NO: 13); an IgG4 CH2-CH3 Domain lacking the C-terminal residue (SEQ ID NO: 4); and a C-terminal.

[00315] The amino acid sequence of the first and third polypeptide chains of DART B is SEQ ID NO: 2 67, where Xi is G; X2 is Μ; X3 is S; and X4 is T.

[00316] The amino acid sequence of the second and fourth polypeptide chains of DART C is SEQ ID NO: 268.

4. DART I

[00317] DART I is a diabody containing the four-chain, bispecific Fe Region, which has two specific binding sites for PD-1, two specific binding sites for LAG-3, a variant IgG4 Fe Region designed for Cysteine-Containing E/K-Helix Heterodimer Promotion Domains, Extended Half-Life. The first and third DART I polypeptide chains comprise, in the N-terminal to C-terminal direction: an N-terminus; a VL domain of a monoclonal antibody capable of binding to LAG-3 (mAb 6

211

VL1 of VLlag-3 hLAG-3) (SEQ ID NO:296); an intervening linker peptide (Linker 1: GGGSGGGG (SEQ ID NO:14)); a VH Domain of a monoclonal antibody capable of binding to PD-1 (VHpd-i hPD-1 mAb 7 VH1) (SEQ ID NO: 147); a cysteine-containing intervening linker peptide (Linker 2: GGCGGG (SEQ ID NO: 15)); a Heterodimer Promoter Domain (E-helix) containing cysteine ​​(EVAACEK-EVAALEK-EVAALEKEVAALEK (SEQ ID NO: 23)) a stabilized IgG4 hinge region (SEQ ID NO: 13); a variant IgG4 CH2-CH3 Domain comprising M252Y/S254T/T256E substitutions and lacking the C-terminal residue (SEQ ID NO: 259); and a Cterminal.

[00318] The amino acid sequence of the first and third polypeptide chains of DART I is SEQ ID NO: 290.

DIQMTQSPSS LSASVGDRVT ITCRASQDVS SVVAWYQQKP GKAPKLLIYS ASYRYTGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ HYSTPWTFGG GTKLEIKGGG SGGGGQVQLV QSGAEVKKPG ASVKVSCKAS GYSFTSYWMN WVRQAPGQGL EWIGVIHPSD SETWLDQKFK DRVTIT VDKS TSTAYMELSS LRSEDTAVYY CAREHYGTSP FAYWGQGTLV TVSSGGCGGG EVAACEKEVA ALEKEVAALE KEVAALEKES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LYITREPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGL PS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG

[00319] The second and fourth polypeptide chains of DART I comprise, in the N-terminal to C-terminal direction: a VL domain, N-terminal, of a monoclonal antibody capable of binding to PD-1 (mAb 7 VL2 of VLPD-1 hPD-1) (SEQ ID NO: 153); an intervention linker peptide (Linker 1: GGGSGGGG (SEQ cnan Ln/zznz/E/YiAi

212

ID NO:14)); a VH Domain of a monoclonal antibody capable of binding to LAG-3 (VHlag-3 hPD-3 mAb 6 VH1) (SEQ ID NO:294); a cysteine-containing intervening linker peptide (Linker 2: GGCGGG (SEQ ID NO: 15)); a Cysteine-containing Heterodimer Promoter Domain (K-helix) (KVAACKEKVAALKE-KVAALKE-KVAALKE (SEQ ID NO:24); and a C-terminus.

[00320] The amino acid sequence of the second and fourth polypeptide chains of DART A is (SEQ ID NO:291):

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVQSGAEV KKPGASVKVS CKASGYTFTD YNMDWVRQAP GQGLEWMGDI NPDNGVTIYN QKFEGRVTMT T DTSTSTAYM ELRSLRSDDT AVYYCAREAD YFYFDYWGQG TTLTVSSGGC GGGKVAACKE KVAALKEKVA ALKEKVAALK E cnan Ln/zznz/E/YiAi

C. Diabodies Containing the Fe Region of Four Exemplary Chains Having CL/CH1 Domains.

[00321] Four exemplars of diabodies containing the Fe Region were generated from four PD-1 X LAG-3 bispecific chains comprising the designated CL/CH1 DART D, DART E, DART J and DART 1 Domains. The structure of these diabodies containing the Fe Region is detailed in the following. These exemplary PD-1 x LAG3 diabodies are intended to illustrate, but in no way limit, the scope of the invention.

213

1. DART D

[00322] DART D is a bispecific, four-chain Fe Region-containing diabody that has two specific binding sites for PD-1, two specific binding sites for LAG-3, CL/CH1 Domains, and one Fe region. of variant IgG4 designed for extended half-life. The first and third DART D polypeptide chains comprise, in the N-terminal to C-terminal direction: an N-terminal; a VL Domain of a monoclonal antibody capable of binding PD-1 (VLpdi hLAG-1 mAb 7 VL2) (SEQ ID NO:153); an intervening linker peptide (Linker 1: GGGSGGGG (SEQ ID NO:14)); a VH Domain of a monoclonal antibody capable of binding to LAG3 (VHlag-3 hPD-3 VH1 mAb 1) (SEQ ID NO: 49); an intervening linker peptide (Linker 2: LGGGSG (SEQ ID NO: 261)); an IgG4 CHI Domain (SEQ ID NO:254); a stabilized IgG4 hinge region; a CHICHI IgG4 Domain variant comprising M252Y/S254T/T256E substitutions and lacking the C-terminal residue (SEQ ID NO: 259); and a Cterminal.

[00323] The amino acid sequence of the first and third polypeptide chains of DART D is (SEQ ID NO: 269):

CbQb ίη/77Π7/Ε/ΥΙΛΙ

214 cnan Ln/zznz/E/YiAi

EIVLTQSPAT LTHAASNQGS YGMNWVRQAP QISSLKAEDT PSVFPLAPCS VLQSSGLYSL CPPCPAPEFL NWYVDGVEVH KGLPSSIEKT DIAVEWESNG SVMHEALHNH

LSLSPGERAT GVPSRFSGSG GQGLEWMGWI AVYYCARESL RSTSESTAAL SSVVTVPSSS GGPSVFLFPP NAKTKPREEQ ISKAKGQPRE QPENNYKTTP YTQKSLSLSL

LSCRASESVD SGTDFTLTIS NTYTGESTYA YDYYSMDYWG GCLVKDYFPE LGTKTYTCNV KPKDTLYITR FNSTYRVVSV PQVYTLPPSQ PVLDSDGSFF G

NYGMSFMNWF SLEPEDFAVY DDFEGRFVFS QGTTVTVSSL PVTVSWNSGA DHKPSNTKVD EPEVTCVVVD LTVLHQDWLN EEMTKNQVSL LYSRLTVDKS

QQKPGQPPKL ^CQQSKEVPY MDTSASTAYL GGGSGASTKG LTSGVHTFPA KRVESKYGPP VSQEDPEVQF GKEYKCKVSN TCLVKGFYPS RWQEGNVFSC

[00324] The second and fourth DART D polypeptide chains comprise, in the N-terminal to C-terminal direction: an N-terminal; a VL Domain of a monoclonal antibody capable of binding LAG-3 (VLlag-3 hLAG-3 mAb 1 VL4) (SEQ ID NO:54); an intervening linker peptide (Linker 1: GGGSGGGG (SEQ ID NO: 14)); a VH domain of a monoclonal antibody capable of binding PD-1 (VHpd-i hPD-1 mAb 7 VH1) (SEQ ID NO:147); an intervening linker peptide (Linker 2: LGGGSG (SEQ ID NO:261)); a Kappa CL Domain (SEQ ID NO:8); and a Cterminal.

[00325] The amino acid sequence of the second and fourth polypeptide chains of DART A is (SEQ ID NO:270):

DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL HSDAKTYLNW LLQKPGQPPE RLIYLVSELD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP YTFGGGTKVE IKGGGSGGGG QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWIGV IHPSDSETWL DQK FKDRVTI TVDKSTSTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSSL GGGSGRTVAA PSVFIFPPSD EQLKSGTASV VCLLNNFYPR EAKVQWKVDN ALQSGNSQES VTEQDSKDST YSLSSTLTLS KADYEKHKVY ACEVTHQGLS SPVTKSFNRG EC

215

2. TO GIVE YOU

[00326] DART E is another diabody containing the bispecific, four-chain Fe Region, which has two specific binding sites for PD-1, two specific binding sites for LAG-3, CL/CH1 Domains, and one Fe Region of IgG4 variant designed for extended half-life. The position of the PD-1 and LAG-3 binding sites of DART E is reversed compared to DART D.

[00327] The first and third DART E polypeptide chains comprise, in the N-terminal to C-terminal direction: an N-terminal; a VL Domain of a monoclonal antibody capable of binding LAG-3 (VLlag-3 hLAG-3 mAb 1 VL4) (SEQ ID NO:54); an intervening linker peptide (Linker 1: GGGSGGGG (SEQ ID NO: 14)); a VH Domain of a monoclonal antibody capable of binding to PD-1 (VHpd-i hPD-1 mAb 7 VH1) (SEQ ID NO:147); an intervening linker peptide (Linker 2: LGGGSG (SEQ ID NO: 261)); an IgG4 CHI Domain (SEQ ID NO:254); a stabilized IgG4 hinge region; an IgG4 CH2-CH3 Domain variant comprising M252Y/S254T/T256E substitutions and lacking the C-terminal residue (SEQ ID NO: 259); and a Cterminal.

[00328] The amino acid sequence of the first and third polypeptide chains of DART E is (SEQ ID NO: 271):

cnan Ln/zznz/E/YiAi

216

DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL HSDAKTYLNW LLQKPGQPPE RLIYLVSELD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP YTFGGGTKVE IKGGGSGGGG QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWIGV IHPSDSETWL DQK FKDRVTI TVDKSTSTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSSL GGGSGASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTKTYTCNV DHKPSNTKVD KRVESKYGPP CPPCPAPEFL GGPSVFLFPP KPKDTLYITR EP EVTCVVVD VSQEDPEVQF NWYVDGVEVH NAKTKPREEQ FNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KGLPSSIEKT ISKAKGQPRE PQVYTLPPSQ EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSRLTVDKS RWQEGNVFSC SVMHEALHNH YTQKSLSLSL G

[00329] The second and fourth DART E polypeptide chains comprise, in the N-terminal to C-terminal direction: an N-terminal; a VL Domain of a monoclonal antibody capable of binding PD-1 (VLPD-1 hPD-1 mAb 7 VL2) (SEQ ID NO: 153); an intervening linker peptide (Linker 1: GGGSGGGG (SEQ

ID NO:14)); a VH Domain of a monoclonal antibody capable of binding to LAG-3 (VHlag-3 hLAG-3 mAb 1 VH1) (SEQ ID NO: 49);

an intervening linker peptide (Linker 2: LGGGSG (SEQ ID NO:261)); a Kappa CL Domain (SEQ ID NO:8); and a Cterminal.

[00330] The amino acid sequence of the second and fourth polypeptide chains of DART E is (SEQ ID NO:272):

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVQSGAEV KKPGASVKVS CKASGYTFTN YGMNWVRQAP GQGLEWMGWI NTYTGESTYA DDFEGRFVFS MDTS ASTAYL QISSLKAEDT AVYYCARESL YDYYSMDYWG QGTTVTVSSL GGGSGRTVAA PSVFIFPPSD EQLKSGTASV VCLLNNFYPR EAKVQWKVDN ALQSGNSQES VTEQDSKDST YSLSSTLTLS KADYEKHKVY ACEVTHQGLS SPVTKSFNRG EC

217

3. DART J

[00331] DART J is a bispecific, four-chain Fe Region-containing diabody that has two specific binding sites for PD-1, two specific binding sites for LAG-3, CL/CH1 Domains, and one Fe region. of variant IgG4 designed for extended half-life. The first and third DART J polypeptide chains comprise, in the N-terminal to C-terminal direction: an N-terminal; a VL Domain of a monoclonal antibody capable of binding LAG-3 (VLlag-3 hLAG-3 mAb 6 VL1) (SEQ ID NO:296); an intervening linker peptide (Linker 1: GGGSGGGG (SEQ ID NO:14)); a VH Domain of a monoclonal antibody capable of binding to PD-1 (VHpd-i hPD-1 mAb 7 VH1) (SEQ ID NO: 147); an intervening linker peptide (Linker 2: LGGGSG (SEQ ID NO: 261)); an IgG4 CHI Domain (SEQ ID NO:254); a stabilized IgG4 hinge region (SEQ ID NO:13); an IgG4 CH2-CH3 Domain variant comprising M252Y/S254T/T256E substitutions and lacking the C-terminal residue (SEQ ID NO: 259); and a C-terminal.

[00332] The amino acid sequence of the first and third polypeptide chains of DART J is (SEQ ID NO: 292):

cnan Ln/zznz/E/YiAi

218 cnan Ln/zznz/E/YiAi

DIQMTQSPSS ASYRYTGVPS GTKLEIKGGG WVRQAPGQGL LRSEDTAVYY LAPCSRSTSE GLYSLSSVVT APEFLGGPSV GVEVHNAKTK SIEKTISKAK WESNGQPENN ALHNHYTQKS

LSASVGDRVT RFSGSGSGTD SGGGGQVQLV EWIGVIHPSD CAREHYGTSP STAALGCLVK VPSSSLGTKT FLFPPKPKDT PREEQFNSTY GQPREPQVYT YKTTPPVLDS LSLSLG

ITCRASQDVS FTLTISSLQP QSGAEVKKPG SETWLDQKFK FAYWGQGTLV DYFPEPVTVS YTCNVDHKPS LYITREPEVT RVVSVLTVLH LPPSQEEMTK DGSFFLYSRL

SVVAWYQQKP EDFATYYCQQ ASVKVSCKAS DRVTITVDKS TVSSLGGGSG WNSGALTSGV NTKVDKRVES CVVVDVSQED QDWLNGKEYK NQVSLTCLVK TVDKSRWQEG

GKAPKLLIYS HYSTPWTFGG GYSFTSYWMN TSTAYMELSS ASTKGPSVFP HTFPAVLQSS KYGPPCPPCP PEVQFNWYVD CKVSNKGLPS GFYPSDIAVE NVFSCSVMHE

[00333] The second and fourth DART J polypeptide chains comprise, in the N-terminal to C-terminal direction: an N-terminal; a VL Domain of a monoclonal antibody capable of binding PD-1 (VLPD-1 hPD-1 mAb 7 VL2) (SEQ ID NO:153); an intervening linker peptide (Linker 1: GGGSGGGG (SEQ ID NO:14)); a VH Domain of a monoclonal antibody capable of binding to LAG-3 (VHlag-3 hLAG-3 mAb 6 VH1) (SEQ ID NO:294); an intervening linker peptide (Linker 2: LGGGSG (SEQ ID NO:261)); a Kappa CL Domain (SEQ ID NO:8); and a C-terminal.

[00334] The amino acid sequence of the second and fourth polypeptide chains of DART J is (SEQ ID NO:293):

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVQSGAEV KKPGASVKVS CKASGYTFTD YNMDWVRQAP GQGLEWMGDI NPDNGVTIYN QKFEGRVTMT T DTSTSTAYM ELRSLRSDDT AVYYCAREAD YFYFDYWGQG TTLTVSSLGG GSGRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC

219

4. DART 1

[00335] DART 1 is a bispecific, four-chain Fe Region-containing diabody that has two specific binding sites for PD-1, two specific binding sites for LAG-3, CL/CH1 Domains, and one Fe region. of variant IgGl designed for reduced FcyR binding. The first and third DART 1 polypeptide chains comprise, in the N-terminal to C-terminal direction: an N-terminal; a VL Domain of a monoclonal antibody capable of binding PD-1 (VLpd-i PD-1 mAb A VL) (SEQ ID NO:65); an intervening linker peptide (Linker 1: GGGSGGGG (SEQ ID NO:14)); a VH Domain of a monoclonal antibody capable of binding to LAG-3 (VHlag-3 LAG-3 mAb A VH1) (SEQ ID NO:39); an intervening linker peptide (Linker 2: LGGGSG (SEQ ID NO:261)); A CHI IgGl Domain (SEQ ID NO: 10); an IgGl hinge region (SEQ ID NO: 32); an IgGl CH2-CH3 Domain variant comprising the L234A/L235A substitutions and lacking the C-terminal residue (SEQ ID NO: 5); and a C-terminal.

[00336] The amino acid sequence of the first and third polypeptide chains of DART 1 is (SEQ ID NO: 284):

cnan Ln/zznz/E/YiAi

220 cnan Ln/zznz/E/YiAi

EIVLTQSPAT LSLSPGERAT LSCRASQSIS SYLAWYQQKP GQAPRLLIYD

ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPLTFGQ

GTNLEIKGGG SGGGGQVQLV ESGGGVVQPG RSLRLDCKAS GITFSNSGMH

WVRQAPGKGL EWVAVIWYDG SKRYYADSVK GRFTISRDNS KNTLFLQMNS

LRAEDTAVYY CATNDDYWGQ GTLVTVSSLG GGSGASTKGP SVFPLAPSSK

STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS

SVVTVPSSSL GTQTYICNVN HKPSNTKVDK RVEPKSCDKT HTCPPCPAPE

AAGGPSVFLF PPKPKDTLYI TREPEVTCVV VDVSHEDPEV KFNWYVDGVE

VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE

KTISKAKGQP REPQVYTLPP SREEMTKNQV SLTCLVKGFY PSDIAVEWES

NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH

NHYTQKSLSL SPG

[00337] The second and fourth polypeptide chains of DART 1 comprise, in the N-terminal to C-terminal direction: an N-terminal; a VL domain of a monoclonal antibody capable of binding to LAG-3 (VLlag-3 LAG-3 mAb A VL) (SEQ ID NO:40); an intervening linker peptide (Linker 1: GGGSGGGG (SEQ ID NO:14)); a VH Domain of a monoclonal antibody capable of binding to PD-1 (VHpd-i PD-1 VH mAb A) (SEQ ID NO: 64); an intervening linker peptide (Linker 2: LGGGSG (SEQ ID NO:261)); a Kappa CL Domain (SEQ ID NO: 8); and a Cterminal.

[00338] The amino acid sequence of the second and fourth polypeptide chains of DART 1 is (SEQ ID NO:285):

EIVLTQSPAT ASNRATGIPA GTKVEIKGGG WIRQPPGKGL TAADTAVYYC IFPPSDEQLK DSKDSTYSLS

LSLSPGERAT RFSGSGSGTD SGGGGQVQLQ EWIGEINHNG AFGYSDYEYN SGTASVVCLL STLTLSKADY

LSCRASQSVS FTLTISSLEP QWGAGLLKPS NTNSNPSLKS WFDPWGQGTL NNFYPREAKV EKHKVYACEV

SYLAWYQQKP EDFAVYYCQQ ETLSLTCAVY RVTLSLDTSK VTVSSLGGGS QWKVDNALQS THQGLSSPVT

GQAPRLLIYD SSNWPRTFGQ GGSFSDYYWN NQFSLKLRSV GRTVAAPSVF GNSQESVTEQ KSFNRGEC

221

D. Diabodies Containing the Faith Region of Five Exemplary Chains

[00339] Two diabodies containing the Fe region of five exemplary PD-1 The structure of these diabodies containing the Fe Region is detailed in the following. These exemplary PD-1 X LAG-3 diabodies are intended to illustrate, but in no way limit, the scope of the invention.

cnan ίη/ζζηζ/Ε/γίΛΐ

1. DART F

[00340] DART F is a diabody containing the five-chain, bispecitic Fe Region, which has three specific binding sites for PD-1, one specific binding site for LAG3, CL/CH1 Domains, an IgGl Fe Region that carries the button/hole variant designed for extended half-life E/K-helix Heterodimer Promotion Domains and bound to the reduced FcyR. The first DART F polypeptide chain comprises, in the direction from the N-terminus to the C-terminus: an N-terminus; a VH Domain of a monoclonal antibody capable of binding PD-1 (VHpd-i hPD-1 mAb 7 VH1) (SEQ ID NO:147); An IgGl CH1 Domain (SEQ ID NO:10); an IgGl hinge region (SEQ ID NO:32); a CH2-CH3 IgGl Domain carrying the hole comprising the substitutions

222

L234A/L235A/M252Y/S254T/T256E/N434A/H435K and lacks the C-terminal residue (SEQ ID NO:260, where Xi is A, X2 is A;

X3 is Y, X4 is T, X5 is E, Χε is S, X7 is A, Xs is V, X9 is A,

X10 is K, and Xn is absent); and a C-terminal.

[00341] The amino acid sequence of the first DART F polypeptide chains is (SEQ ID NO: 273):

QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWIGV IHPSDSETWL DQKFKDRVTI TVDKS'ASTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSL SSVVTV PSSSLGTQTY

ICNVNHKPSN TKVDKRVEPK SCDKTHTCPP CPAPEAAGGP SVFLFPPKPK

DTLYITREPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS

TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV

YTLPPSREEM TKNQVSLSCA VKGFYPSDIA VEWESNGQPE NNYKTTPPVL

DSDGSFFLVS KLTVDKSRWQ QGNVFSCSVM HEALHAKYTQ KSLSLSPG

[00342] The second and fifth DART F polypeptide chains comprise, in the N-terminal to C-terminal direction: an N-terminal; a VL Domain of a monoclonal antibody capable of binding PD-1 (VLPD-1 hPD-1 mAb 7 VL2) (SEQ ID NO: 153); a

Kappa CL domain (SEQ ID NO:8), and a C-terminus.

[00343] The amino acid sequence of the second and fifth polypeptide chains of DART F is (SEQ ID NO:274):

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL

LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY

TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV

QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV

THQGLSSPVT KSFNRGEC

[00344] cnan Ln/zznz/E/YiAi

The third polypeptide chain of DART F

223 comprises, in the N-terminal to C-terminal direction: an Nterminal; a VH Domain of a monoclonal antibody capable of binding PD-1 (VH mAb 7 VH1_P.S.-i hPD-1) (SEQ ID NO:147); a CHI IgGl Domain (SEQ ID NO: 10); an IgGl hinge region (SEQ ID NO: 32); a button-bearing IgGl CH2-CH3 Domain comprising substitutions L234A/L235A/M252Y/S254T/T256E and lacking the C-terminal residue (SEQ ID NO:260, where Xi is A, X2 is A; x₄ is T, X5 is E, Xg is W, X7 is L, Xg is Y, X9 is N, X10 is H, and₄₄ he is absent) ; an intervening linker peptide (GGGSGGGSGGG (SEQ ID NO:262)); a VL Domain of a monoclonal antibody capable of binding LAG-3 (VLlag-3 hLAG-3 mAb 1 VL4) (SEQ ID NO:54); a linker intervention peptide (Linker 1: GGGSGGGG (SEQ ID NO:14)); a VH Domain of a monoclonal antibody capable of binding to PD-1 (VHpd-i hPD-1 mAb 7 VH1) (SEQ ID NO: 147); a cysteine-containing intervening linker peptide (Linker 2: GGCGGG (SEQ ID NO:15)); a Heterodimer promoter Domain (E-helix) (EVAALEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO:21)); and a Cterminal.

[00345] The amino acid sequence of the third polypeptide chain of DART F is (SEQ ID NO: 275):

cnan Ln/zznz/E/YiAi

224

QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWIGV IHPSDSETWL DQKFKDRVTI TVDKSTSTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSS VVTV PSSSLGTQTY ICNVNHKPSN TKVDKRVEPK SCDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLYITREPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM TKNQ VSLWCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGGG GSGGGSGGGD IVMTQTPLSL SVTPGQPASI SCKSSQSLLH SDAKTYLNWL LQKPGQPPER LIYLVSELDS GVPDRFSGSG SGTDFTLKIS RVEAEDVGVY YCWQGTHFPY TFGGGTKVEI KGGGSGGGGQ VQLVQSGAEV KKPGASVKVS CKASGYSFTS YWMNWVRQAP GQGLEWIGVI HPSDSETW LD QKFKDRVTIT VDKSTSTAYM ELSSLRSEDT AVYYCAREHY GTSPFAYWGQ GTLVTVSSGG CGGGEVAALE KEVAALEKEV AALEKEVAAL EK

[00346] The fourth DART F polypeptide chain comprises, in the N-terminal to C-terminal direction: an N-terminal; a

VL domain of a monoclonal antibody capable of binding PD-1 (VLPD-1 hPD-1 mAb 7 VL2) (SEQ ID NO:153); an intervening linker peptide (Linker 1: GGGSGGGG (SEQ ID

NO:14)); a VH Domain of a monoclonal antibody capable of binding to LAG-3 (VHlag-3 hLAG-3 mAb 1 VH1) (SEQ ID NO:49)_; a cysteine-containing intervening linker peptide (Linker 2: GGCGGG (SEQ ID NO: 15)); a Domain (K-helix)

Heterodimer Promoter (KVAALKE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO:22)); and a C-terminal.

[00347] The amino acid sequence of the four DART F polypeptide chains is (SEQ ID NO: 276):

225

EIVLTQSPAT LIHAASNQGS TFGGGTKVEI YGMNWVRQAP QISSLKAEDT KEKVAALKEK

LSLSPGERAT GVPSRFSGSG KGGGSGGGGQ GQGLEWMGWI AVYYCARESL VAALKEKVAA

LSCRASESVD SGTDFTLTIS VQLVQSGAEV NTYTGESTYA YDYYSMDYWG LKE

NYGMSFMNWF SLEPEDFAVY KKPGASVKVS DDFEGRFVFS QGTTVTVSSG

QQKPGQPPKL FCQQSKEVPY CKASGYTFTN MDTSASTAYL GCGGGKVAAL cnan Ln/zznz/E/YiAi

2. DART G

[00348] DART G is a diabody containing the Fe Region of five chains, bispecific, having two specific binding sites for PD-1, two specific binding sites for LAG-3, CL/CH1 Domains, an Fe Region of IgGl carrying the button/hole variant designed for E/K-helix Heterodimer Promotion Domains and extended half-life and linked to the reduced FcyR. The first DART G polypeptide chain comprises, in the N-terminal to C-terminal direction: an N-terminal; a VH Domain of a monoclonal antibody capable of binding to LAG-3 (VHlag-3 hLAG3 mAb 1 VH1) (SEQ ID NO:49); a CHI IgGl Domain (SEQ ID NO:10); an IgGl hinge region (SEQ ID NO:32); a hole-bearing IgGl CH2-CH3 domain comprising substitutions L234A/L235A/M252Y/S254T/T256E/N434A/H435K and lacking the C-terminal residue (SEQ ID NO:260, where Xi is A, X2 is A ; X3 is Y, X4 is T, X5 is E, Xe is S, X7 is A, Xs is V, X9 is A, X10 is K, and and a C-terminal.

[00349] The amino acid sequence of the first DART G polypeptide chain is (SEQ ID NO: 277):

226

QVQLVQSGAE VKKPGASVKV SCKASGYTFT NYGMNWVRQA PGQGLEWMGW INTYTGESTY ADDFEGRFVF SMDTSASTAY LQISSLKAED TAVYYCARES LYDYYSMDYW GQGTTVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPEAAGG PSVFLFPPKP KDTLYITREP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLSC AV KGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLV SKLTVDKSRW QQGNVFSCSV MHEALHAKYT QKSLSLSPG

[00350] The second and fifth polypeptide chains of DART

G comprises, in the N-terminal to C-terminal direction: an N-terminal; a Domino VL of a monoclonal antibody capable of binding to LAG-3 (VLlag-3 hLAG-3 mAb 1 VL4) (SEQ ID NO: 54), a

Kappa CL domain (SEQ ID NO:8), and a C-terminus.

[00351] The amino acid sequence of the second and fifth polypeptide chains of DART G is (SEQ ID NO:278):

DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL HSDAKTYLNW LLQKPGQPPE

RLIYLVSELD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP

YTFGGGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK

VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE

VTHQGLSSPV TKSFNRGEC

[00352] The third DART G polypeptide chains comprise, in the N-terminal to C-terminal direction: an N-terminal; a VH Domain of a monoclonal antibody capable of binding to LAG-3 (VHlag-3 hLAG-3 mAb 1 VH1) (SEQ ID NO: 49); a

IgGl CHI domain (SEQ ID NO: 10); an IgGl hinge region (SEQ ID NO:32); an IgGl CH2-CH3 Domain carrying the knob comprising substitutions L234A/L235A/M252Y/S254T/T256E and lacking the C-terminal residue (SEQ ID NO:260, where Xi is

227

A, X2 is A; X3 is Y, X4 is T, X5 is E, Xg is W, X7 is L, Xs is Y, X9 is N, X10 is H, and an intervening linker peptide (GGGSGGGSGGG (SEQ ID NO:262)); a Domino VL of a monoclonal antibody capable of binding to PD-1 (VLpd-i hPD-1 mAb 7 VL2) (SEQ ID NO: 153); an intervening linker peptide (Linker 1: GGGSGGGG (SEQ ID NO:14)); a VH Domain of a monoclonal antibody capable of binding to PD-1 (VHpd-i hPD-1 mAb 7 VH1) (SEQ ID NO: 147); a cysteine-containing intervening linker peptide (Linker 2: GGCGGG (SEQ ID NO:15)); a Promotion of Heterodimer (Ehelix) Domain (EVAALEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO:21)); and a C-terminal.

[00353] The amino acid sequence of the third polypeptide chains of DART G is (SEQ ID NO:279):

cnan Ln/zznz/E/YiAi

QVQLVQSGAE INTYTGESTY LYDYYSMDYW DYFPEPVTVS YICNVNHKPS KDTLYITREP

STYRVVSVLT VYTLPPSREE LDSDGSFFLY GGSGGGSGGG QQKPGQPPKL FCQQSKEVPY CKASGYSFTS VDKSTSTAYM CGGGEVAALE

VKKPGASVKV ADDFEGRFVF GQGTTVTVSS WNSGALTSGV NTKVDKRVEP EVTCVVVDVS VLHQDWLNGK MTKNQVSLWC SKLTVDKSRW EIVLTQSPAT LIHAASNQGS TFGGGTKVEI YWMNWVRQAP ELSSLRSEDT KEVAALEKEV

SCKASGYTFT SMDTSASTAY ASTKGPSVFP HTFPAVLQSS KSCDKTHTCP HEDPEVKFNW

NYGMNWVRQA LQISSLKAED LAPSSKSTSG GLYSLSSVVT PCPAPEAAGG YVDGVEVHNA

EYKCKVSNKA LPAPIEKTIS LVKGFYPSDI AVEWESNGQP QQGNVFSCSV MHEALHNHYT LSLSPGERAT LSCRASESVD GVPSRFSGSG SGTDFTLTIS KGGGSGGGGQ VQLVQSGAEV GQGLEWIGVT HPSDSETWLD AVYYCAREHY GTSPFAYWGQ AALEKEVAAL EK

PGQGLEWMGW TAVYYCARES GTAALGCLVK VPSSSLGTQT PSVFLFPPKP KTKPREEQYN

KAKGQPREPQ ENNYKTTPPV QKSLSLSPGG NYGMSFMNWF SLEPEDFAVY KKPGASVKVS QKFKDRVTIT GTLVTVSSGG

228

[00354] The fourth DART G polypeptide chains comprise, in the N-terminal to C-terminal direction: an N-terminal; a Domino VL of a monoclonal antibody capable of binding to PD-1 (VLpd-i hPD-1 mAb 7 VL2) (SEQ ID NO: 153); an intervening linker peptide (Linker 1: GGGSGGGG (SEQ ID NO: 14)); a VH Domain of a monoclonal antibody capable of binding to PD-1 (VHpd-i hPD-1 mAb 7 VH1) (SEQ ID NO: 147); a cistern-containing intervening linker peptide (Linker 2: GGCGGG (SEQ ID NO:15)); a Promoting Heterodimer (K-helix) Domain (KVAALKE-KVAALKE-KVAALKEKVAALKE (SEQ ID NO:22); and a C-terminus.

[00355] The amino acid sequence of the fourth polypeptide chains of DART G is (SEQ ID NO:280):

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVQSGAEV KKPGASVKVS CKASGYSFTS YWMNWVRQAP GQGLEWIGVI HPSDSETWLD QKFKDRVTIT VDK STSTAYM ELSSLRSEDT AVYYCAREHY GTSPFAYWGQ GTLVTVSSGG CGGGKVAALK EKVAALKEKV AALKEKVAAL KE

CbQb ίη/77Ω7/Ε/ΥΙΛΙ

E. Diabody Containing the Fe Region of the Third Exemplary Chain Having E/K-Helices

[00356] The present invention additionally provides

PD-1 X LAG-3 bispecific, region-containing diabodies

Fe of three chains and comprise E/K-helix Heterodimer Promoter Domains. An exemplary three-chain bispecific PD-1 X LAG-3 diabody, containing the region

229

Fe comprising E/K-helix Heterodimer Promoter Domains designated DART H were generated. The structure of this diabody containing the Fe Region is detailed in the following. This exemplary PD-1 x LAG-3 diabody is intended to illustrate, but in no way limit, the scope of the invention.

[00357] DART H is a diabody containing the bispecific Fe region, three chains, which has a specific binding site for PD-1, a specific binding site for LAG3, an IgGl Fe Region that carries the knob/hole designed to reduce FcyR binding, and E/K-helix Heterodimer Promoter Domains.

[00358] The first DART H polypeptide chains comprise, in the N-terminal to C-terminal direction: an N-terminal; a Domino VL of a monoclonal antibody capable of binding to PD-1 (VLpd-i hPD-1 mAb 7 VL2) (SEQ ID NO: 153); an intervening linker peptide (Linker 1: GGGSGGGG (SEQ ID NO: 14)); a VH Domain of a monoclonal antibody capable of binding to LAG-3 (VH mAb 1 VH1_lag-3 hLAG-3) (SEQ ID NO:49); a cysteine-containing intervening linker peptide (Linker 2: GGCGGG (SEQ ID NO:15)); a Promotion of Heterodimer (E-helix) Domain (EVAALEK-EVAALEK-EVAALEKEVAALEK (SEQ ID NO:21)); an Intervention Linker (Spacer-Linker 3: GGGDKTHTCPPCP (SEQ ID NO:263)); IgGl CH2-CH3 domain carrying a knob comprising cnan Ln/zznz/E/YiAi

230 substitutions L234A/L235A and having the C-terminal Usine residue (SEQ ID NO:6); and a C-terminal.

[00359] The amino acid sequence of the first polypeptide chain of DART H is (SEQ ID NO:281):

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVQSGAEV KKPGASVKVS CKASGYTFTN YGMNWVRQAP GQGLEWMGWI NTYTGESTYA DDFEGRFVFS MDTS ASTAYL QISSLKAEDT AVYYCARESL YDYYSMDYWG QGTTVTVSSG GCGGGEVAAL EKEVAALEKE VAALEKEVAA LEKGGGDKTH TCPPCPAPEA AGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAP IEK TISKAKGQPR EPQVYTLPPS REEMTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

[00360] The second DART H polypeptide chains comprise, in the N-terminal to C-terminal direction: an N-terminal; a Domino VL of a monoclonal antibody capable of binding to LAG-3 (VLlag-3 hLAG-3 mAb 1 VL4) (SEQ ID NO: 54); an intervening linker peptide (Linker 1: GGGSGGGG (SEQ

ID NO:14)); a VH Domain of a monoclonal antibody capable of binding to PD-1 (VH mAb 7 VH1_P.S-i hPD-1) (SEQ ID NO:147) ; a cysteine-containing intervening linker peptide (Linker 2: GGCGGG (SEQ ID NO: 15)); a Promotion of Heterodimer (K-helix) Domain (KVAALKE-KVAALKE-KVAALKEKVAALKE (SEQ ID NO:22)); and a C-terminal.

[00361] The amino acid sequence of the second polypeptide chains of DART H is (SEQ ID NO:282):

cnan Ln/zznz/E/YiAi

231

DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL HSDAKTYLNW LLQKPGQPPE RLIYLVSELD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP YTFGGGTKVE IKGGGSGGGG QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWIGV IHPSDSETWL DQK FKDRVTI TVDKSTSTAY MELSSLRSRD TAVYYCAREH YGTSPFAYWG QGTLVTVSSG GCGGGKVAAL KEKVAALKEK VAALKEKVAA LKE

[00362] The third DART H polypeptide chains comprise, in the N-terminal to C-terminal direction: an N-terminal; a hinge region (DKTHTCPPCP (SEQ ID NO:31); a hole-bearing IgGl CH2-CH3 Domain comprising L234A/L235A substitutions and having the C-terminal Usine residue (SEQ ID NO:7); and a C-terminal .

[00363] The amino acid sequence of the third polypeptide chains of DART H is (SEQ ID NO:283):

DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYi LPPSREEMTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVMHE ALHNRYTQKS LSLSPGK cnan Ln/zznz/E/YiAi

F. Exemplary Bispecific Antibody

[00364] An exemplary PD-1 X LAG-3 four-chain bispecific antibody designated BSAB A was generated. The structure of this bispecific antibody is detailed in the following. This exemplary bispecific PD-1 x LAG-3 antibody is intended to illustrate, but in no way limit, the scope of the invention.

[00365] BSAB A is a bispecific antibody that has a

232 specific binding site for PD-1, a specific binding site for LAG-3, a Fe Region IgGl variant designed to reduce FcyR binding and to promote complexion between the two different heavy chain polypeptides (see for example, WO 2011 /143545).

[00366] The first BSAB A polypeptide chains comprise, in the N-terminal to C-terminal direction: an N-terminal; a VH Domain of a monoclonal antibody capable of binding to PD-1 (VHpd-i hPD-1 mAb 7 VH1) (SEQ ID NO: 147); an IgGl CHI Domain (SEQ ID NO: 10); a variant IgGl hinged region comprising D221E/P228E substitutions (numbered by the EU index as in Rabat and underlined in SEQ ID NO:286, below); a variant IgGl CH2-CH3 Domain comprising substitutions L234A/L235A/L368E (underlined in SEQ ID NO:286, below) and lacking the C-terminal residue; and a C-terminal.

[00367] The amino acid sequence of the first polypeptide chain of BSAB A is (SEQ ID NO:286):

QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWIGV IHPSDSETWL DQKFKDRVTI TVDKSTSTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSS VVTV PSSSLGTQTY ICNVNHKPSN TKVDKRVEPK SCEKTHTCPE CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM TKNQ VSLTCE VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPG

CbQb ίη/77Ω7/Ε/ΥΙΛΙ

[00368] The second polypeptide chains of BSAB

233 comprises, in the N-terminal to C-terminal direction: an Nterminal; a Domino VL of a monoclonal antibody capable of binding to PD-1 (VLpd-i hPD-1 mAb 7 VL2) (SEQ ID NO: 153); a Kappa CL Domain (SEQ ID NO:8), and a C-terminus.

[00369] The amino acid sequence of the second polypeptide chains of BSAB is (SEQ ID NO:287):

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKV YACEV THQGLSSPVT KSFNRGEC

[00370] The third BSAB A polypeptide chains comprise, in the N-terminal to C-terminal direction: an N-terminal; a VH Domain of a monoclonal antibody capable of binding to LAG-3 (VHlag-3 hLAG-3 mAb 1 VH1) (SEQ ID NO:49); an IgGl CHI Domain (SEQ ID NO: 10); a variant IgGl hinged region comprising D221R/P228R substitutions (underlined in SEQ ID NO:288, below); a variant IgGl CH2-CH3 Domain comprising L234A/L235A/L409R substitutions (underlined in SEQ ID NO:288, below) and lacking the C-terminal residue; and a C-terminal.

[00371] The amino acid sequence of the third polypeptide chain of BSAB A is (SEQ ID NO:288):

cnan Ln/zznz/E/YiAi

2. 3. 4 cnan Ln/zznz/E/YiAi

QVQLVQSGAE INTYTGESTY LYDYYSMDYW DYFPEPVTVS YICNVNHKPS KDTLMISRTP STYRVVSVLT

VKKPGASVKV ADDFEGRFVF GQGTTVTVSS WNSGALTSGV NTKVDKRVEP EVTCVVVDVS VLHQDWLNGK

SCKASGYTFT SMDTSASTAY ASTKGPSVFP HTFPAVLQSS KSCRKTHTCP HEDPEVKFNW EYKCKVSNKA

NYGMNWVRQA LQISSLKAED LAPSSKSTSG GLYSLSSVVT RCPAPEAAGG YVDGVEVHNA LPAPIEKTIS

PGQGLEWMGW TAVYYCARES GTAALGCLVK VPSSSLGTQT PSVFLFPPKP KTKPREEQYN KAKGQPREPQ

VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG

[00372] The fourth polypeptide chains of BSAB A comprise, in the N-terminal to C-terminal direction: an N-terminal; a Domino VL of a monoclonal antibody capable of binding to LAG-3 (VLlag-3 hLAG-3 mAb 1 VL4) (SEQ ID NO: 54); a Kappa CL Domain (SEQ ID NO:8), and a C-terminus.

[00373] The amino acid sequence of the fourth polypeptide chain of BSAB A is (SEQ ID NO:289):

DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL HSDAKTYLNW LLQKPGQPPE RLIYLVSELD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP YTFGGGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSK AD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

IX. Reference antibody

A. Dl Anti-Human Reference Antibodies

[00374] To evaluate and characterize the new molecules of

PD-1 binding anti-human of the present invention, the following reference antibodies are used: nivolumab (also known as 5C4, BMS-936558, ONO-4538, MDX-1106, and

235 marketed as OPDIVO® by Bristol-Myers Squibb), a human IgG4 antibody designated herein as PD-1 mAb A; and pembrolizumab (formerly known as lambrolizumab, also known as MK-3475, SCH-900475, and marketed as KEYTRUDA® by Merck) a humanized IgG4 antibody designated herein as PD-1 mAb B.

cnan Ln/zznz/E/YiAi

1. Nivolumab (PD-1 mAb A)

[00375] The Variable Domain amino acid sequence of

PD-1 Heavy Chain mAb A has the amino acid sequence (SEQ ID NO:64) (hCDR residues are shown underlined):

QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAV IWYDGSKRYY ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSS

[00376] The amino acid sequence of the Variable Domain of

PD-1 mAb A light chain has the amino acid sequence (SEQ ID NO:65) (CDRl residues are shown underlined):

EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ

GTKVEIK

2. Pembrolizumab (PD-1 mAb B)

[00377] The amino acid sequence of the Variable Domain of

PD-1 Heavy Chain mAb B has the amino acid sequence (SEQ ID NO:66) (hCDR residues are shown underlined):

236 cnan Ln/zznz/E/YiAi

QVQLVQSGVE VKKPGASVKV SCKASGYTFT NYYMYWVRQA PGQGLEWMGG INPSNGGTNF NEKFKNRVTL TTDSSTTTAY MELKSLQFDD TAVYYCARRD YRFDMGFDYW GQGTTVTVSS

[00378] The amino acid sequence of the Variable Domain of

PD-1 mAb B light chain has the amino acid sequence (SEQ ID NO:67) (CDRl residues shown underlined):

X. Production Methods

[00379] An anti-human PD-1 polypeptide, and other PD-1 agonists, antagonists and modulators can be created from the polynucleotides and/or sequences of the anti-PD-1 antibodies PD-1 mAb 1-15 by methods known in the art. the technique, for example, synthetically or recombinantly. One method for producing such peptide agonists, antagonists and modulators involves chemical synthesis of the polypeptide, followed by treatment under the appropriate oxidative conditions to obtain the native conformation, i.e., the correct disulfide linkages. This can be achieved using methodologies well known to those skilled in the art, (see for example, Kelley, RF et al. (1990) In: GENETIC ENGINEERING PRINCIPLES AND METHODS, Setlow, JK Ed., Plenum Press, NY, vol. 12, pp 1-19; Stewart, J.M. et al.

(1984) SOLID PHASE Peptide Synthesis, Pierce Chemical Co., Rockford, IL; see also United States Patent Nos. 4,105,603; 3,972,859; 3,842,067; and 3,862,925).

237

[00380] The polypeptides of the invention can be conveniently prepared using solid phase peptide synthesis (Merrifield, B. (1986) Salid Phase Synthesis Science 232(4748):341-347; Houghten, RA (1985) General Method For The Rapid Solid-Phase Synthesis Of Large Numbers Of Peptides: Specificity OfAntigen-Antibody Interaction At The Level Of Individual Amino Acids, Proc. Nati. Acad. Sci. (USA) 82(15) :5131-5135,- Ganesan, A . (2006) Solid-Phase Synthesis In The Twenty-First Century Mini Rev. Med. Chem. 6(1) :3-10). [00381] In yet another embodiment, the fully human antibodies have one or more of CDRs of PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 3, PD-1 mAb 4, PD-1 mAb 5 PD-1, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 8, PD-1 mAb 9, PD-1 mAb 10

PD-1, PD-1 mAb 11, PD-1 mAb 12, PD-1 mAb 13, PD-1 mAb14, or PD-1 mAb 15, or which compete with mAb 1de

PD-1, PD-1 mAb 2, PD-1 mAb 3, PD-1 mAb 4, mAb 5de

PD-1, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 8, mAb 9de

PD-1, PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12, PD-1 mAb13, PD-1 mAb 14, or PD-1 mAb 15, to bind PD -1 human or a soluble form thereof can be obtained through the use of commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals that are designed to produce a more desirable one (e.g., fully human antibodies) or can also be cnan Ln/zznz/E/YiAi cnan Ln/zznz/E/YiAi

238 use a more robust immune response for the generation of humanized or human antibodies. Examples of such technology are XENOMOUSE™ (Abgenix, Inc., Fremont, CA) and HuMAb-Mouse® and TC MOUSE™ (both from Medarex, Inc., Princeton, NJ).

[00382] In an alternative, the antibodies can be made recombinantly and expressed using any method known in the art. Antibodies can be made recombinantly by first isolating the antibodies made from the host animals, obtaining the gene sequence, and using the gene sequence to express the antibody recombinantly in the host cells (e.g., CHO cells). Another method that can be used is to express the antibody sequence in transgenic plants (for example, tobacco) or milk. Suitable methods for expressing antibodies recombinantly in plants or in milk have been described (see for example, Peeters et al (2001) Production Of Antlbodles And Antibody Fragments In Plants f Vaccine 19:2756; Lonberg, N. et al (1995 ) Human Antlbodles From Transgenlc Mlcef Int. Rev. Immunol 13:65-93; and Pollock et al (1999) Transgenlc Milk As A Method For The Production Of Recombinant Antlbodles J. Immunol Methods 231:147- 157). Suitable methods for preparing antibody derivatives, for example, humanized, single chain, etc. are known in the art. In another embodiment, the antibodies can

239 made recombinantly by phage display technology (see for example, United States Patent Nos. 5,565,332; 5,580,717; 5,733,743; 6,265,150; and Winter, G. et al. (1994) Making Antibodies By Phage Display Technology Annu. Rev. Immunol . 12.433-455).

[00383] The antibodies or protein of interest can be subjected to Edman degradation sequencing, which is well known to those skilled in the art. Peptide information generated from mass spectrometry or Edman degradation can be used to design probes or primers that are used to clone the protein of interest.

[00384] An alternative method of cloning the protein of interest is by immunoabsorption using purified PD-1 or portions thereof for cells that express an antibody or protein of interest that possesses one or more of the PD-mAb 1 CDRs. 1, PD-1 mAb 2, PD1 mAb 3, PD-1 mAb 4, PD-1 mAb 5, PD-1 mAb 6, PD1 mAb 7, PD-1 mAb 8, PD-1 mAb 9 PD-1, PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12, PD-1 mAb 13, PD-1 mAb 14, or PD-1 mAb 15, or an antibody that competes with PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 3, PD-1 mAb 4, PD-1 mAb 5, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 8, PD-1 mAb 9, PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12, PD1 mAb 13, PD-1 mAb 14 PD-1, or PD-1 mAb 15, to bind human PD-1.

cnan Ln/zznz/E/YiAi cnan Ln/zznz/E/YiAi

240

The immunosorbent procedure can be carried out by obtaining a cDNA library from tissues or cells that express PD-1, overexpressing the cDNAs in a second cell type, and screening transected cells from the second cell type. for specific binding to PD-1 in the presence or absence of PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 3, PD-1 mAb 4, PD-1 mAb 5, mAb 6 PD-1, PD-1 mAb 7, PD-1 mAb 8, PD-1 mAb 9, PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12, PD1 mAb 13, PD-1 mAb 14, or PD-1 mAb 15. Detailed descriptions of the methods used in cloning mammalian genes encoding cell surface proteins by immunosorbent can be found in the art see for example, Aruffo, A. et al. (1987) Molecular Cloning Of A CD28 cDNA By A Hlgh-Efficiency COS Cell Expression System, Proc. Nati. Academic Sci. (USA) 84:8573-8577 and Stephan, J. et al. (1999) Selective Cloning Of Cell Surface Proteins Involved In Organ Development: Epithelial Glycoprotein is Involved In Normal Epithelial Differentiation Endocrinol. 140:5841-5854).

[00385] Vectors containing the polynucleotides of interest can be introduced into the host cell by any of a number of suitable means, including electroporation, employing calcium chloride, rubidium chloride transfection, calcium phosphate, DEAE-dextran, or others

241 substances; bombardment with microprojectiles; lipofection; and infection (e.g., wherein the vector is an infectious agent such as a vaccine virus). The choice of vectors or polynucleotides to be introduced will often depend on characteristics of the host cell.

[00386] Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating genes encoding the antibody, polypeptide or protein of interest. Non-limiting examples of suitable mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. Preferably, the host cells express the cDNAs at a level about 5-fold, more preferably 10-fold, even more preferably 20-fold, than that of the corresponding endogenous antibody or protein of interest, if present, in the host cells. Detection of host cells for binding to PD-1 is carried out by a specific immunoassay or FACS. A cell that overexpresses the antibody or protein of interest can be identified.

[00387] The invention includes polypeptides comprising an amino acid sequence of the antibodies of this invention. The polypeptides of this invention can be prepared by methods known in the art. Polypeptides can be produced by proteolytic or other degradation of antibodies, by cnan Ln/zznz/E/YiAi methods

242 recombinants (i.e., individual or fusion polypeptides) as described above or by chemical synthesis. Antibody polypeptides, especially shorter polypeptides of up to about 50 amino acids, are conveniently prepared by chemical synthesis. Chemical synthesis methods are known in the art and are commercially available. For example, an anti-human PD-1 polypeptide could be produced by an automated polypeptide synthesizer employing the solid phase method.

[00388] The invention includes antibody variants PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 3, PD-1 mAb 4, PD-1 mAb 5, PD-1 mAb 6? PD-1 mAb 7, PD-1 mAb 8, PD-1 mAb 9, PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12, PD-1 mAb 13, mAb 14 of PD-1, or mAb 15 of PD-1 and their polypeptide fragments that bind to PD-1, including functionally equivalent antibodies and fusion polypeptides that do not significantly affect the properties of such molecules, as well as the variants that have improved or decreased activity. Modification of polypeptides is routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids that do not change

CbQb ίη/77Ω7/Β/ΥΙΛΙ

243 significantly detrimental to the functional activity, or the use of chemical analogues. Amino acid residues that can be conservatively substituted for each other include but are not limited to: glycine/alanine; serine/threonine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; lysine/arginine; and phenylalanine/tyrosine. These polypeptides also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation, phosphorylation. Preferably, the amino acid substitutions would be conserved, that is, the substituted amino acid would have chemical properties similar to those of the original amino acids. Conservative substitutions are known in the art, and examples have been provided above. Amino acid modifications can range from changing or modifying one or more amino acids to complete redesign of a region, such as the Variable Domain. Changes in the variable domain can alter binding affinity and/or specificity. Other modification methods include the use of coupling techniques known in the art, including, but not limited to, enzymatic means, oxidative substitution and chelation. The modifications can be used, for example, for the attachment of labels for immunoassay, cnan Ln/zznz/E/YiAi

244 such as the binding of radioactive moieties for radioimmunoassay. Modified polypeptides are made using procedures established in the art and can be selected using standard assays known in the art.

[00389] The invention encompasses fusion proteins comprising one or more of the polypeptides or antibodies PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 3, PD-1 mAb 4, PD-1 mAb 5 PD-1, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 8, PD-1 mAb 9, PD-1 mAb 10, PD-1 mAb 11, PD mAb 12 -1, PD-1 mAb 13, PD-1 mAb 14, or PD-1 mAb 15 of this invention. In one embodiment, a fusion polypeptide is a condition comprising a light chain, a heavy chain, or both a light and heavy chain. In another embodiment, the fusion polypeptide contains a heterologous immunoglobulin constant region. In another embodiment, the fusion polypeptide contains a variable light chain and a variable Domain Heavy Chain Domain of an antibody produced from a publicly deposited hybridoma. For the purposes of this invention, an antibody fusion protein contains one or more polypeptide domains that specifically bind to PD-1 and another amino acid sequence in which it does not bind to the native molecule, for example, a heterologous sequence. or a homologous sequence from another region.

cnan Ln/zznz/E/YiAi

245

XI. Uses of the PD Binding Molecules of the present invention

[00390] The present invention encompasses compositions, including pharmaceutical compositions, comprising the PD-1 binding molecules of the present invention (e.g., anti-PD-1 antibodies, bispecific anti-PD-1 diabodies, etc. .), polypeptides derived from such molecules, polynucleotides encoding sequences comprising such molecules or polypeptides, and other agents as described herein.

A. Therapeutic Uses

[00391] As discussed above, PD-1 plays an important role in negatively regulating T cell proliferation, function and homeostasis. Some of the PD-1 binding molecules of the Present Invention have the ability to inhibit PD-1 function, and thus reverse PD-1-mediated inhibition of the immune system. As such, PD-1 mAb 1, PD-1 mAb 3, PD-1 mAb 5, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 8, PD-1 mAb 9 1, PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12, PD-1 mAb 13, PD-1 mAb 14, and PD-1 mAb 15, their humanized derivatives, and molecules comprise their PD-1 binding fragments (e.g., bispecific antibodies, bispecific diabodies (including, but not limited to, DART-A, DART-B, DART-C, DART-D, DART-E, cnan Ln/zznz/E/YiAi

246

DART-F, DART-G, DART-H, DART-I, and DART-J), etc.), or that compete for binding with such antibodies, can be used to block immune-mediated inhibition-PD- 1, and thus promote the activation of the immune system.

[00392] Such bispecific PD-1 binding molecules of the present invention that bind PD-1 and another molecule involved in the regulation of an immune checkpoint present on the cell surface (e.g., LAG-3) increase the immune system by blocking the inhibition of the immune system mediated by such immune control molecules PD-1. Therefore, the PD-1 binding molecules of the invention are useful for increasing an immune response (e.g., T cell-mediated immune response) of a subject. In particular, such PD-1 binding molecules of the invention can be used to treat any disease or condition associated with an undesirably suppressed immune system, including cancer and diseases that are associated with the presence of a pathogen (for example, a bacterial, fungal, viral or protozoan infection.

[00393] Cancers that can be treated by such PD-1 binding molecules of the present invention include cancers characterized by the presence of a cancer cell selected from the group consisting of a cell from: an adrenal gland tumor, an associated cancer with cnan Ln/zznz/E/YiAi

247

AIDS, an alveolar soft part sarcoma, an astrocytic tumor, bladder cancer, bone cancer, brain and spinal cord cancer, a metastatic brain tumor, breast cancer, carotid body tumors, cervical cancer, chondrosarcoma, a chordoma, a chromophobe renal cell carcinoma, a clear cell carcinoma, a colon cancer, a colorectal cancer, a cutaneous benign fibrous histiocytoma, a desmoplastic small round cell tumor, an ependymoma, an Ewing tumor, an extraskeletal myxoid chondrosarcoma, an imperfect bone fibrogenesis, a fibrous bone dysplasia, a gallbladder or bile duct cancer, gastric cancer, a gestational trophoblastic disease, a germ cell tumor, a head and neck cancer, hepatocellular carcinoma, an islet cell tumor, a Kaposi sarcoma, a kidney cancer, a leukemia, a lipoma/benign lipomatous tumor, a liposarcoma/malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, a papillary thyroid carcinoma, a parathyroid tumor, a pediatric cancer, a peripheral nerve sheath tumor, a pheochromocytoma, a pituitary tumor, a prostate cancer, a posterior uveal melanoma, a disorder cnan Ln/zznz/E/YiAi

248 rare haematological, metastatic renal cancer, rhabdoid tumour, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell cancer, stomach cancer, synovial sarcoma, testicular cancer, carcinoma thymus, a thymoma, a metastatic thyroid cancer, and a uterine cancer.

[00394] In particular, such PD-1 binding molecules of the present invention can be used in the treatment of colorectal cancer, hepatocellular carcinoma, glioma, kidney cancer, breast cancer, multiple myeloma, bladder cancer, neuroblastoma; sarcoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, and rectal cancer.

[00395]Pathogen-associated diseases that can be treated by the PD-1 binding molecules of the present invention include chronic viral, bacterial, fungal and parasitic infections. Chronic infections that can be treated by the PD-1 binding molecules of the present invention include Epstein Barr Virus, Hepatitis A Virus (HAV); Hepatitis B Virus (HBV); Hepatitis C Virus (HCV); herpes virus (e.g. HSV-1, HSV-2, HHV-6, CMV), Human Immunodeficiency Virus (HIV), Vesicular Stomatitis Virus (VSV) Bacillus, Citrobacter, Cholera, Diphtheria, Enterobacter, Gonococcus, Helicobacter pylori, Klebsiella, Legionella, Meningococcus, Mycobacterium, cnan Ln/zznz/E/YiAi

249

Pseudomonas, Pneumococcus, Rickettsia bacteria, Salmonella, Serratia, Staphylococcus, Streptococcus, Tetanus, Aspergillus (fumigatus, niger, etc.), Blastomyces dermatitidis, Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Mucorales genera ( mucor, absidia, rhizopus), Sporothrix schenkii, Paracoccidioides brasiliensis, Coccidioides immitis, Histoplasma capsulatum, Leptospirosis, Borrelia burgdorferi, helminth parasites (hookworms, cestodes, trematodes, flatworms (e.g. Schistosoma), Giardia lamblia, Trichinella, Dientamoeba fragilis, Trypanosoma brucei, Trypanosoma cruz! and Leishmania donovani

[00396] Such PD-1 binding molecules of the invention can be combined with other anticancer agents, in particular, molecules that specifically bind a cancer antigen (e.g., antibodies, diabodies). Anticancer therapies that can be combined with the PD-1 binding molecules of the invention include molecules that specifically bind one or more cancer antigens including: 19.9 as found in colon cancer, gastric cancer mucins; 4.2; A33 (a colorectal carcinoma antigen; Almqvist, Y. 2006, Nucí Med Biol. Nov; 33(8):991-998); ADAM-9 (United States Patent Publication No. 2006/0172350; PCT Publication No. WO 06/084075); AH6 as found in gastric cancer; ALCAM

CbQb ίη/ΖΖΠΖ/Ε/ΥΙΛΙ

250 (PCT Publication No. WO 03/093443); APO-1 (human malignant lymphocyte antigen) (Trauth et al. (1989) Monoclonal Antibody-Mediated Tumor Regression By Induction Of Apoptosis, Science 245:301-304); B1 (Egloff, AM et al. 2006, Cancer Res. 66(1):6-9), - B7-H3 (Collins, M. et al.

(2005) The B7 Family Of Immune-Regulatory Ligands, Genome Biol. 6:223.1-223.7). Chapoval, A. et al. (2001) B7-H3:A

Costimulatory Molecule For T Cell Activation and IFN-y Production, Nature Immunol. 2:269-274; Sun, M. et al. (2002) Characterization of Mouse and Human B7-H3 Genes, J. Immunol. 168:6294-6297); BAGE (Bodey, B. 2002 Expert Opin Biol Ther. 2(6):577-84); beta catenin (Frange W. et al. 2003 J Pathol. 201(2):250-9); ALe blood group^b/You^and as found in colon adenocarcinoma; Burkitt lymphoma antigen 38.13, C14 as found in colon adenocarcinoma; CA125 (ovarian carcinoma antigen) (Bast, RC Jr. et al. 2005 Int J Gynecol Cancer 15 Suppl 3:274-81 ; Yu et al. (1991) Coexpression Of Different Antigenic Markers On Moietíes That Bear CA 125 Determinants, Cancer Res. 51(2):468-475); Carboxypeptidase M (United States Patent Publication No. 2006/0166291); CD5 (Calin, GA et al. 2006 Semin Oncol. 33(2) : 167-73; CD19 (Ghetie et al. (1994) Anti-CD 19 Inhibits The Growth Of Human B-Cell Tumor Lines In Vitro And. Of Dandi Cells In SCID Mice By Inducing Cell Cycle Arrest, Blood 83:1329-1336; Troussard, X. et al. 1998 cnan Ln/zznz/E/YiAi

251

Hematol Cell Ther. 40(4): 139-48); CD20 (Reff et al (1994) Depletion Of B Cells In Vivo By A Chimeric Mouse Human Monoclonal Antibody To CD20, Blood 83:435-445; Thomas, DA et al. 2006 Hematol Oncol Clin North Am. 20(5): 1125 -36); CD22 (Kreitman, RJ 2006 AAPS J. 18;8 (3): E532-51) ; CD23 cnan Ln/zznz/E/YiAi (Rosati, S. et al. 2005 Curr Top Microbiol Immunol. 5;294:91107); CD25 (Troussard, X. et al. 1998 Hematol Cell Ther. 40 (4): 139-48); CD27 (Bataille, R. 2006 Haematologica 91 (9): 1234-40) ; CD28 (Bataille, R. 2006 Haematologica 91 (9): 1234-40); CD33 (Sgouros et al. (1993) Modeling And Dosimetry Of Monoclonal Antibody MI 95 (Anti- CD 3 3) In Acute Myelogenous Leukemia, J. Nucí. Med. 34:422-430); CD36 (Ge, Y. 2005 Lab Hematol. 11(1):31-7); CD40/CD154 (Messmer,

Such. 2005 Ann NY Acad Sci. 1062:51-60); CD45 (Jurele, JG 2005 Curr Oncol Rep. 7 (5): 339-46); CD56 (Bataille, R. 2006 Haematologica 91 (9): 1234-40); CD46 (US Patent No. 7,148,038; PCT Publication No. WO 03/032814); CD52 (Eketorp, SS et al. (2014) Alemtuzumab (Anti-CD52 Monoclonal Antibody) As Single-Agent Therapy In Patients With Relapsed/Refractory Chronic Lymphocytic Leukaemia (CLL)-A Single Region Experience On Consecutive Patients f Ann Hematol. 93(10): 1725-1733; Suresh, T. et al. (2014) New Antibody Approaches To Lymphoma Therapy, J. Hematol. Oncol. 7:58; Hoelzer, D. (2013) Targeted Therapy With Monoclonal Antibodies In Acute Lymphoblastic Leukemia,

252

Curr. Opinion. Oncol. 25(6):701-706); CD56 (Bataille, R. 2006 Haematologica 91 (9): 1234-40); CD79a/CD79b (Troussard, CD103 (Troussard, X. et al. 1998 Hematol Cell Ther. 40(4):139-48); CD317 (Kawai, S. et al. (2008) Interferon-A Enhances CD317 Expression And The Antitumor Activity Of Anti-CD317 Monoclonal Antibody In Renal Cell Carcinoma Xenograft Models, Cancer Science 99(12):2461-2466; Wang, W. et al. (2009) HM1.24 (CD317) Is A Novel Target Against Lung Cancer For Immunotherapy Using Anti-HM1.24 Antibody f Cancer Immunology, Immunotherapy 58(6):967-976; Wang, W. et al. (2009) Chimeric And Humanized Anti-HM1.24 Antibodies Mediate Antibody-Dependent Cellular Cytotoxicity Against Lung Cancer Cells. Lung Cancer, 63(1):23-31,- Sayeed, A. et al. (2013) Aberrant Regulation Of The BST2 (Tetherin) Promoter Enhances Cell Proliferation And Apoptosis Evasion In High Grade Breast Cancer Cells f PLoS ONE 8(6)e67191, pp. 1-10); CDK4 (Lee, YM etal. 2006 Cell Cycle 5(18):2110-4),- CEA (carcinoembryonic antigen; Foon et al (1995) Immune Responso To The Carcinoembryonic Antigen In Patients Treated With An Antiidiotype Antibody Vaccine, J. Clin Invest. 96(1):33442); Mathelin, C. 2006 Gynecol Obstet Fertile. 34(7-8):638-46; Tellez-Avila, FI et al. 2005 Rev Invest Clin. 57(6):814-9),CEACAM9 / CEACAM6 (Zheng, C. et al. (2011) A Novel AntiCbQb ίη/77Π7/Ε/ΥΙΛΙ

CbQb ίη/77Π7/Ε/ΥΙΛΙ

253

CEACAM5 Monoclonal Antibody, CC4, Suppresses Colorectal Tumor Growth and Enhances NK Cells-Mediated Tumor Immunity PLoS One 6(6):e21146, pp. 1-11); CO17-1A (Ragnhammar et al (1993) Effect Of Monoclonal Antibody 17-1A And GM-CSF In Patients With Advanced Colorectal Carcinoma - Long-Lasting, Complete Remissions Can Be Induced, Int. J. Cancer 53:751- 758); CO43 (blood group Le^b) ; CO-514 (blood group Le^to) as found in adenocarcinoma; CTA- 1; CTLA-4 (Peggs, KS et al 2006 Curr Opin Immunol 18 (2):206-13); Cytokeratin 8 (PCT Publication No. WO 03/024191); Dl.l; D156-22; DR5 (Abdulghani, J. et al (2010) TRAIL Receptor Signaling And Therapeutics f Expert Opin. Ther. Targets 14(10): 1091-1108; Andera, L. (2009) Signaling Activated By The Death Receptors Of The TNFR Family, Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech. Repub. 153 (3):173-180; Carlo-Stella, C. et al (2007) Targeting TRAIL Agonistic Receptors for Cancer Therapy, Clin, Cancer 13(8):2313- 2317; Chaudhari, BR et al (2006) Following the TRAIL to Apoptosis, Immunologic Res. 35 (3):249-262) ; Ei series (blood group B) as found in pancreatic cancer; EGFR (Epidermal Growth Factor Receptor; Adenis, A. et al 2003 Bull Cancer. 90 Spec No:S228-32); Ephrin receptors (and in particular EphA2 (US Patent No. 7,569,672; PCT Publication No. WO 06/084226); Erb (ErbBl; ErbB3; ErbB4; Zhou, H. et al 2002 Oncogene 21(57):8732- 8740; Rimon, E. et al 2004 Int

CbQb ίη/77Ω7/Β/ΥΙΛΙ

254

J Oncol. 24(5):1325-1338); GAGE (GAGE-l; GAGE-2; Akcakanat, A. et al 2006 Int J Cancer. 118 (1): 123-128); GD2/GD3/GM2 (Livingston, PO et al 2005 Cancer Immunol Immunother. 54(10): 1018-1025); ganglioside GD2 (G_d2; Saleh et al. (1993) Generation Of A Human Anti-Idlotypic Antibody That Mimics The GD2 Antigen, J.Immunol., 151, 3390-3398); ganglioside GD3 (Gd3; Shitara et al (1993) A Mouse/Human Chimeric Anti(Ganglioside GD3) Antibody With Enhanced Antitumor

Activities, Cancer Immunol. Immunother. 36:373-380); GM2 ganglioside (Gm2; Livingston et al (1994) Improved Survival In Stage III Melanoma Patients With GM2 Antibodies: A Randomized Trial Of Adjuvant Vaccination With GM2 Ganglioside, J. Clin. Oncol. 12:1036-1044); ganglioside GM3 ( Gm3 ; Hoon et al (1993) Molecular Cloning Of A Human Monoclonal Antibody Reactive To Ganglioside GM3 Antigen On Human Cancera, Cancer Res. 53:5244-5250); GICA 19-9 (Herlyn et al (1982) Monoclonal Antibody Detection Of A Circulating Tumor-Associated Antigen. I. Presence Of Antigen In Sera Of Patients With Colorectal, Gastric, And Pancreatic Carcinoma, J. Clin. Immunol. 2:135- 140); gplOO (Lotem, M. et al. 2006 J Immunother. 2 9(6):616-27),- Gp37 (human leukemia T cell antigen; Bhattacharya-Chatterjee et al. (1988) Idiotype Vaccines Against Human T Cell Leukemia. II. Generation And Characterization Of A Monoclonal Idiotype Cascade (Abl, Ab2, and Ab3), J. Immunol. 141:1398-1403); gp75 (melanoma

255 antigen; Vijayasardahl et al. (1990) The Melanoma Antigen Gp75 Is The Human Homologue Of The Mouse B (Brown) Locus Gene Product, J. Exp. Med. 171 (4): 1375-13 80); gpA33 (Heath, JK et al (1997) The Human A33 Antigen Is A Transmembrane Glycoprotein And A Novel Member Of The Immunoglobulin Superfamily, Proc. Nati. Acad. Sci. (USA) 94 (2):469-474; Ritter, G .et al. (1997) Characterization Of cnan Ln/zznz/E/YiAi

Posttranslational Modifications Of Human A33 Antigen, A Novel

Palmitoylated Surface Glycoprotein Of Human Gastrointestinal

Epithelium f Biochem. Biophys. Commun. Res. 236 (3):682-686;

Wong, N.A. et al. (2006) EpCAM and gpA33 Are Markers Of Barrett's Metaplasiaf J. Clin. Pathol. 59 (3):260-263);

HER2 antigen (HER2/neu, pl85^HER2; Kumar, Pal S et al. 2006 Semin Oncol. 33 (4) :3 86-91); HMFG (human milk fat globule antigens; WO1995015171); human papillomavirus E6/human papillomavirus E7 (DiMaio, D. et al. 2006 Adv Virus Res. 66:125-59; HMW-MAA (high molecular weight melanoma antigen; Natali et al. (1987) Immunohistochemistry Detection Of Antigen In Human Primary And Metastatic Melanomas By The Monoclonal Antibody 140.240

And Its Possible Prognostic Significance, Cancer 59:55-63;

Mittelman et al. (1990) Active Specific Immunotherapy In

Patients With Melanoma. A Clinical Trial With Mouse

Antiidiotypic Monoclonal Antibodies Elicited With Syngeneic

Anti-High-Molecular-Weight-Melanoma-Associated

Antigen

256

Monoclonal Antibodies, J. Clin. Invest. 86:2136-2144); antigen I (differentiation antigen; Feizi (1985) Demonstration By Monoclonal Antibodies That Carbohydrate Structures Of Glycoproteins And Glycolipids Are OncoDevelopmental Antigens, Nature 314:53-57); IL13Ra2 (PCT Publication No. WO 2008/146911; Brown, CE et al. (2013) Glioma IL13Ra2 Is Associated With Mesenchymal Signature Gene Expression And Poor Patient Prognosisf PLoS One. 18;8(10):e77769; Barderas, R. et al. (2012) High Expression Of 1L-13 Receptor A2 In Colorectal Cancer Is Associated With Invasion, Liver Metastasis, And Poor Prognosis, Cancer Res. 72 (11):2780-2790; Kasaian, M.T. et al. (2011) IL-13 Antibodies Influence IL-13 Clearance In

Humans By Modulating Scavenger Activity Of lL-13Ra2, J. Immunol. 187 (1):561-569; Bozinov, O. et al. (2010)

Decreasing Expression Of The Interleukin-13 Receptor IL13Ralpha2 In Treated Recurrent Malignant Gliomas, Neurol. Med. Chir. (Tokyo) 50(8):617-621,- Fujisawa, T. et al. (2009) A novel role of interleukin-13 receptor alpha2 in pancreatic cancer invasion and metastasis, Cancer Res. 69(22):86788685); Integrin β6 (PCT Publication No. WO 03/087340); JAM-3 (PCT Publication No. WO 06/084078); KID3 (PCT Publication No. WO 05/028498); KID31 (PCT Publication No. WO 06/076584); KS 1/4 pan carcinoma antigen (Perez et al. (1989) Isolation And Characterization Of A cnan Ln/zznz/E/YiAi

257 cDNA Encoding The Ksl/4 Epithelial Carcinoma Marker, J. Immunol. 142:3662-3667; Moller et al (1991) Bi-specificMonoclonal-Antibody-Directed Lysis Of Ovarian Carcinoma Cells By Activated Human T Lymphocytes, Cancer Immunol. Immunother. 33(4):210-216; Ragupathi, G. 2005 Cancer Treat Res. 123:157-80); L6 and L20 (human lung carcinoma antigens; Hellstrom et al (1986) Monoclonal Mouse Antibodies Raised Against Human Lung Carcinoma, Cancer Res. 46:39173923); READ; LUCA-2 (US Patent Publication No. 2006/0172349; PCT Publication No. WO 06/083852); Ml:22:25:8; M18; M39; MAGE (MAGE-1; MAGE-3; (Bodey, B. 2002 Expert Opin Biol Ther. 2 (6): 577-84) ; MART (Kounalakis, N. et al. 2005 Curr Oncol Rep. 7 (5) : 377-82;

mesothelin (Chang K, and Pastan I. 1996 Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers f Proc Nati Acad Sci USA 93:136-40), MUC-1 (Mathelin, C. 2006 Gynecol ObstetFertil. 34 (7-8): 638-46) ; MUM- 1 (Castelli, C. et al. 2000 J Cell Physiol.182(3):323-31); Myl; N-acetylglucosaminyltransferase (Dennis, JW 1999 Biochím Biophys Acta. 6; 1473(1):21-34); neoglycoprotein; NS-10 as found in adenocarcinomas; OFA-1; OFA-2; Oncostatin M (Oncostatin Beta Receptor; US Patent No. 7,572,896; PCT Publication No. WO 06/084092); pl5 (Gil, J. et al. 2006 Nat Rev Mol Cell Biol. 7(9):667-77); p97 (melanoma-associated antigen;

cnan Ln/zznz/E/YiAi

258

Estin et al (1989) Transfected Mouse Melanoma Lines That Express Various Levels Of Human Melanoma-Associated Antigen p97, J. Nati. Cancer Institute 81(6):445-454); PEM (polymorphic epithelial mucin; Hilkens et al (1992) Cell MembraneAssociated Mucins And Their Adhesion-Modulating Property, Trends in Biochem. Sci. 17:359-363); ΡΕΜΑ (polymorphic epithelial mucin antigen); PIPA (United States Patent No. 7,405,061; PCT Publication No. WO 04/043239); PSA (prostate specific antigen; Henttu et al (1989) cDNA Coding For The Entire Human Prostate Specific Antigen Shows High Homologies To The Human Tissue Kallikrein Genes, Biochem. Biophys. Res. Comm. 10 (2):903-910; Israeli et al al (1993) Molecular Cloning Of A Complementary DNA Encoding A Prostate-Specific Membrane Antigen, Cancer Res. 53:227-230; Cracco, CM et al. 2005 Minerva Urol Nefrol. 57 (4): 301-11) ; PSMA (prostate specific membrane antigen; Ragupathi, G.2005 Cancer Treat Res. 123:157-180); prosthetic acid phosphate (Tailor et al (1990) Nucleotide Sequence OfHuman Prostatic Acid Phosphatase Determined From A Full-Length cDNA Clone, Nucí. Acids Res. 18 (16): 4928); R24 as found in melanoma; ROR1 (United States Patent No. 5,843,749); sphingolipids; SSEA-1; SSEA-3; SSEA-4; sTn (Holmberg, LA 2001 Expert Opin Biol Ther. 1 (5):881-91) ;

receptor peptide derived from the T cells of a cutaneous lymphoma of cnan Ln/zznz/E/YiAi T cells (see Edelson (1998) Cutaneous T-Cell

259

Lymphoma: A Model For Selective Immunotherapy, Cancer J Sci Am. 4:62-71); T5A7 found in myeloid cells; TAG-72 (Yokota et al (1992) Rapid Tumor Penetration Of A SingleChain Fv And Comparison With Other Immunoglobulin Forms, Cancer Res. 52:3402-3408); TL5 (blood group A); TNF receptor (TNF-a receptor, TNF-B receptor; TNF-y receptor (van Horssen, R. et al. 2006 Oncologist. 11 (4): 397-408; Gardnerova, M. et al. 2000 Curr Drug Targets. 1(4):327-64),TRA-1-85 (blood group H) ; Transferrin Receptor (US Patent No. 7,572,895; PCT Publication No. WO 05/121179); 5T4 (TPBG, trophoblastic glycoprotein; Boghaert, ER et al. (2008) The Oncofetal Protein, 5T4, Is A Suitable Target For Antibody-Guided AntiCancer Chemotherapy With Callcheamicin, Int. J. Oncol. 32(1):221-234; Eisen, T. et al. (2014) Naptumomab Estafenatox: Targeted Immunotherapy with a Novel

Immunotoxin, Curr. Oncol. Rep. 16:370, pp. 1-6); TSTA (transplantation specific antigen) such as virally induced tumor antigens including T antigen DNA tumor viruses and RNA tumor virus envelope antigens, oncofetal alpha antigen fetoprotein such as colon CEA, oncofetal antigen bladder tumor (Hellstrom et al (1985) Monoclonal Antibodies To Cell Surface Antigens Shared By Chemically Induced Mouse cnan Ln/zznz/E/YiAi

Bladder Carcinomas, Cancer. Res. 45:2210-2188); VEGF

260 (Pietrantonio, F. et al. (2015) Bevacizumab-BasedNeoadjuvant Chemotherapy For Colorectal Cancer Liver Metastases: Pitfalls And Help ful Tricks In A Review For Clinicians, Crit. Rev. Oncol. Hematol. 95 (3):272-281; ​​Grabowski , JP (2015) Current Management Of Ovarian Cancer, Minerva Med. :997-1007;Suh, DH et al. (2015) Major Clinical Research Advances In Gynecologic Cancer In 2014f J. Gynecol. Oncol. 26(2): 156-167; Liu, K.J. et al. (2015) Bevacizumab In Combination With Anticancer Drugs For Previously Treated Advanced Non-Small Cell Lung Cancer, Tumor Biol. 36(3): 1323-1327; Di Bartolomeo, M. et al. (2015) Bevacizumab treatment in the elderly patient with metastatic colorectal cancer, Clin. Interv. Aging 10:127133); VEGF Receptor (O'Dwyer. PJ 2006 Oncologist. 11(9):992-998); VEP8; VEP9; VIM-D5; and Y hapten, Le^ as found in embryonal carcinoma cells.

[00397] In certain embodiments, such anti-PD-1 binding molecules of the invention are used in combination with one or more molecules that specifically bind 5T4, B7H3, CD 19, CD20, CD51, CD123, DR5, EGFR , EpCam, GD2, gpA33, HER2, ROR-1, TAG-72, VEGF-A antibody, and/or VEGFR2.

[00398] Such PD-1 binding molecules of the invention can be combined with an immunogenic agent such as a

CbQb ίη/77Ω7/Ε/ΥΙΛΙ

261 tumor vaccine. Such vaccines may comprise purified cnan Ln/zznz/E/YiAi tumor antigens (including recombinant proteins, peptides and carbohydrate molecules), autologous or allogeneic tumor cells. A number of tumor vaccine strategies have been described (see, for example, Palena, C., et al, (2006) Cancer vaccines: preclinical studies and novel strategies Adv. Cancer Res. 95, 115-145; Mellman, I., et al (2011) Cancer immunotherapy comes of age, Nature 480, 480-489; Zhang, XM et al (2008) The anti-tumor immune response induced by a combination of MAGE-3/MAGE-n-derived peptides, Oncol. Rep. 20, 245-252; Disis, ML et al (2002) Generation of T-cell immunity to the HER-2/neu protein after active immunization with HER-2/neu peptide-based vaccines, J. Clin. Oncol. 20, 2624-2632; Vermeij, R. et al (2012)

Potentiation of a p53-SLP vaccine by cyclophosphamide in ovarian cancer: a single-arm phase II study. Int. J. Cancer 131, E670-E680). Such PD-1 binding molecules of the invention can be combined with chemotherapeutic regimens. In these cases, it may be possible to reduce the dose of chemotherapeutic reagent administered (Mokyr et al (1998) Cancer Research 58: 5301-5304).

[00399] Such PD-1 binding molecules of the invention can be combined with other immunostimulatory molecules such as antibodies that activate the immune response of the carrier to provide increased levels of

262 T cell activation. In particular, anti-PD-1 antibodies, anti-PD-Ll antibodies and/or an anti-CTLA-4 antibody have been shown to activate the immune system (see, for example, del Rio, ML. et al ( 2005) Antibody-Mediated Signaling Through PD-1 Costimulates T Cells And Enhances CD28-Dependent Proliferation, Eur. J. Immunol 35:3545-3560; Barber, DL et al (2006) Restoringfunction in exhausted CD8 Tcells during chronic viral infection, Nature 439, 682-687; Iwai, Y. et al (2002) Involvement Of PD-L1 On Tumor Cells In The Escape From Host Immune System And Tumor Immunotherapy By PDL1 Blockade, Proc. Nati Acad. Sci. USA 99, 12293-12297; Leach, DR, et al, (1996) Enhancement Of Antitumor Immunity By CTLA-4 Blockade, Science 271, 1734-1736). Immunostimulatory molecules that can be combined with the PD-1 binding molecules of the invention include antibodies to molecules on the surface of dendritic cells that activate dendritic cell (DC) function and antigen presentation, anti- CD40 capable of substituting T cell helper activity, and activating antibodies on T cell costimulatory molecules such as PD-L1, CTLA-4, OX-40 4-1BB, and ICOS (see, for example, Ito et al (2000) Effective Priming Of Cytotoxic T Lymphocyte Precursors By Subcutaneous Administration Of Peptide Antigens In Liposomes Accompanied By Anti-CD40 And Anti-CTLA-4 Antibodies, Immunology 201:527 cnan Ln/zznz/E/YiAi

263

40; US Pat. No. 5,811,097; Weinberg et al (2000) Engagement of the OX-40 Receptor In Vivo Enhances Antitumor Immunity, Immunol 164:2160-2169; Melero et al. (1997) Monoclonal Antibodies Against The 4-1BB T-Cell Activation Molecule Eradicate Established Tumors Nature Medicine 3: 682-685; Hutloff et al. (1999) ICOS Is An Induced T-Cell Co-Stimulator Structurally And Functionally Related To CD28, Nature 397: 263-266; and Moran, A.E. et al (2013) The TNFRs 0X40, 4-1BB, and CD40 As Targets For Cancer Immunotherapy Curr Opin Immunol. 2013 Apr; 25(2):

10.1016/j.coi.2013.01.004), and/or stimulatory Chimeric Antigen Receptors (CARs) comprising a direct antigen binding domain against a disease antigen fused to one or more intracellular signaling domains from various receptors costimulatory protein (for example, CD28, 4-1BB, ICOS, 0X40, etc.) that serve to stimulate T cells after antigen binding (see, for example, Tettamanti, S. et al (2013) Targeting Of Acute Myeloid Leukaemia By Cytokine-Induced Killer Cells Redirected With A Novel CD 123-Specific Chimeric Antigen Receptor, Br. J. Haematol. 161:389-401; Gilí, S. et al (2014) Efficacy Against Human Acute Myeloid Leukemia And Myeloablation Of Normal Hematopoiesis In A Mouse Model Using Chimeric Antigen Receptor-Modified T Cells, Blood 123(15): 2343-2354; Mardiros, A. et al (2013) T Cells Expressing CD cnan Ln/zznz/E/YiAi cnan Ln/zznz/E/YiAi

264

123-Specific Chimeric Antigen Receptors Exhibit Specific Cytolytic Effector Functions And Antitumor Effects Against Human Acute Myeioid Leukemia, Blood 122:3138-3148; Pizzitola, I. et al (2014) Chimeric Antigen Receptors Against CD33/CD123 Antigens Efficiently Target Primary Acute Myeioid Leukemia Cells in vivo, Leukemia doi: 10.103 8/leu.2014.62).

[00400] Such PD-1 binding molecules of the invention can be combined with Chimeric Antigen Receptors (iCAR) to divert responses of the target immunotherapy. Antigen-binding domain iCARs are directed against a disease antigen fused to one or more intracellular signaling domains from various inhibitory protein receptors (e.g., CTLA-4, PD-1, etc.) that serve to limit T cell responses upon antigen binding (see, for example, Fedorov VD (2013) PD-1— and CTLA-4-Based Inhibitory Chimeric Antigen Receptors (iCARs) Divert Off-Target Immunotherapy Responses, Sci.

Transl. Med. 5:215ral72 doi: 10.1126/ scitranslmed.3006597.

[00401] In particular, such anti-PD-1 binding molecules of the invention are used in combination with an anti-CD 137 antibody, an anti-CTLA-4 antibody, an anti-0X40 antibody, an anti-LAG-3 antibody , an anti-PD-Ll antibody, an anti-TIGIT antibody, a TIM-3 antibody and/or a cancer vaccine

265 cnan Ln/zznz/E/YiAi

B. Diagnostic and Theranostic Utility

[00402] Some of the PD-l-binding molecules of the present invention have little or no ability to block the binding between PD-1 and the PD-1L ligand. As such, PD-1 mAb 2 and PD-1 mAb 4 antibodies, their humanized derivatives, and molecules that comprise their PD-1 binding fragments (e.g., bispecific diabodies, etc.) or that compete for The binding with such antibodies can be detectably labeled (for example, with radioactive, enzymatic, fluorescent, chemiluminescent, paramagnetic, diamagnetic, moieties). or other labeled) and is used in the detection of PD-1 in samples or in obtaining images of PD-1 on cells. Since such molecules do not affect the biological activity of PD-1, they are particularly useful in methods for determining the extent, location, and change in PD-1 expression in subjects (e.g., subjects receiving treatment for cancer-associated cancer). with PD-1 expression or targeting).

XII. Pharmaceutical Compositions

[00403] The compositions of the invention include bulk pharmaceutical compositions useful in the manufacture of pharmaceutical compositions (for example, compositions

266 impure or non-sterile) and pharmaceutical compositions (i.e., compositions that are suitable for administration to a subject or patient) that can be used in the preparation of unit dosage forms. Such compositions comprise a prophylactically or therapeutically effective amount of the PD-1 binding molecules of the present invention, or a combination of such agents and a pharmaceutically acceptable carrier. Preferably, the compositions of the invention comprise a prophylactically or therapeutically effective amount of the PD-1 binding molecules of the present invention and a pharmaceutically acceptable carrier. The invention particularly encompasses such pharmaceutical compositions in which the PD-1 binding molecule is: a PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 3, PD-1 mAb 4, mAb PD-1 5, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 8, PD-1 mAb 9, PD-1 mAb 10, PD-1 mAb 11, mAb 12 of PD-1, mAb 13 of PD-1, mAb 14 of PD-1, or mAb 15 of PD-1; a humanized PD-1 mAb 1; PD-1 mAb 2, PD-1 mAb 3, PD-1 mAb 4, PD-1 mAb 5, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 8, mAb PD-1 9, PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12, PD-1 mAb 13, PD-1 mAb 14, or PD-1 mAb 15; a PD-1 binding fragment of any such antibody; or in the PD-1 binding molecule is a bispecific PD-1 diabody (eg, a PD-1 x LAG-3 bispecific diabody). It is especially open to such cnan Ln/zznz/E/YiAi

267 molecules comprising 3CDRls and the 3CDRhs antibody of PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 3, PD-1 mAb 4, PD-1 mAb 5, PD-1 mAb 6 , PD-1 mAb 7, PD-1 mAb 8, PD-1 mAb 9, PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12, PD-1 mAb 13, PD-1 mAb 14, or PD-1 mAb 15; a PD-1 mAb 1; PD-1 mAb 2, PD-1 mAb 3, PD-1 mAb 4, PD-1 mAb 5, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 8, mAb 9 of PD-1, mAb 10 of PD-1, mAb 11 of PD-1, mAb 12 of PD-1, mAb 13 of PD1, PD-1 mAb 14, or mAb 15 of humanized PD-1.

cnan ίη/ζζηζ/Ε/γίΛΐ

[00404] The invention also encompasses such pharmaceutical compositions that additionally include a second therapeutic antibody (e.g., tumor-specific monoclonal antibody) that is specific for a particular cancer antigen, and a pharmaceutically acceptable carrier.

[00405] In a specific embodiment, the term pharmaceutically acceptable means approved by a federal or state government regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term carrier refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers may be sterile liquids, such as water and oils,

268 including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry skim milk, glycerol, propylene, glycol , water, ethanol and the like. If desired, the composition may also contain minor amounts of wetting or emulsifying agents, or pH regulating agents. These compositions may take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained release formulations and the like.

[00406] Generally, the ingredients of the compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as a vial. or sachet indicating the amount of active agent. When the composition is to be administered by infusion, cnan Ln/zznz/E/YiAi

CbQb ίη/ΖΖΠΖ/Ε/ΥΙΛΙ

269 can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, a vial of sterile water for injection or saline may be provided so that the ingredients can be mixed before administration.

[00407] The compositions of the invention can be formulated as neutral or saline forms. Pharmaceutically acceptable salts include, but are not limited to, those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric, etc. acids, and those formed with cations such as those derived from hydroxides of sodium, potassium, ammonium, calcium, ferric, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[00408] The invention also provides a pharmaceutical package or kit comprising one or more containers with a PD-1 binding molecule of the present invention (and more preferably, a PD-1 mAb 1, PD-1 mAb 2 1, PD-1 mAb 3, PD-1 mAb 4, PD-1 mAb 5, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 8, PD-1 mAb 9 , PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12, PD-1 mAb 13, PD-1 mAb 14, or PD-1 mAb 15; an antibody PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 3, PD-1 mAb 4, PD-1 mAb 5, PD-1 mAb 6, PD-1 mAb 7 , PD-1 mAb 8, PD-1 mAb 9, PD-1 mAb 10,

270

PD-1, PD-1 mAb 12, PD-1 mAb 13, PD-1 mAb 14, or humanized PD-1 mAb 15; a PD-l-binding fragment of any antibody; or wherein the PD-1 binding molecule is a bispecific PD-1 diabody (e.g., a PD-1 LAG-3 bispecific diabody x)). Especially encompassed are such molecules comprising the 3 CDRls and the 3 CDRhs of PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 3, PD-1 mAb 4, PD-1 mAb 5, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 8, PD-1 mAb 9, PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12, mAb PD-1 13, PD-1 mAb 14, or PD-1 mAb 15, alone or with such pharmaceutically acceptable carrier. Additionally, one or more different prophylactic or therapeutic agents useful for the treatment of a disease may also be included in the pharmaceutical package or kit. The invention also provides a pharmaceutical package or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such containers may be a notice in the form prescribed by a government agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects the approval by the agency of the manufacture, use or sale for administration in humans. .

[00409] The present invention provides kits that can be used in the above methods. One kit can comprise cnan Ln/zznz/E/YiAi

271 any of the PD-1 binding molecules of the present invention. The kit may further comprise one or more different prophylactic and/or therapeutic agents useful for the treatment of cancer, in one or more containers; and/or the kit may further comprise one or more cytotoxic antibodies that bind one or more cancer antigens associated with cancer. In certain embodiments, the other prophylactic or therapeutic agent is a chemotherapeutic. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic.

cnan Ln/zznz/E/YiAi

XIII. Administration Methods

[00410] The compositions of the present invention can be provided for the treatment, prophylaxis and improvement of one or more symptoms associated with a disease, disorder or infection by administering to a subject an effective amount of a fusion protein or a conjugate molecule of the invention, or a pharmaceutical composition comprising a fusion protein or conjugate molecule of the invention. In a preferred aspect, such compositions are substantially purified (i.e., substantially free of substances that limit their effect or produce undesirable side effects). In a specific embodiment, the subject is an animal, preferably a mammal such as a non-primate (for example, bovine, equine, feline, canine, rodent,

272 etc.) or a primate (for example, monkeys such as, a cynomolgus monkey, human, etc.). In a preferred embodiment, the subject is a human.

[00414] The lyophilized PD-1 binding molecules of the present invention should be stored between 2°C and 8°C in their original container and the molecules should be administered within 12 hours, preferably at 6 hours, at 5 hours, 3 hours, or 1 hour after reconstitution. In an alternative embodiment such molecules are supplied in liquid form in a hermetically sealed container indicating the amount and concentration of the molecule, fusion protein, or conjugate molecule. Preferably, when such PD-1 binding molecules are provided in liquid form, they are provided in a hermetically sealed container.

[00415] The amount of the composition of the invention that will be effective in treating, preventing or improving one or more symptoms associated with a disorder can be determined by standard clinical techniques. The precise dose used in the formulation will also depend on the route of administration, and the severity of the condition, and should be decided according to the professional's judgment and the circumstances of each patient. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

As used herein,

[00416] a quantity cnan Ln/zznz/E/YiAi

CbQb ίη/77Π7/Ε/ΥΙΛΙ

273 Effectiveness of a pharmaceutical composition, in one embodiment, is an amount sufficient to effect beneficial or desired results including, without limitation, clinical results such as reduction in symptoms resulting from the disease, attenuation of a symptom of infection (for example, load virus, fever, pain, sepsis, etc.) or a symptom of cancer (for example, proliferation of cancer cells, presence of tumor, tumor metastasis, etc.), thereby increasing the quality of life of those suffering from the disease, reducing the dose of other medications required to treat the disease, intensifying the effect of another medication such as through targeting and/or internalization, delaying the progression of the disease , and/or prolong the survival of individuals.

[00417] An effective amount may be administered in one or more administrations. For purposes of this invention, an effective amount of drug, compound or pharmaceutical composition is an amount sufficient to reduce the proliferation of (or the effect of) viral presence and reduce and/or delay the development of viral disease, whether direct or indirectly. In some embodiments, an effective amount of a drug, compound or pharmaceutical composition may or may not be obtained together with another drug, compound or pharmaceutical composition. In this way, a

274 effective amount may be considered in the context of administering one or more chemotherapeutic agents, and a single agent may be considered to be provided in an effective amount if, together with one or more other agents, a desirable result can or will be achieved. Although individual needs vary, determining

CbQb ίη/77Ω7/Β/ΥΙΛΙ Optimal ranges of effective quantities of each component are within the skill in the art.

[00418] For the PD-1 binding molecules encompassed by the invention, the dosage administered to a patient is preferably determined based on the body weight (kg) of the recipient subject. For the PD-1 binding molecules encompassed by the invention, the dosage administered to a patient is typically at least about 0.01 pg/kg, at least about 0.05 pg/kg, at least about 0.1 pg/kg. kg, at least about 0.2 pg/kg, at least about 0.5 pg/kg, at least about 1 pg/kg, at least about 2 pg/kg, at least about 5 pg/kg kg, at least about 10 pg/kg, at least about 20 pg/kg, at least about 50 pg/kg, at least about 0.1 mg/kg, at least about 1 mg/kg, at least about 3 mg/kg, at least about 5 mg/kg, at least about 10 mg/kg, at least about 30 mg/kg, at least about 50 mg/kg, at least about 75 mg/kg, at least

275 about 100 mg/kg, at least about 125 mg/kg, at least about 150 mg/kg or more of the subject's body weight.

[00419] The dosage and frequency of administration of a PD-1 binding molecule of the present invention can be reduced or altered by enhancing the uptake and penetration of the molecule into the tissue by modifications such as, for example, lipidation.

[00420] The dosage of a PD-1 binding molecule of the invention administered to a patient can be calculated for use as a single agent therapy. Alternatively, the molecule may be used in combination with other therapeutic compositions and the dosages administered to a patient are lower than when the molecules are used as a single agent therapy.

[00421] The pharmaceutical compositions of the invention can be administered locally to the area in need of treatment; this may be obtained, for example, and not by way of limitation, by local infusion, by injection or by means of an implant, the implant being of a porous, non-porous or gelatinous material, including membranes, such as sialastic membranes or fibers. Preferably, when administering a molecule of the invention, care should be taken to use materials into which the cnan Ln/zznz/E/YiAi molecule is not absorbed.

276

[00422] The compositions of the invention can be delivered in a vesicle, in particular a liposome (See, Langer (1990) New Methods Of Drug Delivery, Science 249:1527-1533); Treat et al., in LIPOSOMES IN THE THERAPY OF INFECTIOUS DISEASE AND CANCER, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); LopezBerestein, ibid., pp. 3 17-327).

[00423] The compositions of the invention can be delivered in a controlled release or sustained release system. Any technique known to one skilled in the art can be used to produce sustained release formulations comprising one or more of the PD-1 binding molecules of the invention. See, for example, United States Patent No. 4,526,938; PCT Publication WO 91/05548; PCT Publication WO 96/20698; Ning et al (1996) Intratumoral Radioimmunotheraphy Of A Human Colon Cancer Xenograft Using A Sustained-Release Gel, Radiotherapy &amp; Oncology 39:179-189, Song et al (1995) Antibody Mediated Lung Targeting Of Long-Circulating Emulsions, PDA Journal of Pharmaceutical Science &amp; Technology 50:372-397; Cleek et al (1997) Biodegradable Polymeric Carrlers For A bFGF Antibody For Cardiovascular Application, Pro. Int'l. Symp. Control. I laughed. Bioact. Mater. 24:853-854; and Lam et al (1997) Microencapsula tion Of Recombinant Humanized Monoclonal Antibody For Local Delivery, cnan Ln/zznz/E/YiAi

277

Proc. Int'l. Symp. Control Laughter. Bioact. Mater. 24:759-760, each of which is incorporated herein by reference in its entirety. In one embodiment, a pump may be used in a Controlled Release System (See Langer, supra; Sefton, (1987) Implantable Pumps, CRC Crit. Rev. Biomed. Eng. 14:201-240; Buchwald et al (1980) Long-Term, Continuous Intravenous Heparin Administration By An Implantable Infusion Pump In Ambulatory Patients with Recurrent Venous Thrombosis, Surgery 88:507-516; and Saudek et al (1989) 'Ά Preliminary Trial Of The Programmable Implantable Medication System For Insulin Delivery, N. Engl. J. Med. 321:574-579). In another embodiment, polymeric materials can be used to obtain controlled release of the molecules (see, for example, MEDICAL APPLICATIONS OF CONTROLLED RELEASE, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); CONTROLLED DRUG BIOAVAILABILITY , DRUG PRODUCT DESIGN AND PERFORMANCE, Smolen and Ball (eds.), Wiley, New York (1984); Levy et al (1985) Inhibition Of Calcification Of Bioprosthetic Heart Valves By Local Controlled-Release Diphosphonate, Science 228:190-192; During et al (1989) Controlled Release Of Dopamine From A Polymeric Brain Implant: In Vivo Characterization, Ann. Neurol. 25:351-356; Howard et al (1989) Intracerebral Drug Delivery In Rats with Lesion-Induced Memory Deficits, J. Neurosurg. 7(1): 105-112); United States Patent No.

cnan Ln/zznz/E/YiAi

278

5,679,377; United States Patent No. 5,916,597; United States Patent No. 5,912,015; United States Patent No. 5,989,463; United States Patent No. 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(acid methacrylic), polyglycolides (PLG), polyanhydrides, poly (N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly (ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. A controlled release system can be placed in proximity to the therapeutic target (e.g., the lungs), thereby only providing a fraction of the systemic dose (see, e.g., Goodson, in MEDICAL APPLICATIONS OF CONTROLLED RELEASE, supra, vol. 2, pp. 115138 (1984)). Polymeric compositions useful as controlled release implants can be used according to Dunn et al (See US 5,945,155). This particular method is based on the therapeutic effect of the in situ controlled release of the bioactive material from the polymeric system. Implantation can generally occur onto any part within the patient's body in need of therapeutic treatment. Non-cnan Ln/zznz/E/YiAi System can be used

CbQb ίη/ΖΖΠΖ/Ε/ΥΙΛΙ

279 sustained release polymeric, whereby a non-polymeric implant in the subject's body is used as a drug delivery system. After implantation into the body, the organic solvent of the implant will dissipate, disperse or leach from the composition into the surrounding tissue fluid, and the non-polymeric material will gradually coagulate or precipitate to form a solid, microporous matrix (See US 5,888,533 ).

[00424] Controlled release systems are discussed in the review by Langer (1990, New Methods Of Drug Delivery, Science 249:1527-1533). Any technique known to one skilled in the art can be used to produce sustained release formulations comprising one or more therapeutic agents of the invention. See, for example. United States Patent No. 4,526,938; International Publications Nos. WO 91/05548 and WO 96/20698; Ning et al. (1996) Intratumoral Radioimmunotheraphy Of A Human Colon Cancer Xenograft Uslng A Sustained-Release Gel, Radiotherapy &amp; Oncology 39:179-189, Song et al (1995) Antibody Mediated Lung Targeting Of Long-Circulating Emulsions, PDA Journal of Pharmaceutical Science &amp; Technology 50:372-397; Cleek et al (1997) Biodegradable Polymeric Carriers For A bFGF Antibody For Cardiovascular Application, Pro. Int'l. Symp. Control. I laughed. Bioact. Mater.

24:853-854;

and Lam et al (1997)

Microencapsulation Of

280

Recombinant Humanized Monoclonal Antibody For Local Delivery, Proc. Int'l. Symp. Control Laughter. Bioact. Mater. 24:759-760, each of which is incorporated herein by reference in its entirety.

[00425] When the composition of the invention is a nucleic acid encoding a PD-1 binding molecule of the present invention, the nucleic acid can be administered in vivo to promote the expression of its encoded PD1 binding molecule by constructing it as a portion of an appropriate nucleic acid vector and administering it so that it becomes intracellular, for example, by use of a retroviral vector (See United States Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell surface receptors or transection agents, or by administering it in conjunction with a homeoblock-like peptide known to enter the nucleus (See for example, Joliot et al. (1991) Antennapedia Homeobox Peptide Regulates Neural Morphogenesis, Proc. Nati. Acad. Sci. (USA) 88:18641868), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated into the DNA of the host cell for expression by homologous recombination.

[00426] Treating a subject with a cnan quantity Ln/zznz/E/YiAi

281 therapeutically or prophylactically effective PD-1 binding molecule of the present invention may include a single treatment or, preferably, may include a series of treatments. In a preferred example, a subject is treated with such a diabody once a week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. The pharmaceutical compositions of the invention may be administered once a day, twice a day, or three times a day. Alternatively, the pharmaceutical compositions may be administered once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, twice a year or once a year. anus. It should also be appreciated that the effective dosage of the molecules used for treatment may be increased or decreased during the course of a particular treatment.

CbQb ίη/77Ω7/Β/ΥΙΛΙ

Examples

[00427] The following examples illustrate various methods for the compositions in the diagnostic or treatment methods of the invention. The examples are intended to illustrate, but not to limit, the scope of the invention.

282

Example 1

Characterization of Anti-Human PD-1 Monoclonal Antibodies

[00428] Fifteen murine monoclonal antibodies were isolated as being capable of specifically binding to both human and cynomolgus monkey PD-1 and according to the designations PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 3 PD1, PD-1 mAb 4, PD-1 mAb 5, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 8, PD-1 mAb 9, PD-1 mAb 10 , PD-1 mAb 11, PD-1 mAb 12, PD-1 mAb 13, PD-1 mAb 14, and PD-1 mAb 15. The CDRs of these antibodies were found to differ and were provided above. Binding to the extracellular domain of human and cynomolgus monkey PD-1 was evaluated as follows: Maxisorb flat-bottom 96-well plates were coated with soluble human or cynomolgus monkey PD-1 (the extracellular domain of human or cynomolgus monkey PD-1). human fused to a His tag (shPD-1 His) or to a human Fe Region (shPD-1 hFc), or to the extracellular domain of cynomolgus monkey PD-1 fused to a human Fe Region (scyno-Fe PDl)), each at 0.5 or 1 pg/mL, The plates were washed and incubated with one of the isolated anti-PD-1 antibodies mAb 1-15 of PD-1. For these studies, anti-PD-1 antibodies were used at 3, 1.0, 0.3333, 0.1111, 0.0370, 0.0123, or 0.0041 pg/mL (serial three-fold dilutions). The amount of antibodies bound to PD-1 cnan ίη/ζζηζ/Ε/γίΛΐ

283 immobilized (from human or cynomolgus monkey) was evaluated using a goat anti-mouse IgG-HRP secondary antibody. All samples were analyzed in a plate reader (Victor 2 Wallac, Perkin Elmers). Representative binding curves for human and soluble cynomolgus monkey PD-1 are shown in Figures 7A-7D and Figures 8A-8C, respectively.

[00429] The results of these binding assays (Figures 7A-7D and Figures 8A-8C) show that all anti-PD-1 mAb 1-15 antibodies bound both to human PD-1 soluble like soluble cynomolgus monkey.

[00430] To further characterize the murine anti-PD-1 antibodies, their ability to block the binding of soluble human PD-L1 to soluble human PD-1 was evaluated in two different assays. In one assay, the ability of antibodies to block the binding of PD-1 to human PD-L1 immobilized on a surface was examined. For this assay, each of the anti-PD-1 mAb 1-15 antibodies, or a reference anti-PD-1 antibody (PD-1 mAb A) was mixed with the protein. shPD-1 His fusion, (at 2.5 pg/mL) and incubated separately with biotin-soluble human PD-L1 (the extracellular domain of PD-L1 fused to human Fe (sPD-Ll)) at 1 pg/mL mL immobilized on a streptavidin-coated layer. For these studies, anti-PD-1 antibodies were used at 10, 5.0, 2.5

1.25

0.625

0.3125

CbQb ίη/77Π7/Ε/ΥΙΛΙ

284 or 0.1563 pg/mL (serial two-fold dilutions). The amount of shPD-1 His bound on the immobilized sPD-Ll was assessed by His tag using an anti-His-Tag-HRP secondary antibody. All samples were analyzed in a plate reader (Victor 2 Wallac, Perkin Elmers). The results of this experiment are shown in Figures 9A9D.

[00431] The results of these inhibition assays (Figures 9A-9D) show that anti-PD antibodies PD-1 mAb 1, PD-1 mAb 3, PD-1 mAb 5, PD-1 mAb 6 , PD-1 mAb 7, PD-1 mAb 8, PD-1 mAb 9, PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12, PD-1 mAb 13, PD-1 mAb 14, and PD-1 mAb 15, were able to block binding of soluble human PD-L1 to soluble human PD-1 to varying degrees while PD-1 mAb 2 and PD-1 mAb 4 showed little or no blocking activity in this assay format.

[00432] In the second assay, the ability of PD-1 mAb 1-15 murine anti-PD-1 antibodies to block PD-1 ligand binding (i.e., human PD-L1 or Human PD-L2) to PD-1 expressed on the surface of NSO cells. For this assay, each of the anti-PD-1 mAb 1-15 antibodies, or a reference anti-PD-1 antibody (PD-1 mAb A or PD-1 mAb B ) was mixed separately with a soluble human biotinylated PD-L1 (shPD-Ll fusion protein) or cnan biotinylated PD-L2 Ln/zznz/E/YiAi

285 human soluble- muIgFc fusion protein (shPD-L2; Ancell

Cat# 573-030), each at 0.1 pg/test, and incubated with NSO cells expressing human PD-1 (~250,000 cells/well) in blocking buffer (FACS + 10% human serum albumin). ). For these studies, anti-PD-1 antibodies were used at 4.0, 1.0, 2.5 xlO^-1, 6.25 xl0“²,

1.56 x ΙΟ'², 3.90 x 10'³, 9.76 x ΙΟ'⁴, 2.4 x 10'⁴, 0.6 x 10'⁴pg/test (serial four-fold dilutions). The amount of shPD-Ll (or shPD-L2) bound on the surface of NSO 10 cells was determined using a secondary antibody with

PE-conjugated streptavidin by FACS analysis. IC50 values ​​for inhibition of PD-1/PD-L1 binding were determined and the sample mean (□) from at least two experiments (except where noted) is provided in Table 6.

cnan Ln/zznz/E/YiAi

<td colspan="5">Table 6</td>

<td>anti-PD-1 antibody</td><td>IC50 (ug/test)</td><td></td><td>anti-PD-1 antibody</td><td>IC50 (pg/test)</td>

<td>PD-1 mAb A</td><td> 0.0044</td><td></td><td>PD-1 mAb 8</td><td> 0.6611 *</td>

<td>PD-1 mAb B</td><td> 0.0064</td><td></td><td>PD-1 mAb 9</td><td> 0.0154 *</td>

<td>PD-1 mAb 1</td><td> 0.0048</td><td></td><td>PD-1 mAb 10</td><td> 0.0057</td>

<td>PD-1 mAb 2</td><td> 0.0110</td><td></td><td>PD-1 mAb 11</td><td> 0.0259 *</td>

<td>PD-1 mAb 3</td><td> 0.0361 *</td><td></td><td>PD-1 mAb 12</td><td> 0.0238 *</td>

<td>PD-1 mAb 4</td><td> 0.0156 *</td><td></td><td>PD-1 mAb 13</td><td> 0.0117</td>

<td>PD-1 mAb 5</td><td> 0.0039</td><td></td><td>PD-1 mAb 14</td><td> 0.0149 *</td>

<td>PD-1 mAb 6</td><td> 0.0051</td><td></td><td>PD-1 mAb 15</td><td> 0.0060</td>

<td>PD-1 mAb 7</td><td> 0.0024</td><td></td><td></td><td></td>

* Results from a single experiment

286

[00433] The results of the shPD-Ll inhibition assays (Table 6) show that the antibodies PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 3, PD-1 mAb 4, PD-1 mAb PD-1 5, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 9, PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12, mAb 13 of PD-1, PD-1 mAb 14, and anti-PD-1 PD-1 mAb 15, were able to block the binding of human PD-L1 to human PD-1 expressed on the surface of cells NSO. In particular, PD-1 mAb 1, PD-1 mAb 5, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 10, and PD-1 mAb 15 blocked shPD binding. -Ll as well as or better than the reference PD-1 antibodies (PD-1 mAb A, PD-1 mAb B), while PD-1 mAb 8 essentially did not block in this assay format. Both PD-1 mAb 2 and PD-1 mAb 4 were able to block PD-1/PD-L1 binding in this assay format.

[00434] Similarly, PD-1 mAb 1, PD-1 mAb 2, and PD-1 mAb 3, PD-1 mAb 4, PD-1 mAb 5, PD-1 mAb 6 1, PD-1 mAb 7, PD-1 mAb 9, PD-1 mAb 10, PD-1 mAb 12, PD-1 mAb 13, anti-PD-1 PD-1 mAb 14, were able to block the binding of human PD-L2 to human PD-1 expressed on the surface of NSO cells, while PD-1 mAb 8 essentially did not block in this assay format. In particular, PD-1 mAb 1, PD-1 mAb 5, PD-1 mAb 6, PD-1 mAb 7, and PD-1 mAb 10 blocked shPD-L2 binding as well as, or better than cnan Ln/zznz/E/YiAi PD-1 antibodies

287 reference (PD-1 mAb A, PD-1 mAb B). PD-1 antibodies PD-1 mAb 11 and PD-1 mAb 15 were not tested in this assay. Results for various humanized anti-PD-1 antibodies that included hPD-1 mAb 15 are provided below.

cnan Ln/zznz/E/YiAi

Example 2 Humanization and Additional Characterization [00435] The Variable Domains of the antibodies PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 7, PD-1 mAb 9, and PD-1 mAb 15 were humanized. 1 of anti-PD-1, when the antigenic epitopes were identified the antibodies were further deimmunized to generate final humanized Variable Domains. Humanization of PD-1 mAb 1, PD-1 mAb 2, and PD-1 mAb 15 produced a humanized Domain V and a humanized Domain VL for each antibody designated herein as hPD-1 mAb 1 VH1 , and hPD-Ι mAb 1 VL1; hPD-11 mAb 2 VH1 and hPD-11 mAb 2 VL1; and VH1 of hPD-1 mAb 15 and VL1 of hPD-1 mAb 15. Humanization of PD-1 mAb 7 produced two humanized VH Domains, designated herein as hPD-1 mAb 7 VH1, and hPD-1 mAb 7 VH2, and three humanized VL Domains designated herein as VL1 of hPD-1 mAb 1, VL2 of hPD-1 mAb 7, and VL3 of hPD-1 mAb 7.

Humanization of PD-1 mAb 9 produced two VH Domains

288 humanized, designated herein as VH1 of hPD-1 mAb 9, and VH2 of hPD-1 mAb 9, and two humanized VL Domains designated herein as VL1 of hPD-1 mAb 9, and VL2 of mAb 9 of hPD-1. When multiple humanized Variable Domains have been generated, the humanized light and heavy chain Variable Domains of a particular anti-PD-1 antibody (e.g., PD1 mAb 7) can be used in any combination and particular combinations of humanized chains are designated by Reference to specific VH/VL Domains, for example, a humanized antibody comprising hPD-1 mAb 7 VH1 and hPD-1 mAb 7 VL2 is specifically referred to as hPD-1 mAb 7(1.2). Full-length humanized antibodies were generated with either an IgGl constant region comprising the L234A/L235A substitutions (IgGl(AA)) or a human IgG4 constant region comprising the S228P substitution (IgG4(P)).

[00436] Full-length humanized IgGl antibody heavy chains were constructed as follows: the humanized VH Domain Terminus was fused to the N-terminus of a human IgGl Constant Region having a Variant CH2-CH3 Domain (comprising substitutions L234A/L235A (AA)) and lacks the C-terminal lysine residue (SEQ ID

NO:255) :

ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYR VVSVLT VLHQDWLNGK cnan Ln/zznz/E/YiAi

289 cnan Ln/zznz/E/YiAi

EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG

[00437] In SEQ ID NO:255, amino acid residues 1-98 correspond to the CHI Domain of IgGl (SEQ ID NO: 10), amino acid residues 99-113 correspond to the hinge region of IgGl (SEQ ID NO: 10). : 32) and amino acid residues 114-329 correspond to the CH2-CH3 Domain of IgGl which comprises the L234A/L235A substitutions (underlined) (SEQ ID NO:5) but lacks the C-terminal lysine residue.

[00438] The amino acid sequence of a heavy chain of an exemplary humanized antibody ((hPD-Ι mAb 7(1.2)) having an IgGl heavy chain constant region comprises the L234A/L235A mutation and lacks the lysine residue Cterminal which is (SEQ ID NO:265):

QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWIGV IHPSDSETWL DQKFKDRVTI TVDKSTSTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSS VVTV PSSSLGTQFY ICNVNHKPSN TKVDKRVEPK SCDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMTSRTPF, VTCVVVDVñH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM T KNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPG

[00439] In SEQ ID NO: 265, amino acid residues 1119 correspond to the VH domain of VH1 of hPD-Ι mAb 7 (SEQ

290

ID NO: 147), amino acid residues 120-217 correspond to the CHI Domain of IgGl (SEQ ID NO: 10), residues 218-232 correspond to the hinge region of IgGl (SEQ ID NO: 32) and residues 233-448 correspond to the CH2-CH3 Domain of IgGl that comprises the L234A/L235A substitutions (underlined) (SEQ ID NO: 5) but lacks the C-terminal Usine residue.

[00440] The heavy chains of the humanized antibody of

Full-length IgG4 were constructed as follows: the humanized VH Domain Cterminus was fused to the N-terminus of a humanized IgG4 Constant Region that has a stabilized hinge region (comprising the S228P substitution) and lacks the C-Usin residue. terminal (SEQ ID NO: 256):

ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV

HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES

KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED

PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK

CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK

GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG

NVFSCSVMHE ALHNHYTQKS LSLSLG

[00441] In SEQ ID NO:256, amino acid residues 1-98 correspond to the CHI Domain of IgG4 (SEQ ID NO:254), amino acid residues 99-110 correspond to the stabilized IgG4 hinge region comprising the S228P substitutions (underlined) (SEQ ID NO: 13) and amino acid residues 111-326 correspond to the CH2-CH3 Domain of IgG4 (SEQ ID NO: 4) but lack the C-terminal Usine residue.

[00442] The amino acid sequence of a cnan heavy chain Ln/zznz/E/YiAi

291 An exemplary humanized antibody (hPD-1 mAb 7(1.2)) had an IgG4 heavy chain constant region comprising a stabilized hinge region that has the S228P mutation and lacks the C-terminal lysine residue (SEQ ID

NO:266):

QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWIGV IHPSDSETWL DQKFKDRVTI TVDKSTSTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSSA STKGPSVFPL APCSRSTSES TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSV VTV PSSSLGTKTY TCNVDHKPSN TKVDKRVESK YGPPCPPCPA PEFLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSQEDP EVQFNWYVDG VEVHNAKTKP REEQFNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKGLPSS IEKTISKAKG QPREPQVYTL PPSQEEMTKN QVñLTCLV KG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLG

[00443] In SEQ ID NO:266, amino acid residues 1-119 correspond to the VH domain of VH1 of mAb 7 of hPD-1 (SEQ ID

NO:147), amino acid residues 120-217 correspond to

CH1 domain of IgG4 (SEQ ID NO:254), amino acid residues 218-229 correspond to the stabilized IgG4 hinge region comprising substitutions S228P (underlined) (SEQ ID NO:13) and amino acid residues 230- 445 correspond to the CH2-CH3 Domain of IgG4 (SEQ ID NO:4) but lack the C-terminal lysine residue.

[00444] Full-length humanized antibody light chains were constructed as follows: the C-terminus of the humanized VL Domain was fused to the N-terminus of a human light chain kappa region (SEQ ID NO:8). The

CbQb ίη/77Π7/Ε/ΥΙΛΙ

292 The same light chain was paired with the heavy chains of IgGl (AA) and IgG4 (P).

[00445] The amino acid sequence of a light chain of an exemplary humanized PD-1 antibody (hPD1 mAb 7(1.2)) having a kappa constant region is (SEQ ID NO:264):

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKV YACEV THQGLSSPVT KSFNRGEC

[00446] In SEQ ID NO:264, amino acid residues 1-111 correspond to the VL domain of VL2 of mAb 7 of hPD-1 (SEQ ID NO:153), and amino acid residues 112-218 correspond to the region kappa light chain constant (SEQ ID NO: 8).

[00447] Anti-PD-1 antibodies having alternative Constant Regions, for example, the Engineered Fe Regions, were easily generated by incorporating different Constant Regions and/or by introducing one or more amino acid substitutions, additions or deletions. For example, when CH2-CH3 domains of a support button and support hole type bispecific antibody are desired, they are used to facilitate heterodimerization. Chimeric anti-PD-1 antibodies comprising murine Variable Domains and human Constant Regions are generated as described above.

[00448] Humanized antibodies (IgGl (AA) and/or IgG4 cnan Ln/zznz/E/YiAi cnan Ln/zznz/E/YiAi

293 (Ρ)) were tested for binding and blocking activity as described above. Binding to human PD-1 (shPD-1 His, and shPD-1 hFc) and cynomolgus monkey PD-1 (shPDLl hFc) of the humanized antibodies was comparable to that of the corresponding murine antibody. Furthermore, the humanized antibodies retained the ability to block the binding of human PD-L1 to human PD-1 in an ELISA assay.

[00449] Binding kinetics of murine PD-1 mAb 2, PD-1 mAb 7, PD-1 mAb 9, murine PD-1 mAb 2, hPD-1 mAb 2, mAb hPD-1 mAb 7(1.2), hPD-1 mAb 9(1.1), humanized hPD-1 mAb 15, and the reference anti-PD-1 antibodies PD-1 mAb A and PD-1 mAb B were investigated. using a Biacore analysis. Anti-PD-1 antibodies were captured on immobilized Protein A and incubated with His-tagged soluble human PD-1 (shPD-l-His) or soluble cynomolgus monkey PD-1 fusion human Fe (hFc of scyno-PDl) cleaved to remove the Fe moiety, and the binding kinetics were determined by Biacore analysis. In additional studies, hPD1 mAb 7(1.2) IgGl (AA), hPD-1 mAb 7(1.2) IgG4 (P), hPD-1 mAb 9(1.1) IgGl (AA), hPD-1 mAb 7(1.2) IgGl (AA), (P) of hPD-1 mAb 9(1.1), IgGl (AA) of PD-1 mAb A, IgG4 (P) of PD-1 mAb A, IgGl (AA) of PD1 mAb B, and IgG4 (P) of anti-PD-1 mAb B, captured in

294

Fe immobilized goat anti-human F(ab)2 and the binding kinetics were determined by a Biacore analysis as described above. The ka, kd and KD calculated from these studies are presented in Table 7.

CbQb ίη/77Ω7/Β/ΥΙΛΙ

<td colspan="8">Table 7</td>

<td colspan="8">Protein A Capture</td>

<td rowspan="2">AntiPD-1 antibody</td><td colspan="3">Human³</td><td></td><td colspan="3">Cynomolgus monkey^b</td>

<td>ka(xlO^)</td><td>k_d(xl0⁴)</td><td>KD (nM)</td><td></td><td>ka (xlO⁴)</td><td>k_d(xl0⁴)</td><td>KD (nM)</td>

<td>PD-1 mAb A</td><td> 60</td><td> 18</td><td> 3</td><td></td><td> 14</td><td> 9.6</td><td> 6.9</td>

<td>PD-1 mAb B</td><td> 140</td><td> 35</td><td> 2.5</td><td></td><td> 37</td><td> 12</td><td> 3.2</td>

<td>PD-1 mAb 7</td><td> 21</td><td> 2.8</td><td> 1.3</td><td></td><td> 17</td><td> 6</td><td> 3.5</td>

<td>hPD-1 mAb 7(1.2)</td><td> 110</td><td> 4.3</td><td> 0.39</td><td></td><td> 37</td><td> 6.4</td><td> 1.7</td>

<td>hPD-1 mAb 9</td><td> 4 . 3</td><td> 4.2</td><td> 9.8</td><td></td><td> 2.2</td><td> 16</td><td> 72.7</td>

<td>hPD-1 mAb 9(1.1)</td><td> 1.8</td><td> 6.5</td><td> 36.1</td><td></td><td> 1.5</td><td> 11</td><td> 73.3</td>

<td>PD-1 mAb 15</td><td> 4.5</td><td> 1.3</td><td> 2.9</td><td></td><td> 2.7</td><td> 11</td><td> 40.7</td>

<td>hPD-1 mAb 15</td><td> 2.4</td><td> 3.2</td><td> 13.3</td><td></td><td> 2.3</td><td> 18</td><td> 78.3</td>

<td>PD-1 mAb 2</td><td> 5.5</td><td> 5.6</td><td> 10.2</td><td></td><td> 4.2</td><td> 6.0</td><td> 14.3</td>

<td>hPD-1 mAb 2</td><td> 3.2</td><td> 1.6</td><td> 5.0</td><td></td><td> 2.3</td><td> 3.9</td><td> 17</td>

<td colspan="8">Capture of goat anti-human Fe F(ab&gt;2</td>

<td>PD-1 mAb A IgGl (AA)</td><td> 13</td><td> 8.4</td><td> 6.5</td><td></td><td> 8 . 1</td><td> 4.5</td><td> 5.6</td>

<td>PD-1 mAb A IgG4 (P)</td><td> 13</td><td> 7.9</td><td> 6.1</td><td></td><td> 8.4</td><td> 5.0</td><td> 6.0</td>

<td>PD-1 mAb B IgGl (AA)</td><td> 25</td><td> 28</td><td> 11.2</td><td></td><td> 20</td><td> 6.4</td><td> 3.2</td>

<td>PD-1 mAb B IgG4 (P)</td><td> 26</td><td> 25</td><td> 9.6</td><td></td><td> 20</td><td> 7.9</td><td> 4.0</td>

<td>hPD-1 mAb 7(1.2) IgGl (AA)</td><td> 25</td><td> 3.8</td><td> 1.5</td><td></td><td> 16</td><td> 7.8</td><td> 4.9</td>

<td>hPD-1 mAb 7(1.2) IgG4 (P)</td><td> 27</td><td> 4.1</td><td> 1.5</td><td></td><td> 17</td><td> 7.8</td><td> 4.6</td>

<td>hPD-1 mAb 9(1.1) IgGl (AA)</td><td> 5.6</td><td> 6.1</td><td> 10.9</td><td></td><td> 5.6</td><td> 5.2</td><td> 9.3</td>

<td>hPD-1(1.1) mAb 9 IgG4 (P)</td><td> 6.1</td><td> 5.8</td><td> 9.5</td><td></td><td> 4.9</td><td> 7.4</td><td> 15.1</td>

^toHis-tagged soluble human PD-l (shPD-1 His)^bSoluble cynomolgus monkey PD-l (scyno PD-1 hFc) cleaved

[00450] The results demonstrated that PD-1 mAb 7 and humanized hPD-1 mAb 7(1.2) showed greater kinetics of

295 binding relative to the reference anti-PD-1 antibodies PD-1 mAb A and PD-1 mAb B. PD-1 mAb 2, and hPD-1 mAb 2 showed binding kinetics about twice the reference anti-PD-1 antibodies while PD-1 mAb 9, hPD mAb 9(1.1) -1, PD-1 mAb 15, and hPD-1 mAb 15 showed binding kinetics for about 2-6 times the reference anti-PD-1 antibodies.

[00451] The tissue specificity of the PD-1 anti-human mAb 7 antibody was investigated. Normal tissue was contacted with PD-1 mAb 7 or a control isotope (0.313 pg/mL) and the degree of staining was visualized. Bloxall was used for endogenous enzyme blockade to reduce nonspecific mucin staining in colon tissue. As shown in Figure 10A, Panels i-xii, PD-1 mAb 7 and the control isotope did not label cells from normal colon, liver, lung, pancreas, kidney and heart tissue. Furthermore, PD-1 mAb 7 and the control isotope did not stain normal skin (Figure 10B, Panels i-ii). In contrast, PD-1 mAb 7 was found to strongly label lymphocytes present in normal tonsil tissue and PD-1-transfected NSO cells expressing PD-1 (Figure 10B, Panels iii and v), while the control isotope did not. labeled none (Figure 10B, Panels iv and vi). The results presented in Figures 10A-10B indicated clearly

CbQb ίη/77Ω7/Β/ΥΙΛΙ cnan Ln/zznz/E/YiAi

296 Thus, PD-1 mAb 7 was able to bind specifically to lymphocytes and cells expressing PD-1. [00452] Saturation profiles of binding to IgGl (AA) of hPD-1 mAb 2, IgGl (AA) of hPD-1 mAb 7(1.1), IgGl (AA) of hPD mAb 7(1.2) -1, hPD-1 mAb 7(1.2) IgG4 (P), hPD-1 mAb 9(1.1) IgGl (AA), hPD-1 mAb 9(1.1) IgG4 (P) IgG4, hPD-1 mAb 9(1.1) IgG4 (P) (AA) hPD-1 mAb 15, and the reference anti-PD-1 antibodies PD-1 mAb A and PD-1 mAb B were examined. Briefly, each of the anti-PD1 antibodies, PD-1 mAb 1-15, or the reference anti-PD-1 antibodies (PD-1 mAb A and PD-1 mAb B) were mixed with NSO cells expressing human PD-1 (-250,000 cells/well) by blocking the regulator (FACS + 10% human serum albumin). For these studies, anti-PD-1 antibodies were used at 50, 12.5, 3.13, 2.0 xlO^-1, 4.9 x ΙΟ'², 1.2 x ΙΟ'², 3.0 x ΙΟ'³, 1.9 x 10'⁴, 7.6 x 10'⁴, 4.75 x 10'⁵, or 1.19 x 10^-5 pg/test (serial four-fold dilutions). The amount of antibody bound to the surface of NSO cells was determined using goat anti-human-APC secondary antibody by FACS analysis. Representative saturation curves are shown in Figure 11. EC50 and EC90 values ​​were determined and the sample mean (SM) and standard deviation (SD σ) of four separate experiments are provided in Table 8.

297 cnan Ln/zznz/E/YiAi

<td colspan="5">Table 8</td>

<td></td><td colspan="4">Union Saturation</td>

<td></td><td colspan="2">EC50 (pg/test)</td><td colspan="2">EC90 (pg/test)</td>

<td>Anti-PD-1 antibodies</td><td>YE</td><td>SD to</td><td>YE</td><td>SD σ</td>

<td>PD-1 mAb A IgGl (AA)</td><td> 0.1991</td><td> 0.1309</td><td> 1.4528</td><td> 0.8040</td>

<td>PD-1 mAb A IgG4 (P)</td><td> 0.1581</td><td> 0.1161</td><td> 1.5464</td><td> 1.7690</td>

<td>PD-1 mAb B IgGl (AA)</td><td> 0.1347</td><td> 0.0681</td><td> 1.3917</td><td> 0.9573</td>

<td>PD-1 mAb B IgG4 (P)</td><td> 0.1398</td><td> 0.0951</td><td> 1.1619</td><td> 1.2681</td>

<td>PD-1 mAb 2 IgGl (AA)</td><td> 0.4431</td><td> 0.1997</td><td> 2.4374</td><td> 1.2637</td>

<td>hPD-1 mAb 7(1.1) IgGl (AA)</td><td> 0.1069</td><td> 0.0500</td><td> 0.9102</td><td> 0.5476</td>

<td>hPD-1 IgGl (AA) mAb 7(1.2)</td><td> 0.1872</td><td> 0.1553</td><td> 0.6810</td><td> 0.3226</td>

<td>PD-1(1.2) mAb 7 IgG4 (P)</td><td> 0.1376</td><td> 0.0926</td><td> 0.6609</td><td> 0.3437</td>

<td>PD-1(1.1) mAb 9 IgGl (AA)</td><td> 0.3123</td><td> 0.2291</td><td> 1.6486</td><td> 0.9117</td>

<td>hPD-1 mAb 9(1.1) IgG4 (P)</td><td> 0.5128</td><td> 0.2228</td><td> 3.0563</td><td> 0.9437</td>

<td>PD-1 mAb 15 IgGl (AA)</td><td> 0.2927</td><td> 0.1333</td><td> 2.0640</td><td> 0.6096</td>

[00453] Binding saturation studies demonstrated that humanized versions of PD-1 mAb 2, PD-1 mAb 7

1, PD-1 mAb 9, and PD-1 mAb 15 had a favorable profile for binding to cell surface PD-1. In particular, humanized PD-1 mAb 7 (hPD-1 mAb 7(1.1), and hPD-1 mAb 7(1.2) had either an IgGl Fe Region (AA) or an IgG4 Fe Region (P). ) had the lowest EC90 values ​​of all antibodies examined.

298

[00454] To further characterize humanized antiPD-1 antibodies hPD-1 mAb 2 IgGl (AA), mAb 7(1.1) IgGl (AA), mAb 7(1.2) IgGl (AA) ) of hPD-1, IgG4 (P) of mAb 7(1.2) of hPD-1, IgGl (AA) of mAb 9(1.1) of hPD-1, 5 IgG4 (P) of mAb 9(1.1) of hPD-1 1, and hPD-1 mAb 15 IgGl (AA), were examined for their ability to block the binding of human PD-L1 (shPD-L1) and human PD-L2 (shPD-L2) to expressed PD-1. on the surface of NSO cells. These assays were performed essentially as described above.

Representative curves for inhibition of sPD-Ll and sPD-L2 binding to PD-1 expressed in NSO cells are shown in Figures 12A and 12B, respectively. The IC50 and IC90 values ​​were determined and the sample mean (SM) and standard deviation (SD σ) from three separate experiments are provided in Table 9.

CbQb ίη/ΖΖΠΖ/Ε/ΥΙΛΙ

299

CbQb ίη/77Π7/Ε/ΥΙΛΙ

<td colspan="9">Table 9</td>

<td></td><td colspan="4">sPD-Ll</td><td colspan="4">SPD-L2</td>

<td></td><td colspan="2">IC50 (pg/test)</td><td colspan="2">IC90 (pg/test)</td><td colspan="2">IC50 (pg/test)</td><td colspan="2">IC90 (pg/test)</td>

<td>Antibodies Anti-PD-1</td><td>YE</td><td>SD σ</td><td>YE</td><td>SD σ</td><td>YE</td><td>SD σ</td><td>YE</td><td>SD σ</td>

<td>IgGl (AA) PD-1 mAb A</td><td> 0.0203</td><td> 0.0089</td><td> 0.2985</td><td> 0.3279</td><td> 0.0414</td><td> 0.0124</td><td> 0.1601</td><td> 0.066</td>

<td>mAb IgG4(P) A for PD-1</td><td> 0.0156</td><td> 0.0096</td><td> 0.0776</td><td> 0.0208</td><td> 0.0280</td><td> 0.0070</td><td> 0.1594</td><td> 0.1153</td>

<td>IgGl (AA) PD-1 mAb B</td><td> 0 . 0148</td><td> 0.0008</td><td> 0.1034</td><td> 0.0100</td><td> 0.0280</td><td> 0.0059</td><td> 0.1190'</td><td> 0.060</td>

<td>mAb IgG4(P) PD-1 B</td><td> 0.0143</td><td> 0.0013</td><td> 0.0798</td><td> 0.0239</td><td> 0.0280</td><td> 0.0055</td><td> 0.0924</td><td> 0.0065</td>

<td>IgGl (AA) hPD-1 mAb 2</td><td> 0.0578</td><td> 0.0124</td><td> 0.2480</td><td> 0.050</td><td> 0.1294</td><td> 0.0143</td><td> 0.3813</td><td> 0.0656</td>

<td>hPD-1 mAb 7(1.1) IgGl (AA)</td><td> 0.0166</td><td> 0.0032</td><td> 0.0674</td><td> 0.0041</td><td> 0.0283</td><td> 0.0147</td><td> 0.0886</td><td> 0.0166</td>

<td>IgGl (AA) mAb 7(1.2) hPD-1</td><td> 0.0118</td><td> 0.0027</td><td> 0.0678</td><td> 0.0031</td><td> 0.0212</td><td> 0.0031</td><td> 0.0672</td><td> 0.0043</td>

<td>hPD-1 mAb 7(1.2) IgG4 (P)</td><td> 0.0103</td><td> 0.0023</td><td> 0.0520</td><td> 0.0033</td><td> 0.0213</td><td> 0.0019</td><td> 0.0616</td><td> 0.0063</td>

<td>hPD-1 mAb 9(1.1) IgGl (AA)</td><td> 0.0593</td><td> 0.0036</td><td> 0.3238</td><td> 0.0508</td><td> 0.4002</td><td> 0.5000</td><td> 0.4573</td><td> 0.1805</td>

<td>mAb IgG4(P) 9(1.1) of hPD-1</td><td> 0.0460</td><td> 0.0118</td><td> 0.2461</td><td> 0.0513</td><td> 0.1105</td><td> 0.0146</td><td> 0.2914</td><td> 0.0526</td>

<td>mAb IgG4(P) 15 hPD-1</td><td> 0.0440</td><td> 0.0092</td><td> 0.2068</td><td> 0.035</td><td> 0.0945</td><td> 0.0022</td><td> 0.3093</td><td> 0.0588</td>

300

[00455] Ligand binding inhibition studies demonstrated that humanized versions of PD-1 mAb 2, PD-1 mAb 7, PD-1 mAb 9, and PD-1 mAb 15 were able to inhibit the binding of sPD-Ll and sPD-L2 to PD-1 on the cell surface. In particular, humanized PD-1 mAb 7 (hPD-1 mAb 7(1.1), and hPD-1 mAb 7(1.2)) had the lowest IC90 values ​​of all antibodies examined.

cnan Ln/zznz/E/YiAi

Example 3

PD-1/PD-L1 Endpoint Blockade by Humanized Anti-Human PD-1 Antibodies

[00456] The capacity of IgGl (AA) of mAb 2 of hPD-1, IgGl (AA) of mAb 7(1.1) of hPD-1, IgGl (AA) of mAb 7(1.2) of hPD1, IgG4 (P) hPD-1 mAb 7(1.2), hPD-1 mAb 9(1.1) IgGl (AA), hPD-1 mAb 9(1.1) IgG4 (P), hPD mAb 15 IgG4 (P) -1, and the reference anti-PD-1 PD-1 mAb A and PD-1 mAb B antibodies to antagonize the PD-1/PD-L1 axis (i.e. blocking the PD-1/PD-L1 interaction and preventing deregulation of T cell responses) was examined in a Jurkat-luc-NFAT/CHO-PD-Ll luciferase reporter assay. Briefly, CHO cells expressing PD-L1 (CHO/PD-L1) were plated at 40,000/well in 100 pL of culture medium (RPMI + 10% FBS + 100 pg/mL of

Hygromycin B + 100 pg/mL G418) and incubated overnight. The next day, the medium was removed and added in

301 each well Jurkat MNFAT-luc2/PD-l cells (Promega) at 50,000 cells/well in 40 pL of assay buffer (RPMI + 2% FBS), and anti PD-1 mAb 1-15 antibodies. -PD1, or reference anti-PD-1 antibodies (PD-1 mAb A and PD-1 mAb B) (0-25 pg/mL; eight 2.5-fold serial dilutions in assay buffer) and They were incubated for 6 hours at 37°C followed by 5-10 minutes incubated at room temperature. Afterwards, 80 pL of BioGlo Substrate (Promega) was added to each well and the plate was incubated for an additional 5-10 minutes at room temperature, luminescence was measured in a Victor Plate Reader. Representative saturation curves are shown in Figure 13. EC50 and EC90 values ​​were determined and the sample mean (SM) and standard deviation (SD σ) of four separate experiments are provided in Table 10.

ctat Ln/zznz/E/YiAi

<td colspan="5">Table 10</td>

<td rowspan="3">anti-PD-1 antibody</td><td colspan="4">Reporter Signage</td>

<td colspan="2">EC50 (pg/test)</td><td colspan="2">EC90 (pg/test)</td>

<td>YE</td><td>SD σ</td><td>YE</td><td>SD σ</td>

<td>PD-1 mAb A IgGl (AA)</td><td> 0.2549</td><td> 0.0480</td><td> 2.4474</td><td> 1.2228</td>

<td>PD-1 mAb A IgG4 (P)</td><td> 0.2049</td><td> 0.0719</td><td> 2.5535</td><td> 1.2139</td>

<td>PD-1 mAb B IgGl (AA)</td><td> 0.2119</td><td> 0.1781</td><td> 2.2036</td><td> 2.0118</td>

<td>PD-1 mAb B IgG4 (P)</td><td> 0.1142</td><td> 0.0323</td><td> 0.9418</td><td> 0.2863</td>

<td>hPD-1 mAb 2 IgGl (AA)</td><td> 0.3539</td><td> 0.0983</td><td> 3.8975</td><td> 2.0054</td>

<td>hPD-1 mAb 7(1.1) IgGl (AA)</td><td> 0.1080</td><td> 0.0386</td><td> 1.1992</td><td> 0.5103</td>

302

<td>hPD-1 mAb 7(1.2) IgGl (AA)</td><td> 0.0944</td><td> 0.0153</td><td> 0.6452</td><td> 0.2615</td>

<td>hPD-1 mAb 7(1.2) IgG4 (P)</td><td> 0.0965</td><td> 0.0169</td><td> 0.6885</td><td> .01858</td>

<td>hPD-1 mAb 9 IgGl (AA)</td><td> 0.2835</td><td> 0.0530</td><td> 2.9968</td><td> 0.8866</td>

<td>hPD-1 mAb 9 IgG4 (P)</td><td> 0.3154</td><td> 0.0872</td><td> 5.0940</td><td> 4.0496</td>

<td>hPD-1 mAb 15 IgGl (AA)</td><td> 0.2585</td><td> 0.0592</td><td> 3.3138</td><td> 1.0532</td>

cnan Ln/zznz/E/YiAi

[00457] Reporter signaling studies demonstrated that humanized versions of PD-1 mAb 2, PD-1 mAb 7, PD-1 mAb 9, and PD-1 mAb 15 were able to block the PD-1 axis /PD-L1 and will prevent deregulation of T cell responses. In particular, humanized PD-1 mAb 7 (hPD-1 mAb 7(1.1), and hPD-1 mAb 7(1.2) had either a Fe region of IgGl (AA) or IgG4 (P)) had the lowest EC50/EC90 values.

Example 4

Functional Activity of Anti-Human PD-1 Antibodies

[00458] Enterotoxin type B (SEB) from Staphylococcus aureus is a microbial superatigen capable of activating a large proportion of T cells (5-30%) in SEB-sensitive donors. SEB binds to MHC II outside the peptide binding notch and is thus MHC II dependent, but is not restricted and mediated by TCR. SEB stimulation of T cells results in oligoclonal T cell proliferation and cytokine production (although cnan Ln/zznz/E/YiAi can be observed

303 donor variability and some donors will not respond).

At 48 hours of SEB stimulation, PMBCs upregulate PD-1 and LAG-3 with a further enhancement observed at day 5, after secondary culture in 96-well plates with SEB stimulation. Upregulation of immune feedback proteins PD-1 and LAG-3 followed by SEB stimulation of PBMCs limits cytokine release after restimulation. We examined the ability of anti-PD-1 antibodies alone and in combination with anti-LAG-3 antibodies to enhance cytokine release through titration inhibition.

[00459] Briefly, PBMCs were purified using the FicollPaque Plus density gradient centrifugation method (GE Healthcare) according to the manufacturer's instructions from whole blood obtained from healthy donors under informed consent (Biological Specialty Corporation) and then T cells were purified using the Dynabeads® Untouched Human T Cell Kit (Life Technologies) according to the manufacturer's instructions. Purified PBMCs were cultured in RPMI medium + 10% heat-inactivated FBS + 1% Penicillin/Streptomycin in T-25 volumetric flasks for 2-3 days alone or with SEB (Sigma-Aldrich) at 0.1 ng/mL (stimulation primary). At the end of the first round of

304 SEB stimulation, PBMCs were washed twice with PBS and immediately plated into 96-well tissue culture plates at a concentration of 1-5 x 10⁵ cells/well in medium alone, medium with a control or an anti-PD1 antibody, medium with SEB at 0.1 ng/mL (secondary stimulation) and without antibody or medium with SEB and a control IgG or an anti-PD antibody -1 +/- an anti-LAG-3 mAb, and were cultured for an additional 2-3 days. At the end of the second stimulation, supernatants were harvested to measure cytokine secretion using Human DuoSet ELISA Kits for ΙΕΝγ, TNFα, IL-10, and IL-4 (R&amp;D Systems) according to the manufacturer's instructions.

[00460] PD-1 mAb 2, PD-1 mAb 7, PD-1 mAb 9, and PD-1 mAb 15 alone or in combination with the anti-LAG-3 single mAb were examined for their ability to 1 of LAG-3 to enhance cytokine release through titration inhibition. These studies also included one or more of the following reference anti-PD-1 antibodies: PD-1 mAb A; PD-1 mAb B; and LAG-3 mAb A, alone or in combination. Figure 14 shows the IFNy secretion profiles of PBMC stimulated with SEB (0.1 ng/mL) from a representative sensitive donor (D:38941), treated with: no antibody; control antibody isotype; PD-1 mAb 7 and/or LAG-3 mAb 7; PD-1 mAb 9 and/or LAG-3 mAb 1; PD-1 mAb 15 and/or LAG-3 mAb 1; mAb 2 of PD-1 and/or cnan Ln/zznz/E/YiAi

305 LAG-3 mAb 1; or the reference anti-PD-1 antibodies PD-1 mAb B and/or LAG-3 mAb A (antibodies were used at 10 pg/mL).

[00461] In additional studies, the ability of humanized versions of PD-1 mAb 2, PD-1 mAb 7, PD-1 mAb 9, and PD-1 mAb 15 (comprising a Region of Fe of human IgGl (AA) or one of human IgG4 (P) as well as the reference anti-PD-1 mAb A and PD-1 mAb B antibodies to enhance cytokine release through of titration inhibition. For these studies, antibodies were used at 0.625, 2.5, and 10 pg/mL. Figures 15A-15B show the secretion profiles of IFNy (Figure 15A) and TNFα (Figure 15B), from PBMC stimulated with SEB (0.2 ng/mL) from a representative sensitive donor (D: 5 7709), treated no antibody or one of the following antibodies: isotype control; hPD-1 mAb 2 IgGl (AA); hPD-1 mAb 7(1.2) IgGl (AA); hPD-1 mAb 7(1.2) IgG4 (P); hPD-1 mAb 9(1.1) IgGl (AA); hPD-1 mAb 9(1.1) IgG4 (P); hPD-1 mAb 15 IgG4 (P); or the reference anti-PD-1 antibodies PD-1 mAb A IgGl (AA), PD-1 mAb A IgG4 (P), PD-1 mAb B IgGl (AA), IgG4 (P ) of PD-1 mAb B. The total pg/mg of IFNy in the samples treated with SEB+Ab were determined for the samples treated with the anti-PD-1 antibodies at 0.625, 2.5 and 10 pg/mL and the sample mean (SM) and the cnan deviation Ln/zznz/E/YiAi

306 standard (SD. σ) from 3 different sensitive donors (except where observed) are provided in Table 11. The ratio of IFNy secreted in the sample treated with the humanized versions of PD-1 mAb 2, PD-1 mAb 7, PD-1 mAb 9, and 5 PD-1 mAb 15 (comprising an Fe Region of human IgGl (AA) or one of human IgG4 (P) over the reference anti-PD-1 PD-1 mAb A and PD-1 mAb B antibodies (i.e., anti-PD mAb A -l/humanized PD-1, and antiPD-l/humanized PD-1 mAb B) are presented in Table 12 and Table 13, 10 respectively.

ctat Ln/zznz/E/YiAi

<td colspan="7">Table 11</td>

<td></td><td colspan="6">IFNy secretion (pg/mL)</td>

<td>pg/mL of anti-PDl antibody</td><td colspan="2">0.625 pg/mL</td><td colspan="2">2.5 pg/mL</td><td colspan="2">10 pg/mL</td>

<td>anti-PD-1 antibody</td><td>YE</td><td>SD σ</td><td>YE</td><td>SD σ</td><td>YE</td><td>SD σ</td>

<td>PD-1 mAb A IgGl (AA)</td><td> 221.18</td><td> 110.89</td><td> 341.13</td><td> 247.93</td><td> 347.46</td><td> 144.72</td>

<td>PD-1 mAb A IgG4 (P)</td><td> 281.36</td><td> 132.65</td><td> 495.15</td><td> 190.57</td><td> 399.41</td><td> 117.56</td>

<td>PD-1 mAb B IgGl (AA)</td><td> 366.69</td><td> 196.64</td><td> 387.682</td><td> 215.51</td><td> 387.32</td><td> 282.81</td>

<td>PD-1 mAb B IgG4 (P)</td><td> 348.40</td><td> 185.96</td><td> 433.382</td><td> 163.23</td><td> 551.68</td><td> 125.08</td>

<td>mAb 7(1.2) IgGl (AA) hPD-1</td><td> 302.05</td><td> 185.71</td><td> 610.70</td><td> 209.77</td><td> 414.63</td><td> 272.65</td>

<td>hPD-1 mAb 7(1.2) IgG4 (P)</td><td>384.57φ</td><td>323.79φ</td><td> 411.40</td><td> 398.59</td><td> 370.06</td><td> 108.12</td>

<td>hPD-1 mAb 9(1.1) IgGl (AA)</td><td> 340.81</td><td> 207.76</td><td> 442.598</td><td> 303.70</td><td> 655.29</td><td> 567.91</td>

<td>mAb 9(1.1) IgG4 (P) hPD-1</td><td> 309.82</td><td> 130.30</td><td> 468.62</td><td> 350.15</td><td> 424.35</td><td> 288.95</td>

<td>hPD-1 mAb 15 IgG4 (P)</td><td> 360.00</td><td> 274.28</td><td> 373.32</td><td> 160.25</td><td> 541.83</td><td> 444.22</td>

<td>hPD-1 mAb 2 IgGl (AA)</td><td> 275.88</td><td> 135.23</td><td> 372.73</td><td> 53.53</td><td> 496.70</td><td> 235.37</td>

<td>control IgG</td><td> 137.14</td><td> 76.61</td><td> 100.65</td><td> 48.67</td><td> 138.10</td><td> 120.81</td>

<td>No antibody</td><td> 120.05</td><td> 73.90</td><td> 120.05</td><td> 73.90</td><td> 109.46</td><td> 85.18</td>

307 *Results from two sensitive donors

CbQb ίη/77Ω7/Ε/ΥΙΛΙ

<td colspan="7">Table 12</td>

<td></td><td colspan="6">IFNy secretion ratio (New anti-PD-1/PD-1 mAb A)</td>

<td>pg/mL anti-PDl antibody</td><td colspan="2">0.625 pg/mL</td><td colspan="2">2.5 pg/mL</td><td colspan="2">10 pg/mL</td>

<td>anti-PD-1 antibody</td><td>YE</td><td>SD σ</td><td>YE</td><td>SD σ</td><td>YE</td><td>SD σ</td>

<td>PD-1 mAb A IgGl (AA)</td><td> 1.00</td><td> 0.00</td><td> 1.00</td><td> 0.00</td><td> 1.00</td><td> 0 . 00</td>

<td>PD-1 mAb A IgG4 (P)</td><td> 1.00</td><td> 0.00</td><td> 1.00</td><td> 0.00</td><td> 1.00</td><td> 0.00</td>

<td>PD-1 mAb B IgGl (AA)</td><td> 1.77</td><td> 0.92</td><td> 1.28</td><td> 0.36</td><td> 1.07</td><td> 0.42</td>

<td>PD-1 mAb B IgG4 (P)</td><td> 1.23</td><td> 0.16</td><td> 0.92</td><td> 0.27</td><td> 1.40</td><td> 0 . 12</td>

<td>mAb 7(1.2) IgGl (AA) hPD-1</td><td> 1.36</td><td> 0.37</td><td> 2.46</td><td> 1.85</td><td> 1.17</td><td> 0.41</td>

<td>mAb 7(1.2) IgG4 (P) hPD-1</td><td>1.20f</td><td>0.35φ</td><td> 0.79</td><td> 0.54</td><td> 0.95</td><td> 0.22</td>

<td>mAb 9(1.1) IgGl (AA) hPD-1</td><td> 1.48</td><td> 0.19</td><td> 1.46</td><td> 0.71</td><td> 1.70</td><td> 0.84</td>

<td>mAb 9(1.1) IgG4 (P) hPD-1</td><td> 1.13</td><td> 0.13</td><td> 0.91</td><td> 0.42</td><td> 1.02</td><td> 0.46</td>

<td>hPD-1 mAb 15 IgG4 (P)</td><td> 1.50</td><td> 0.39</td><td> 1.51</td><td> 1.23</td><td> 1.48</td><td> 0.71</td>

<td>hPD-1 mAb 2 IgGl (AA)</td><td> 1.32</td><td> 0.53</td><td> 1.48</td><td> 0.86</td><td> 1.42</td><td> 0.12</td>

<td>control IgG</td><td> 0.63</td><td> 0.2</td><td> 0.33</td><td> 0.08</td><td> 0.39</td><td> 0.24</td>

<td>No antibody</td><td> 0.54</td><td> 0.12</td><td> 0.39</td><td> 0.14</td><td> 0.31</td><td> 0 . 17</td>

í Results from two sensitive donors

<td colspan="7">Table 13</td>

<td></td><td colspan="6">Relationship of IFNy secretion (new anti-PD-1 mAb/PD-1 mAb B)</td>

<td>pg/mL anti-PDl antibody</td><td colspan="2">0.625 pg/mL</td><td colspan="2">2.5 pg/mL</td><td colspan="2">10 pg/mL</td>

<td>anti-PD-1 antibody</td><td>YE</td><td>SD σ</td><td>YE</td><td>SD σ</td><td>YE</td><td>SD σ</td>

<td>PD-1 mAb A IgGl (AA)</td><td> 0.37</td><td> 0.37</td><td> 0.82</td><td> 0.20</td><td> 1.06</td><td> 0.48</td>

<td>PD-1 mAb A IgG4 (P)</td><td> 0.82</td><td> 0.12</td><td> 1.16</td><td> 0.38</td><td> 0.72</td><td> 0 . 07</td>

<td>PD-1 mAb B IgGl (AA)</td><td> 1.0</td><td> 0.00</td><td> 1.0</td><td> 0.00</td><td> 1.0</td><td> 0 . 00</td>

308

<td>PD-1 mAb B IgG4 (P)</td><td> 1.0</td><td> 0.00</td><td> 1.0</td><td> 0.00</td><td> 1.0</td><td> 0 . 00</td>

<td>mAb 7(1.2) IgGl (AA) hPD-1</td><td> 0.84</td><td> 0.22</td><td> 1.77</td><td> 0.81</td><td> 1.11</td><td> 0 . 07</td>

<td>mAb 7(1.2) IgG4 (P) hPD-1</td><td> 0.911</td><td> 0.2 61</td><td> 0.83</td><td> 0.50</td><td> 0.68</td><td> 0 . 17</td>

<td>mAb 9(1.1) IgGl (AA) hPD-1</td><td> 1.04</td><td> 0.59</td><td> 1 . 12</td><td> 0.29</td><td> 1.60</td><td> 0.42</td>

<td>mAb 9(1.1) IgG4 (P) hPD-1</td><td> 0.92</td><td> 0.09</td><td> 0.99</td><td> 0.36</td><td> 0.75</td><td> 0.39</td>

<td>hPD-1 mAb 15 IgG4 (P)</td><td> 1.01</td><td> 0.48</td><td> 1.07</td><td> 0.57</td><td> 1.34</td><td> 0 . 15</td>

<td>hPD-1 mAb 2 IgGl (AA)</td><td> 0.78</td><td> 0.12</td><td> 1.10</td><td> 0.38</td><td> 1.46</td><td> 0.53</td>

<td>control IgG</td><td> 0.39</td><td> 0.08</td><td> 0.27</td><td> 0.08</td><td> 0.34</td><td> 0 . 13</td>

<td>No antibody</td><td> 0.34</td><td> 0.11</td><td> 0.31</td><td> 0.03</td><td> 0.28</td><td> 0 . 08</td>

cnan Ln/zznz/E/YiAi

Results from two sensitive donors

[00462] The results of these studies demonstrate that PD-1 mAb 2, PD-1 mAb 7, PD-mAb 9 antibodies

1, and PD-1 mAb 15 dramatically enhanced the production of IFNy (Figures 14 and 15A, and Tables 11-13), and TNFα (Figure 15B) from SEB-stimulated PBMCs after restimulation. Furthermore, the combination of anti-PD-1 antibodies with anti-LAG-3 antibodies resulted in a further enhancement of cytokine release (Figure

14) from PBMCs stimulated with SEB after restimulation. In particular, the combination of PD1 mAb 2, PD-1 mAb 7, PD-1 mAb 9, or PD-1 mAb 15 with the single anti-LAG3 antibody LAG-3 mAb 1 provided the greatest enhancement .

Example 5

309

Binding Studies of PD-1 x LAG-3 Bispecific Molecules [00463] A number of PD-1 x LAG-3 bispecific molecules were generated, including Fe Region-containing diabodies comprising three, four, and five chains and a bispecific antibody. Four diabodies that had four chains and comprised heterodimer-promoting E/K helix domains were generated and the designations DART A, DART B, DART C, and DART I were agreed upon. Four diabodies were generated that had four chains and comprised CH1/CL domains and the designations DART D, DART E, DART J, and DART 1 were agreed upon. Two diabodies were generated that had five chains and comprised promoter E/K helix domains. of heterodimers and CH1/CL domains were generated and the designations DART F, and DART G were agreed upon. A diabody that had three chains and comprised heterodimer-promoting E/K helix domains was generated and the designation DART H was agreed upon. A bispecific antibody having four chains was generated and the designation BSAB A was agreed upon. The structure and amino acid sequences of these bispecific PD-1 x LAG-3 molecules are provided above and summarized in Table 14 below.

cnan Ln/zznz/E/YiAi

310

<td colspan="6">Table 14</td>

<td>Name</td><td>original mAbs</td><td>Faith*</td><td>Chains</td><td>SEQ ID NOs:</td><td>Other components</td>

<td>DART A</td><td>hPD-1 mAb 7(1.2) hLAG-3 mAb 1(1.4)</td><td>IgG4 (YTE)</td><td> 4</td><td>267 (Xi=A; x₂=Y; x₃=T; x₄=E) and 268</td><td>E/K propellers; see Figure 3B</td>

<td>DART B</td><td>hPD-1 mAb 7(1.2) hLAG-3 mAb 1(1.3)</td><td>IgG4 (YTE)</td><td> 4</td><td>267 (Xt=G; x₂=Y; x₃=T; x₄=E) and 268</td><td>E/K propellers; see Figure 3B</td>

<td>DART C</td><td>hPD-1 mAb 7(1.2) hLAG-3 mAb 1(1.3)</td><td>IgG4</td><td> 4</td><td>267 (Xi=G; x₂=M; x₃=S; x₄=T) and 268</td><td>E/K propellers; see Figure 3B</td>

<td>DART D</td><td>hPD-1 mAb 7(1.2) hLAG-3 mAb 1(1.4)</td><td>IgG4 (YTE)</td><td> 4</td><td>269 ​​and 270</td><td>CL/CH1; see Figure 3C</td>

<td>TO GIVE YOU</td><td>hPD-1 mAb 7(1.2) hLAG-3 mAb 1(1.4)</td><td>IgG4 (YTE)</td><td> 4</td><td>271 and 272</td><td>CL/CH1; see Figure 3C</td>

<td>DART F</td><td>hPD-1 mAb 7(1.2) hLAG-3 mAb 1(1.4)</td><td>IgGl (AA/YTE)</td><td> 5</td><td>273,274,275, and 27 6</td><td>CL/CH1 and E/K Helices; see Figure 5</td>

<td>DART G</td><td>hPD-1 mAb 7(1.2) hLAG-3 mAb 1(1.4)</td><td>IgGl (AA/YTE)</td><td> 5</td><td>277, 278, 279, and 280</td><td>CL/CH1 and E/K Helices; see Figure 5</td>

<td>DART H</td><td>hPD-1 mAb 7(1.2) hLAG-3 mAb 1(1.4)</td><td>IgGl (AA)</td><td> 3</td><td>281,282, and 283</td><td>E/K propellers; see Figure 4A</td>

<td>DART I</td><td>hPD-1 mAb 7(1.2) hLAG-3(1.1) mAb 6</td><td>IgG4 (YTE)</td><td> 4</td><td>290 and 291</td><td>E/K propellers; see Figure 3B</td>

<td>DART J</td><td>hPD-1 mAb 7(1.2) hLAG-3(1.1) mAb 6</td><td>IgG4 (YTE)</td><td> 4</td><td>292 and 293</td><td>CL/CH1; see Figure 3C</td>

<td>DART 1</td><td>PD-1 MAb A LAG-3 MAb A</td><td>IgGl (AA)</td><td> 4</td><td>284 and 285</td><td>CL/CH1; see Figure 3C</td>

<td>BSAB A</td><td>hPD-1 mAb 7(1.2) hLAG-3 mAb 1(1.4)</td><td>IgGl (AA)</td><td> 4</td><td>286, 287, 288, and 289</td><td>Fe region of mAb with designed charge</td>

CbQb ίη/ΖΖΠΖ/Ε/ΥΙΛΙ

Φ Molecules that incorporate Fe Regions of IgG4 also incorporate a stabilized IgG4 hinge region.

[00464] Additional PD-1 x LAG-3 bispecific molecules comprising alternative PD-1 and/or LAG-3 binding sites to the epitope could be easily generated by incorporating different VH and VL Domains. Similarly, molecules that bound an antigen other than LAG-3 were able to cnan Ln/zznz/E/YiAi

311 be generated by incorporating the VH and VL that had the desired specificity.

[00465] The binding saturation profiles of the PD-1 X LAG-3 specific diabody constructs: DART A, DART B, DART D, DART E, DART F, DART G, DART H, DART I, and DART 1; anti-PD-1 antibodies: hPD-1 mAb 7(1.2) IgG4 (P), hPD-1 mAb 7(1.2) IgGl (AA), PD-1 mAb A IgGl (AA) and PD-1 mAb A IgG4 (P); and anti-LAG-3 antibodies: IgG4 (P) of mAb 1(1.4) of hLAG-3, IgG4 (P) of mAb A of hLAG-3, IgGl (AA) of mAb 1(1.4) of hLAG-3 , and hLAG-3 mAb A (1.4) IgGl (AA) were examined essentially as described above. PD-1 x LAG-3 bispecific diabody constructs were tested for binding of PD1 and LAG-3, while anti-PD-1 and antiLAG-3 antibodies were only tested for binding of their respective antigens. For those studies, NSO cells expressing PD-1 or LAG-3 were used. Diabodies and antibodies (170.0-0.013 μΜ or 85.0-0.0021 μΜ (serial four-fold dilutions) were used. EC50 and EC90 values ​​were determined and presented in Tables 15-16. The sample mean (SM) was provided. ) and the standard deviation (SD. σ) where 2 or more separate experiments were performed.

312 cnan Ln/zznz/E/YiAi

<td colspan="5">Table 15</td>

<td rowspan="3">Molecule</td><td colspan="4">PD-1 Saturation Binding</td>

<td colspan="2">EC50 (μΜ)</td><td colspan="2">EC90 (μΜ)</td>

<td>YE</td><td>SD σ</td><td>YE</td><td>SD σ</td>

<td>DART A</td><td> 1.9297</td><td> 0.4324</td><td> 9.6027</td><td> 0.4801</td>

<td>DART B</td><td> 1.7640®</td><td></td><td> 12.2700®</td><td></td>

<td>DART D</td><td> 2.2267</td><td> 0.4140</td><td> 10.9313</td><td> 2.6351</td>

<td>TO GIVE YOU</td><td> 3.2180</td><td> 0.5742</td><td> 23.840</td><td> 3.2385</td>

<td>DART F</td><td> 1.4320®</td><td></td><td> 14.5800®</td><td></td>

<td>DART G</td><td> 1.1488</td><td> 0.6227</td><td> 3.4220</td><td> 2.4600</td>

<td>DART H</td><td> 4.5310®</td><td></td><td> 22.6600®</td><td></td>

<td>DART I</td><td> 1.3232</td><td> 0.4890</td><td> 7.8135</td><td> 4.0821</td>

<td>DART 1</td><td> 2.1329</td><td> 1.4850</td><td> 13.8113</td><td> 9.0256</td>

<td>hPD-1 mAb 7(1.2) IgG4 (P)</td><td> 1.2083</td><td> 0.8112</td><td> 3.9340</td><td> 1.8746</td>

<td>PD-1 mAb A IgG4 (P)</td><td> 2.3470</td><td> 1.2362</td><td> 22.7770</td><td> 15.0690</td>

<td>hPD-1 mAb 7(1.2) IgGl (AA)</td><td> 1.0879</td><td> 0.3958</td><td> 7.4153</td><td> 3.0794</td>

<td>PD-1 mAb A IgGl (AA)</td><td> 1.6733</td><td> 0.5464</td><td> 9.9543</td><td> 6.6569</td>

§ results from a single experiment

<td colspan="5">Table 16</td>

<td rowspan="3">Molecule</td><td colspan="4">LAG-3 Union Saturation</td>

<td colspan="2">EC50 (μΜ)</td><td colspan="2">EC90 (μΜ)</td>

<td>YE</td><td>SD σ</td><td>YE</td><td>SD σ</td>

<td>DART A</td><td> 0.8402</td><td> 0.2231</td><td> 4.4448</td><td> 2.4770</td>

<td>DART B</td><td> 1.0750®</td><td></td><td> 9.8580®</td><td></td>

<td>DART D</td><td> 0.8985</td><td> 0.5326</td><td> 5.7967</td><td> 4.7329</td>

<td>TO GIVE YOU</td><td> 0.9250</td><td> 0.8075</td><td> 5.6450</td><td> 5.6809</td>

<td>DART F</td><td> 5.0090</td><td> 0.5770</td><td> 19.3350</td><td> 4.7447</td>

<td>DART G</td><td> 0.9396</td><td> 0.3045</td><td> 8.5507</td><td> 4.7448</td>

<td>DART H</td><td> 2.3840®</td><td></td><td> 9.7810</td><td> 4.2412</td>

<td>DART I</td><td> 0.5321</td><td> 0.0547</td><td> 4.198</td><td> 3.2188</td>

<td>DART 1</td><td> 20.0233</td><td> 2.1454</td><td> 115.97</td><td> 15.2425</td>

<td>hLAG-3 mAb 1(1.4) IgG4 (P)</td><td> 1.0057</td><td> 0.1969</td><td> 5.1360</td><td> 4.7904</td>

<td>LAG-3 mAb A IgG4 (P)</td><td> 0.5968</td><td> 0.1376</td><td> 2.0833</td><td> 0.3244</td>

<td>hLAG-3 mAb 1(1.4) IgGl (AA)</td><td> 0.6069</td><td> 0.3430</td><td> 3.6373</td><td> 2.4762</td>

<td>LAG-3 mAb A IgGl (AA)</td><td> 0.4523</td><td> 0.1660</td><td> 2.0187</td><td> 0.7035</td>

§ results from a single experiment

313

[00466] Binding saturation studies demonstrated that PD-1 x LAG-3 bispecific diabody constructs retained binding to PD-1 and had binding profiles that were similar to the binding profiles of anti- Original PD-1. Similarly, PD-1 x LAG-3 bispecific diabody constructs retained binding to LAG-3 and, with the exception of DART 1, had binding profiles that are similar to the binding profiles of anti-LAG antibodies. -3 originals.

cnan Ln/zznz/E/YiAi

Example 6

Inhibition Studies of PD-1 X LAG-3 Bispecific Molecules

[00467] The capacity of PD-1 x LAG-3 bispecific molecules: DART A, DART B, DART D, DART E, DART F, DART G, DART H, DART I, DART 1 and BSAB A; and anti-PD-1 antibodies: IgG4 (P) of mAb 7(1.2) of hPD-1, IgGl (AA) of mAb 7(1.2) of hPD-1, IgGl (AA) of mAb A of PD-1 and PD-1 mAb A IgG4 (P), to block binding to human PD-L1 (shPD-L1) and human PD-L2 (shPD-L2) on PD-1 expressed on the cell surface NSO was examined essentially as described above. Diabodies and antibodies were used at 33,750,002 μΜ or 107.5-0.0001 μΜ (serial four-fold dilutions).

[00468] The IC50 and IC90 values ​​were determined and

314 presented in Table 17. Sample mean (SM) and standard deviation (SD σ) were provided where 2 or more separate experiments were performed.

cnan Ln/zznz/E/YiAi

<td colspan="9">Table 17</td>

<td rowspan="3">Molecule</td><td colspan="4">Blockade of the sPDLl/PD-1 junction</td><td colspan="4">Blockade of the sPDL2/PD-1 junction</td>

<td>IC50</td><td>(μΜ)</td><td colspan="2">IC90 (pM)</td><td colspan="2">IC50 (pM)</td><td colspan="2">IC90 (pM)</td>

<td>YE</td><td>SD or</td><td>YE</td><td>SD or</td><td>YE</td><td>SD or</td><td>YE</td><td>SD or</td>

<td>DART A</td><td> 0.9645</td><td> 0.1485</td><td> 5.6312</td><td> 1.5247</td><td> 1.6273</td><td> 0.4285</td><td> 6.9335</td><td> 3.9849</td>

<td>DART B</td><td> 1.1515</td><td> 0.0007</td><td> 4.8615</td><td> 0.2199</td><td> 2.1150</td><td> 0.3154</td><td> 7.9550</td><td> 0.0933</td>

<td>DART D</td><td> 1.5548</td><td> 0.1692</td><td> 7.8950</td><td> 2.5135</td><td> 3.1255</td><td> 0.5869</td><td> 9.2973</td><td> 5.5426</td>

<td>TO GIVE YOU</td><td> 1.6533</td><td> 0.3307</td><td> 7.8470</td><td> 1.1642</td><td> 2.9460</td><td> 0.7736</td><td> 6.6135</td><td> 0.0177</td>

<td>DART F</td><td> 0.5697</td><td> 0.1729</td><td> 2.0360</td><td> 0.1174</td><td> 0.8389</td><td> 0.0846</td><td> 1.7995</td><td> 0.2171</td>

<td>DART G</td><td> 1.6013</td><td> 0.3581</td><td> 8.1953</td><td> 1.5708</td><td> 2.5540</td><td> 0.7891</td><td> 7.4810</td><td> 0.2333</td>

<td>DART H</td><td> 3.3950</td><td> 0.1018</td><td> 18.640</td><td> 9.5742</td><td> 6.2065</td><td> 3.6847</td><td> 29.395</td><td> 3.8679</td>

<td>DART I</td><td> 0.8363</td><td> 0.1302</td><td> 5.3115</td><td> 0.3125</td><td> 1.286</td><td> 0.3125</td><td> 6.2485</td><td> 1.3951</td>

<td>DART 1</td><td> 1.7467</td><td> 0.3097</td><td> 5.4533</td><td> 1.0214</td><td> 2.8355</td><td> 1.8250</td><td> 7.2735</td><td> 3.9831</td>

<td>BSAB A</td><td> 2.1590</td><td> 0.3097</td><td> 11.075</td><td> 0.8132</td><td> 4 . 8775</td><td> 0.5438</td><td> 15.580</td><td> 1.3294</td>

<td>IgG4 (P) mAb 7 (1.2) hPD-1</td><td> 0.5186</td><td> 0.1668</td><td> 3.8050</td><td> 1.2227</td><td> 1.0425</td><td> 0.2563</td><td> 3.4880</td><td> 0.5459</td>

<td>IgG4 (P) PD-1 mAb A</td><td> 0.9209</td><td> 0.3256</td><td> 4.3023</td><td> 0.7069</td><td> 1.3859</td><td> 0.3882</td><td> 5.1675</td><td> 0.2943</td>

<td>hPD-1 mAb 7(1.2) IgGl (AA)</td><td> 0.7320</td><td> 0.2337</td><td> 3.2048</td><td> 1.1479</td><td> 0.9769</td><td> 0.2893</td><td> 2.8437</td><td> 1.4801</td>

<td>IgGl (AA) PD-1 mAb A</td><td> 1.0765</td><td> 0.2393</td><td> 5.2775</td><td> 0.9933</td><td> 1 . 9510</td><td> 0.8814</td><td> 5.0880</td><td> 1.3831</td>

[00469]

cnan Ln/zznz/E/YiAi ligand binding inhibition studies

315 demonstrated that PD-1 x LAG-3 bispecific diabody constructs retained the ability to inhibit the binding of sPD-Ll and sPD-L2 to PD-1 on the cell surface.

[00470] Additionally, the capacity of PD-1 x LAG-3 bispecific molecules was examined: DART A, DART B, DART D, DART E, DART F, DART G, DART H, DART I, DART 1 and BSAB TO; and the anti-LAG-3 antibodies: IgG4 (P) of mAb 1 of hLAG-3(1.4), IgG4 (P) of mAb A of hLAG-3, IgGl (AA) of mAb 1 of hLAG3(1.4), and hLAG-3(1.4) mAb A IgGl (AA), to block the binding of human LAG-3 to natural MHC Class II on the surface of Daudi cells. Briefly, each bispecific molecule of PD-1 x LAG-3 and control anti-LAG3 antibody was mixed with a soluble biotinylated human Fc-LAG3 (shLAG-3) fusion protein, (at 0.5 pg/mL) and where incubated separately with MHC II-positive Daudi cells (2.5 x 10⁶ cells). The amount of LAG-3 bound on the surface of Daudi cells was determined using a PE-conjugated Streptavidin secondary antibody by FACS analysis. Diabodies and antibodies were used at 27.5-0.026 μΜ (serial two-fold dilutions) or 107.50.0001 μΜ (serial four-fold dilutions), or 35-0.002 μΜ (serial four-fold dilutions).

[00471] IC50 and IC90 values ​​were determined and presented in Table 18. The sample mean (SM) and standard deviation (SD σ) were provided where

316 performed 2 or more separate experiments.

cnan Ln/zznz/E/YiAi

<td colspan="5">Table 18</td>

<td rowspan="3">Molecule</td><td colspan="4">shLAG-3/MHC class II junction block</td>

<td colspan="2">EC50 (μΜ)</td><td colspan="2">EC90 (μΜ)</td>

<td>YE</td><td>SD σ</td><td>YE</td><td>SD σ</td>

<td>DART A</td><td> 1.3835</td><td> 1.6465</td><td> 8.396102</td><td> 8.3962</td>

<td>DART B</td><td> 0.4081</td><td> 0.1104</td><td> 3.0645</td><td> 0.3924</td>

<td>DART D</td><td> 1.1843</td><td> 1.1398</td><td> 8.0041</td><td> 7.3317</td>

<td>TO GIVE YOU</td><td> 3.2706</td><td> 2.9177</td><td> 28.9683</td><td> 24.1694</td>

<td>DART F</td><td> 1.5347</td><td> 1.2674</td><td> 10.3920</td><td> 11.2555</td>

<td>DART G</td><td> 2.0618</td><td> 3.3552</td><td> 11.4422</td><td> 12.4964</td>

<td>DART H</td><td> 2.8967</td><td> 4.9817</td><td> 17.2533</td><td> 21.1420</td>

<td>DART I</td><td> 0.4864</td><td> 0.1549</td><td> 2.339</td><td> 1.1780</td>

<td>DART 1</td><td> 15.9610</td><td> 14.0883</td><td> 87.1486</td><td> 109.533</td>

<td>BSAB A</td><td> 0.7101</td><td> 0.0571</td><td> 7.2470</td><td> 1.0706</td>

<td>hLAG- mAb 1 IgG4 (P) 3(1.4)</td><td> 0.4815</td><td> 0.2176</td><td> 3.4837</td><td> 1.7564</td>

<td>LAG- mAb A IgG4 (P) 38</td><td> 0.7011</td><td> 0.1900</td><td> 2.4232</td><td> 0.3481</td>

<td>mAb 1 IgGl (AA) hLAG-3(1.4)</td><td> 0.3637</td><td> 0.1409</td><td> 9.4422</td><td> 7.9319</td>

<td>LAG-mAb A IgGl (AA) 3</td><td> 0.5923</td><td> 0.3407</td><td> 2.1451</td><td> 1.1139</td>

[00472] Ligand binding inhibition studies demonstrated that bispecific diabody constructs of

PD-1 x LAG-3 retained the ability to inhibit the binding of a shLAG-3-Fc fusion protein to MHC class II in the

317 cell surface. With the exception of DART 1, the PD-1 x LAG-3 bispecific molecules had similar inhibition profiles as the original anti-LAG-3 antibodies.

cnan Ln/zznz/E/YiAi

Example 7

Blockade of the PD-1/PD-L1 Endpoint by PD-1 x LAG-3 Bispecific Molecules

[00473] The capacity of PD-1 x LAG-3 bispecific molecules: DART A, DART B, DART D, DART E, DART F, DART G, DART H, DART I, DART 1 and BSAB A; and anti-PD-1 antibodies: IgG4 (P) of mAb 7(1.2) of hPD-1, IgGl (AA) of mAb 7(1.2) of hPD-1, IgGl (AA) of mAb A of PD-1 and PD-1 mAb A IgG4 (P), to antagonize the PD-1/PD-L1 axis (i.e., block PD-1/PD-L1 interaction and prevent deregulation of T cell responses). examined in a Jurkat-luc2-NFAT/CHO-PD-Ll luciferase reporter assay (using CHO/PD-L1 cells and Jurkat MNFAT-luc2/PD-l cells) essentially as described above. Diabodies and antibodies were used at 100-0.0065 μΜ (serial four-fold dilutions) or 100-0.0013 μΜ (serial five-fold dilutions).

[00474] The IC50 and IC90 values ​​were determined and presented in Table 19. The sample mean (SM) and standard deviation (SD σ) were provided where 2 or more separate experiments were performed.

318 cnan Ln/zznz/E/YiAi

<td colspan="5">Table 19</td>

<td rowspan="3">Molecule</td><td colspan="4">Reporter signage</td>

<td colspan="2">IC50 (μΜ)</td><td colspan="2">IC90 (μΜ)</td>

<td>YE</td><td>SD σ</td><td>YE</td><td>SD σ</td>

<td>DART A</td><td> 0.8804</td><td> 0.1949</td><td> 7.9115</td><td> 1.3232</td>

<td>DART B</td><td> 1.079</td><td> 0.1535</td><td> 7.5413</td><td> 3.1483</td>

<td>DART D</td><td> 1.4044</td><td> 0.2584</td><td> 12.0786</td><td> 3.6616</td>

<td>TO GIVE YOU</td><td> 1.4060</td><td> 0.1222</td><td> 13.7867</td><td> 1.4981</td>

<td>DART F</td><td> 0.3404</td><td> 0.0103</td><td> 1.8710</td><td> 0.481</td>

<td>DART G</td><td> 0.6914</td><td> 0.0206</td><td> 4.2090</td><td> 0.7331</td>

<td>DART H</td><td> 36.6167</td><td> 20.8078</td><td> 968.300</td><td> 811.8471</td>

<td>DART I</td><td> 1.3335</td><td> 0.3641</td><td> 12.146</td><td> 6.8787</td>

<td>DART 1</td><td> 11.8807</td><td> 3.4905</td><td> 1048.2000</td><td> 1508.9992</td>

<td>BSAB A</td><td> 9.7825</td><td> 1.0288</td><td> 113.3350</td><td> 22.2951</td>

<td>hPD-1 mAb 7(1.2) IgG4 (P)</td><td> 0.6460</td><td> 0.3035</td><td> 6.0736</td><td> 2.5513</td>

<td>mAb A IgG4 (P) PD-1</td><td> 1.328</td><td> 0.7439</td><td> 16.5138</td><td> 9.7149</td>

<td>hPD-1 mAb 7(1.2) IgGl (AA)</td><td> 0.5214</td><td> 0.1541</td><td> 4.7592</td><td> 2.1044</td>

<td>PD-1 mAb A IgGl (AA)</td><td> 1.4514</td><td> 1.0049</td><td> 35.7382</td><td> 40.9858</td>

[00475] Reporter signaling studies demonstrated that the majority of PD-1 x LAG-3 bispecific diabody constructs retained the ability to inhibit sPD-L1 binding to PD-1 on the cell surface.

The tetravalent PD-1 x LAG-3 bispecific diabody constructs, DART A, DART B, DART D, DART-E, DART F, DART G, and DART I were the strongest inhibitors in this assay. Similar results were obtained for several of these 10 bispecific constructs examined in a PD-L2 reporter assay.

319

Example 8 Functional Activity of PD-1 x LAG-3 Bispecific Molecules

[00476] The ability of PD-1 x LAG-3 bispecific molecules to enhance cytokine release through titration inhibition was examined in SEB-stimulated PBMCs after restimulation essentially as described above, except where observed.

[00477] In initial studies, the capacity of PD-1 x LAG-3 bispecific molecules was examined: DART A, DART D, DART E, DART F, DART G, DART H; and anti-PD-1 and anti-LAG antibodies: IgG4 (P) of PD-1 mAb A and IgG4 (P) of LAG-3 mAb A, alone or in combination to enhance cytokine release through checkpoint inhibition. In these studies, PD-1 x LAG-3 bispecific molecules and antibodies were used at a total concentration of 3.125, 12.5, or 50 nM, and PBMCs were stimulated with 0.2 ng/mL SEB (previous studies used 0.1 ng/mL). For these studies, a combination of antibodies was provided at one-half the total concentration, (i.e., 1.563, 6.25, or 25 nM). Figures 16A and 16B show the IFNy secretion profiles from SEB-stimulated PBMCs from two representative sensitive donors, D: 35644 and D: 59697, respectively.

cnan Ln/zznz/E/YiAi

320

[00478] As noted, not all donors responded to SEB at 0.1 or 0.2 ng/mL. To enhance SEB stimulation of PBMCs from a large number of donors, SEB was used at a high concentration of 85 ng/mL, or at a medium concentration of 0.5 ng/mL in additional studies. At these concentrations, SEB stimulation was more robust across more donors, although donor-to-donor variability could still be observed.

[00479] In one such study, the capacity of PD-1 x LAG-3 bispecific molecules was examined: DART A, DART B; anti-PD-1 antibody: LAG-3 mAb 7(1.2) IgG4 (P); anti-LAG-3 antibody: LAG-3 mAb 1(1.4) IgG4 (P); and the combination of: IgG4 (P) of PD-1 mAb A and IgG4 (P) of LAG-3 mAb A, to intensify cytokine release through checkpoint inhibition. In these assays, PD-1 x LAG-3 bispecific molecules and antibodies were used at a concentration of 0.019, 0.078, 0.3125, 1.25, 5, or 20 nM and PBMCs were stimulated with 85 ng/mL SEB. For this assay where a combination of the antibodies is used, each antibody was provided at the indicated concentration and thus, the total antibody concentration was twice the concentration used for each antibody (i.e., 0.038, 0.156, 0.625, 2.5 , 10, or 40 nM). Figures 17A and 17B show the IFNy secretion profiles from the cnan ίη/ζζηζ/Ε/γίΛΐ

321

PBMC stimulated with SEB from two representative donors, D: 55515 and D: 54024, respectively.

[00480] In another study, PD-1 x LAG-3 bispecific molecules were examined: DART A, DART B, DART C; anti-PD-1 antibody: hPD-1 mAb 7(1.2) IgG4 (P); anti-LAG-3 antibody: LAG-3 mAb 1(1.4) IgG4 (P); and the combination of: IgG4 (P) of PD-1 mAb A and IgG4 (P) of LAG-3 mAb A, to intensify cytokine release through checkpoint inhibition. In these assays, PD-1 x LAG-3 bispecific molecules and antibodies were used at a total concentration of 0.048, 0.195, 0.78, 3.125, 12.5, or 50 nM and PBMCs were stimulated with 0.5 ng/mL SEB. For these studies where a combination of the antibodies is used, each antibody was provided at one-half the total concentration (i.e., 0.024, 0.098, 0.39, 1.563, 6.25, or 25 nM). Figures 18A and 18B show the IFNy secretion profiles of PBMCs stimulated with SEB from two representative donors, D: 20990 and D: 54947, respectively).

[00481] In a further study, the release of the cytokine IL-2 is examined. Specifically, PD-1 x LAG-3 bispecific molecules were examined: DART D, DART H; anti-PD-1 antibodies: IgG4 (P) of mAb A of PD-1, IgG4 (P) of mAb 7(1.2) of hPD-1; anti-LAG-3 antibodies: IgG4 (P) of LAG-3 mAb A and IgG4 (P) of LAG-3 mAb 1(1.4); and the

CbQb ίη/77Ω7/Β/ΥΙΛΙ

322 combination of: IgG4 (P) of mAb A of PD-1 and IgG4 (P) of mAb A of LAG-3, and IgG4 (P) of mAb 7(1.2) of hPD-1 and IgG4 (P) of mAb 1 (1.4) of LAG-3, to enhance IL-2 release through checkpoint inhibition. In these assays, PD-1 x LAG-3 bispecific molecules and antibodies were used at a total concentration of 3.125, 12.5, or 50 nM and PBMCs were stimulated with the high concentration of 85 ng/mL SEB. For these studies, where a combination of the antibodies was used, each antibody was provided at one-half the total concentration (i.e., 1.563, 6.25, or 25 nM). Figure 19 shows the IL-2 secretion profile from SEB-stimulated PBMCs from a representative donor (D: 54024). [00482] In additional studies, PD-1 x LAG-3 bispecific molecules were examined: DART B, and DART I; anti-PD-1 antibodies: IgG4 (P) of mAb A of PD-1, and IgG4 (P) of mAb 7(1.2) of hPD-1; anti-LAG-3 antibodies: IgG4 (P) of mAb A of LAG-3, IgG4 (P) of mAb 1(1.4) of hLAG-3, and IgG4 (P) of mAb 6(1.1) of hLAG-3 ; and the combinations of: IgG4 (P) of mAb A of PD-1 and IgG4 (P) of mAb A of LAG-3, IgG4 (P) of mAb 7(1.2) of hPDl and IgG4 (P) of mAb 1( 1.4) of hLAG-3, and IgG4 (P) of mAb 7(1.2) of hPDl and IgG4 (P) of mAb 6(1.1) of hLAG-3 to enhance cytokine release through checkpoint inhibition . In these assays, bispecific PD-1 x LAG-3 molecules and antibodies were

CbQb ίη/77Π7/Ε/ΥΙΛΙ

323 used at a concentration of 0.0061, 0.024, 0.09, 0.39, 1.56, 6.25 or 25 nM and PBMCs were stimulated with 0.5 ng/mL SEB. For these studies, where a combination of the antibodies was used, each antibody was provided at the indicated concentration and thus the total antibody concentration was twice the concentration used for each antibody (i.e., 0.0122, 0.048, 0.18, 0.78 , 3.12, 12.5 or 50 nM). Figure 20 shows the IFNy secretion profiles from SEB-stimulated PBMCs from a representative donor D: 56041).

[00483] The ability of the PD-1 x LAG-3 bispecific molecule DART I; the combination of anti-PD-1 PD-1 mAb A IgG4 antibody and anti-LAG-3 LAG-3 mAb A IgG4 (P) antibody; and a negative control antibody to enhance antigen-specific T cell responses were examined using a Tetanus Toxoid Recovery Assay. In particular, the response of enhanced antigen-specific secretion of cytokines was measured using tetanus toxoid as a recovery antigen in a co-culture assay system. Briefly, CD4 memory T cells (0.5 1.0X10⁵ cells/well) were isolated using negative selection isolation kits (Miltenyi Biotec, San Diego, CA and Invitrogen, Carlsbad, CA) from human peripheral blood and cultured for 5-7 days with cnan Ln/zznz/E /YiAi

324 irradiated monocytes (0.01 - 0.05X10⁵ cells/well, 3500 rads) from the same donor in the presence or absence of 5 pg/mL recovery antigen tetanus toxoid (TTd) and dilution (starting at 25nM) of DART I, PD-1 mAb A IgG4 + IgG4 (P) from LAG-3 mAb A, or a control isotype. In parallel plates, proliferation was measured through the incorporation of tritiated thymidine and IL-2 and IFNy were measured using ELISA (R&amp;D systems, Minneapolis, MN) at days 57. Figures 21A-D show the secretion profiles of IFNy (Figures 21A, 21C) and IL-2 (Figures 21B, 21D) on day 7, from two representative donors (D50702 and D54267).

[00484] The results of these studies demonstrated that bispecific PD-1 x LAG-3 molecules dramatically enhanced the production of IFNy (Figures 16A-16B, 17A17B, 18A-18B, 20), and IL-2 (Figure 19) from PBMCs stimulated with SEB after restimulation. Furthermore, bispecific PD-1 x LAG-3 molecules dramatically enhanced IFNy production (Figures 21A and 21C) from CD4 memory T cells stimulated with tetanus toxoid. In particular, bispecific tetravalent PD-1 x LAG-3 molecules provided greater enhancement than the combination of anti-PD-1 antibodies with anti-LAG-3 antibodies.

cnan Ln/zznz/E/YiAi

Example 9

325

Pharmacokinetics of PD-1 x LAG-3 Bispecific Molecules

[00485] The pharmacokinetics of a representative PD-1 x LAG-3 bispecific molecule, DART I, and a representative anti-PD-1 antibody, PD-1 mAb A, were examined in Cynomolgus monkeys. Briefly, two cynomolgus monkeys (one male and one female) were infused with a single dose of DART I (5 mg/kg) or PD-1 mAb A (10 mg/kg) and the concentration of the molecules was monitored. in serum over time using a stratification ELISA assay. Briefly, maxisorb 96-well assay plates were coated with soluble human PD-1 (shPD-1), blocked with bovine serum albumin, washed, and incubated with calibration standards, quality control standards, and serum samples. diluted. The amount of captured PD-1 DART I and mAb A was assessed by the sequential addition of a secondary goat anti-human IgG Fc-biotin and horseradish peroxidase streptavidin (SA-HRP). HRP activity was detected using TMB substrate. All samples were analyzed on a microplate reader (SpectraMax M2e, Molecular Device, Sunnyvale, CA) and the OD signals produced by the standard calibrators were used in the four-parameter logistic model using SofitMax Pro software (Version 5.4, Molecular Devices ). PD-1 mAb A concentrations, or DART I were determined from cnan Ln/zznz/E/YiAi

CbQb ίη/77Π7/Ε/ΥΙΛΙ

326 the interpolation of the OD signal data of the samples with the equation that described the standard curve. The lower limit of quantification (LLOQ) for this assay was estimated to be 9,775 ng/mL.

[00486] Figure 22 shows serum concentration over time, lines represent the mean of male (filled symbols) and female (open symbols) monkeys infused with DART I (solid line, triangles) or mAb A PD1 (dotted line, circles). These data demonstrated that the pharmacokinetics of a PD-1 x LAG-3 bispecific molecule were comparable to those of an anti-PD-1 antibody in cynomolgus monkeys.

Example 10

Toxicology Study of PD-1 Antibodies and PD-1 x LAG-3 Bispecific Molecules

[00487] The safety profile of a representative anti-PDl antibody, hPD-1 mAb 7(1.2) IgG4(P), and a representative PD1 x LAG3 bispecific molecule, DART I, was evaluated in a dosing study without GLP (Good Laboratory Practices) in cynomolgus monkeys.

[00488] In this study, the potential toxicity and toxicokinetics of anti-PD-1 antibody (hPD-1 mAb 7(1.2) IgG4(P) were evaluated, when administered by multiple intravenous infusions. In addition, we also evaluated the

327 Potential toxicity and pharmacokinetics of cnan Ln/zznz/E/YiAi DART molecule

PD-1 x LAG-3 (DART I) when administered as a single intravenous infusion. The study design is presented in

Table 20.

<td colspan="8">Table 20</td>

<td rowspan="2">Group No.</td><td rowspan="2">Test material</td><td rowspan="2">Dose level (mg/kg)</td><td rowspan="2">Dosing days</td><td rowspan="2">Dose volume</td><td rowspan="2">Dose (mg/mL)</td><td colspan="2">No. of Animals</td>

<td>Males</td><td>Females</td>

<td> 1</td><td>Control</td><td> 0</td><td> 1, 8,15</td><td> 5</td><td> 0</td><td>Yo^to</td><td>Yo^to</td>

<td>2A</td><td>hPD1 mAb 7 IgG4 (P) (1.2)</td><td> 1</td><td> 1, 8, 15</td><td> 5</td><td> 0.2</td><td>Yo^to</td><td>Yo^to</td>

<td>2B</td><td>hPD1 mAb 7 d.2 IgG4 (P)</td><td> 1</td><td> 1, 8, 15</td><td> 5</td><td> 0.2</td><td>Ib</td><td>l^b</td>

<td>3A</td><td>hPD-1 mAb 7(1.2) IgG4 (P)</td><td> 100</td><td> 1, 8, 15</td><td> 5</td><td> 20</td><td>Yo^to</td><td>Yo^to</td>

<td>3B</td><td>hPD-1 mAb 7(1.2) IgG4 (P)</td><td> 100</td><td> 1, 8, 15</td><td> 5</td><td> 20</td><td>l^b</td><td>l^b</td>

<td> 4</td><td>DART I</td><td> 5</td><td> 1</td><td> 5</td><td> 1</td><td>l^c</td><td>l^c</td>

<td colspan="8">^to Groups 1, 2A, and 3A were dosed beginning on Day 1 and necropsied 72 hours after their last (third) dose on Day 18.^b Groups 2B and 3B were dosed beginning on Day 1 and necropsied 7 days after their last (third) dose on Day 22.^c Group 4 was dosed beginning on Day 1 and 28 days after a single dose administration (on Day 29); then the animals returned to the colony.</td>

[00489] The following parameters and endpoints were evaluated in this study: clinical signs, body weight, food consumption, body temperature, clinical pathology parameters (hematology, coagulation, and clinical chemistry), bioanalysis and toxicokinetic parameters, analysis of anti-drug antibody, flow cytometry, cytokine, autopsy findings, organ weight, and histopathological examinations.

328

[00490] All animals survived until scheduled euthanasia on Day 18 or 22 or release from the study on Day 29. For hPD1 mAb 7(1.2) IgG4 (P), there were no changes related to the test article on clinical signs, feed intake, body weight, body temperature, hematology, coagulation, or clinical chemistry parameters, or findings at autopsy. On Days 18 and 22, increases in spleen weight and a dose-dependent mild to moderate lymphohistiocytic infiltrate of the red pulp were evident in animals receiving allocate hPD-1 mAb 7(1.2) IgG4(P). 100 mg/kg. When compared with surrounding lymphocytes, lymphohistiocytic cells had pale cytoplasm and irregular nuclei. Rare mitotic figures were evident. The infiltrate was a microscopic correlation of the increased weight of the spleen.

[00491] Serum concentration time profiles for animals provided by hPD-1 mAb 7(1.2) IgG4(P) showed the expected profile for an antibody in this species, with few exceptions. The slopes of the curves after the third dose fell more steeply than after the first dose for two animals in the 1 mg/kg dose group and two animals in the 100 mg/kg dose group, indicating possible emergence. of anti-drug antibodies (ADA) in subsequent cycles. cnan analysis Ln/zznz/E/YiAi

329 showed that 2/4 animals developed ADA in the 1 mg/kg group and 1/4 animals developed ADA in the 100 mg/kg group.

/*/*/* [00492] In conclusion, administration of hPD-Ι mAb 7(1.2) IgG4 (P) by intravenous infusion once a week for 3 weeks (Days 1, 8, and 15) was well tolerated in cynomolgus monkeys at levels of 1 and 100 mg/kg. A dose-dependent mild to moderate lymphohistiocytic cellular infiltrate of the splenic red pulp occurred in hPD-Ι mAb 7(1.2) IgG4 (P) at 1 and 100 mg/kg.

[00493] For DART I, there were no test article-related changes in clinical signs, food intake, body weight, body temperature, hematology, or coagulation parameters. DART I-related changes in clinical chemistry parameters included transient non-adverse elevations in aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) on Day 2. The mean AST change was 3.2x in vehicle-treated control animals and 7.8x at levels from the previous study, with levels above the control reference range 2. The average LDH change was 2.5x in vehicle-treated animals and 6.9x at previous study levels. Both parameters returned to near baseline levels on Day 8. In conclusion, simple administration of DART-I by intravenous infusion was well tolerated in cynomolgus monkeys at cnan Ln/zznz/E/YiAi

330 a level of 5 mg/kg.

cnan Ln/zznz/E/YiAi

Example 11

Single-Dose PK Study with Anti-PD-1 Antibodies

[00494] A single-dose PK study with selected toxicological checkpoints was carried out in cynomolgus monkeys. In this study, IgG4 (P) of hPD-1 mAb 7(1.2) was compared with two other anti-PDl IgG4 (P), mAbs k: IgG4 (P) of PD-1 mAb A and IgG4 (P ) of PD-1 mAb B. Each antibody was administered to 2 monkeys by intravenous infusion of 10 mg/kg over one hour (1 M, IF) and the animals were monitored for 65 days.

[00495] There were no clinical signs related to the test article, changes in body weight, food consumption, cytokine, or immunophenotyping associated with the administration of hPD-1 mAb 7(1.2) IgG4 (P) or IgG4 ( P) of PD-1 mAb A. Data were similar for PD-1 mAb B IgG4(P) with the exception that elevations in IL-5 were observed following administration of PD-1 mAb B IgG4(P).

[00496] Anti-PD-1 antibody binding to PD-1 on the surface of T cells was determined by flow cytometry using a competition method in which the mean fluorescence intensity (MFI) of IgG4 ( P) of fluorescently labeled hPD-1 mAb 7(1.2) that binds to

331 T cells in the absence (PBS control) or presence of excess competitor (unlabeled hPD-1 mAb 7(1.2) IgG4(P)) over the entire time course of blood samples collected from treated cynomolgus monkeys with IgG4 (P) of hPD-1 mAb 7(1.2), IgG4 (P) of PD-1 mAb A or IgG4 (P) of PD-1 mAb B. As shown in Figures 23A-23C, IgG4 (P) of hPD-1 mAb 7(1.2) and IgG4 (P) of PD-1 mAb B demonstrated prolonged binding to PD-1 on the surface of T cells. of CD4 + and CD8 + (binding to PD-1 maintained at > 80% for 28 days or more) (Figures 23A and 23C, respectively) compared to IgG4 (P) of PD-1 mAb A (binding to PD-1 maintained to > 80% for 21 days or less) (Figure 23B). For each of the anti-PD-1 antibodies, the T cell PD-1 binding data were correlated with their serum concentrations.

cnan Ln/zznz/E/YiAi

Example 12

Repeat Dose Toxicology Studies

[00497] To evaluate the safety in the toxicokinetic and pharmacodynamic profile of the therapeutic molecules of the present invention, an exemplary molecule (hPD-1 mAb 7(1.2) IgG4 (P)) was administered to cynomolgus monkeys and a dosage study with LPG (Good Laboratory Practices). In this study, four groups of animals (10 per group, 5 males, and 5 females) were

332 treated with hPD-1 mAb 7(1.2) IgG4 (P) or a control article, once a week by infusion at 3 dose levels. Animals were evaluated for any potential toxicity during a 4-week drug dosing period followed by monitoring during a 10-week drug-free period. The experimental design of this study is presented in Table 21. Animals were dosed once a week by a one-hour intravenous infusion using a calibrated infusion pump on Days of

Study 1, 8, 15 and 22. One male and one female from each group were sacrificed on Day 25, the remaining animals were sacrificed on Study Day 95. The effects of administration of IgG4(P) mAb were evaluated. 7(1.2) of hPD-1 in circulating leukocyte subpopulations, including PD-1 receptor occupancy on T lymphocytes. Additionally, anti-drug antibody (ADA) profiles were determined.

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333

<td colspan="9">Table 21</td>

<td rowspan="3">Group No.</td><td rowspan="3">Test Material^to</td><td rowspan="3">Dose Level (mg/kg)</td><td rowspan="3">Dose Volume (mL/kg)</td><td rowspan="3">Dose (mg/mL)</td><td colspan="4">No. of Animals^b</td>

<td colspan="2">Main Studio</td><td colspan="2">Recovery Study</td>

<td>M</td><td>F</td><td>M</td><td>F</td>

<td> 1</td><td>Control</td><td> 0</td><td> 5.88</td><td> 0</td><td> 3</td><td> 3</td><td> 2</td><td> 2</td>

<td> 2</td><td>hPD-1 mAb 7(1.2) IgG4 (P)</td><td> 10</td><td> 5.88</td><td> 1.7</td><td> 3</td><td> 3</td><td> 2</td><td> 2</td>

<td> 3</td><td>hPD-1 mAb 7(1.2) IgG4 (P)</td><td> 40</td><td> 5.88</td><td> 6.8</td><td> 3</td><td> 3</td><td> 2</td><td> 2</td>

<td> 4</td><td>hPD-1 mAb 7(1.2) IgG4 (P)</td><td> 150</td><td> 5.88</td><td> 25.5</td><td> 3</td><td> 3</td><td> 2</td><td> 2</td>

^to Control and hPD-1 mAb 7 (1.2) IgG4 (P) were administered weekly by intravenous infusion^b Six monkeys (3M/3F) per group underwent necropsy on Day 25, while the remaining monkeys in the¹⁰ recovery (2M/2F) underwent necropsy on Day 95

[00498] Once weekly intravenous (IV) infusions of hPD-1 mAb 7(1.2) IgG4 (P) at 0, 10, 40, and 15 150 mg/kg in cynomolgus monkeys were well tolerated and all Monkeys were scheduled for euthanasia on Days 25 or 95. There were no changes in clinical signs, food consumption, body weights, physical, ophthalmic, and neurological examinations, electrocardiology, body temperatures, respiratory rates, blood pressure and heart rates, coagulation parameters, clinical chemistry, and urinalysis, organ weight, or autopsy findings related to hPD-1 mAb 7(1.2) IgG4 (P).

[00499] hPD-1 mAb 7(1.2) IgG4 (P)-related changes in hematology parameters included reductions cnan ίη/ζζηζ/Ε/γίΛΐ

334 transients in lymphocyte titers. Lymphocyte titers were moderately reduced compared to the previous study (Day 1 predose) on Day 2 (23 hours post-infusion) in males and females at > 10 mg/kg, statistically significant for males at 10 and 40 mg/kg and females at 40 and 150 mg/kg compared to controls. Lymphocyte titers returned to near pre-study levels at Day 8 predosing but were slightly reduced for some males and females individually at all dose levels (0.47x to 0.68x pre-study) on Day 9 ( 23 hours after infusion). Lymphocyte titers increased before dosing on Days 15 and 22, but decreased for some males and females individually (0.36x to 0.54x from the previous study) on Days 16 and 23 (23 hours after infusion).

[00500] A transient dose-independent drop in circulating immune cell populations, including total leukocytes, T cells, B cells, and NK cells, was observed 23 hours after the end of the infusion in animals treated with IgG4 (P) of mAb 7(1.2) of hPD-1 compared with the control group. The greatest changes in magnitude were observed after administration of the first dose on Day 1; were observed transiently after subsequent doses on Days 8, 15, or 22.

335 Immune cell populations generally recovered at or near baseline values ​​by 72 hours after EOI and throughout the recovery phase. No changes in circulating monocytes were observed in animals treated with hPD-1 mAb 7(1.2) IgG4 (P) compared to the control group.

[00501] Maximal IgG4 binding (P) of hPD-1 mAb 7(1.2) to PD-1+/CD4+ and PD-1+/CD8+ cells was observed during the IgG4 treatment phase of the study (P ) of hPD-1 mAb 7(1.2) at all doses tested (10, 40 or 150 mg/kg). At recovery, in animals that did not develop anti-drug antibody (ADA) responses, serum hPD-1 mAb 7(1.2) IgG4 (P) concentrations remained above 29 pg/mL and maximum binding of IgG4 (P) from hPD-1 mAb 7(1.2) to PD-1+/CD4+ and PD-1+/CD8+ T cells was maintained throughout the 10-week recovery period. In these animals, there was no evidence of PD-1 modulation in T cells. In recovery, animals that developed ADA responses, the frequency of PD-1+ T cells bound to MGD012 fell to baseline levels. Drops in maximal IgG4 binding (P) of hPD-1 mAb 7(1.2) in PD-1+/CD4+ and PD-1+/CD8+ cells from ADA-positive animals generally occurred when IgG4 concentrations ( P) of apparent serum hPD-1 mAb 7(1.2) fell below approximately 25 pg/mL. However, Ln/zznz/E/YiAi are not cnan

336 It is not known whether this apparent threshold relationship applies to ADA-negative animals, since the presence of ADA in ADA-positive animals may contribute to blocking PD-1 antibody binding to PD-1.

[00502] There were minimal sex-associated differences in the IgG4 pharmacokinetic (P) responses of hPD-1 mAb 7(1.2), which were linear across the dose range tested (10 to 150 mg/kg). . For hPD-1 mAb 7(1.2) IgG4 (P) at 10, 40, and 150 mg/kg, the combined mean gender Cmax was 240 pg/mL (0.240 mg/mL), 1078 pg/mL (1.08 mg/mL), and 3938 pg/mL (3.94 mg/mL) and the AUC was 47310 h*pg/mL (47.3 h*mg/mL), 205723 h*pg/mL (206 h*mg/mL) , and 745681 h*pg/mL (746 h*mg/mL), respectively. The average separation by non-compartmental analysis (NCA) of the first cycle of IgG4 (P) of hPD-1 mAb 7(1.2) before it emerged from ADA was 0.21 mL/h/kg, substantially lower than the rate of glomerular filtration of cynomolgus monkeys, as would be expected for a large molecular weight protein. The average steady-state volume of distribution per NCA of the first cycle of IgG4 (P) of hPD-1 mAb 7(1.2) was 68 mL/kg, approximately 1.5 times the volume of serum, but less than the extracellular space of water. This suggests that IgG4 (P) from hPD-1 mAb 7(1.2) extravasates from the vascular compartment into the extracellular space of the tissue, but that not all of the extracellular space was accessible to this molecule. The value

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337 The average mean residence time (MRT) per NCA of the first cycle of hPD-1 mAb 7(1.2) IgG4 (P) was 335 hours or approximately 14 days. Emergence of ADA reduced hPD-1 mAb 7(1.2) IgG4 (P) concentrations in Cycles 2 to 4. Evidence of reduced hPD-1 mAb 7(1.2) IgG4 (P) serum concentrations was observed after repeated doses of hPD-1 mAb 7(1.2) IgG4 (P) in 7/10, 4/10 , and 3/10 animals in the 10, 40 and 150 mg/kg dose groups, respectively. The presence of ADA against hPD-1 mAb 7(1.2) IgG4 (P) was confirmed in 4, 2, and 1 of these animals in the 10, 40, and 150 mg/kg dose groups, respectively; all animals in which ADA was not confirmed were in the terminal necropsy group during which hPD-1 mAb 7(1.2) lgG4(P) serum concentrations likely interfered with the ability to detect ADA. Therefore, in subsequent TK analyses, when the trough concentration was lower than the preceding trough concentration, data were censored from this point forward. From two-compartment modeling of data across all cycles for all 3 dose groups, excluding points that were affected by ADA, the average values ​​for the primary TK parameters for a 2-compartment model were 0.22 mL. /h/kg for its clearance, 38.5 mL/kg for the initial volume of distribution (Vi), and 33.8 mL/kg for V₂, which produced a

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338 average volume at steady state of distribution (V_H.H) of 72.3 mL/kg, and an MRT of 329 hours. These values ​​were consistent with the parameters obtained from NCA of the first dose. In the absence of ADA, simulations predicted that with weekly dosing, steady state would be achieved in cynomolgus monkeys after the 5th dose and the accumulation index would be 2.4.

[00503] On Day 25, minimal multifocal perivascular mononuclear cell infiltrates related to hPD-1 mAb 7(1.2) IgG4 (P) occurred in the superficial dermis of the IV injection site in males at > 40 mg/kg and in females at > 10 mg/kg and was an expected reaction to repeated injection of a foreign protein (monoclonal antibody). On Day 95, no microscopic changes related to hPD1 mAb 7(1.2) IgG4 (P) were observed, indicating recovery of the change related to the test article presented on Day 25.

[00504] In summary, The results of this study indicate that the administration of hPD-1 mAb 7(1.2) IgG4 (P) by intravenous infusion once a week (Days 1, 8, 15 and 22) was well tolerated clinically in cynomolqus monkeys at levels of 10, 40 or 150 mg/kg. The observed effects were limited to transient reductions in circulating lymphocytes and minimal changes at the injection site related to the injection of a foreign protein. With

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339 Based on these results, the no observed adverse effect level (NOAEL) was considered to be 150 mg/kg (combined mean of genus C_max of 3.94 mg/mL and AUC of 746 h*mg/mL).

[00505] All publications and patents mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and individually indicated as incorporated by reference in its entirety. Although the invention has been described together with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, generally, the principles of the invention and including such departures from the present description as fall within known or customary practice within the art to which the invention belongs and which can be applied to the essential characteristics set forth above.