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Sister Ar'a's background

This application includes one or more lists of sequences pursuant to 37 CFR 1.821 et .seq, which are disclosed in both paper and computer-readable media, and inventions in both paper and computer-readable media are included herein. For reference 5 in full.

The present invention relates to monovalent CD123 x CD3 bispecific antibodies that can immediately bind to CD123 and CD3, and to uses of these molecules in the treatment of hematological malignancies.

CD123.I

<p dir="rtl">10 CD123 (interleukin 3 receptor alpha, IL-3Ra) is a 40 kDa molecule and part of</p>

Stomski, F. C. et al. (1996) “Human Interleukin-3 (3) Interleukin (IL-3) Receptor Complex Induces Disulfide-Linked IL-3 Receptor Alpha-And Beta-Chain Heterodimerization, Which Is Required For Receptor Activation But Not High-Affinity Binding.” ", Mol. Cell. Biol. 16(6):3035-3046

<p dir="rtl">15 Interleukin 3 (IL-3) drives early differentiation of pluripotent stem cells</p>

Differentiation of multipotent stem cells

Red cells of the erythroid, progenitors of myeloid cells, and progenitors of lymphoid cells. CD123 is expressed genetically on conservative cell progenitors

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Taussig, D. C. et al. (2005) ( CD34+ committed progenitors “Hematopoietic Stem Cells Express Multiple Myeloid Markers: Implications For The Origin And Targeted Therapy Of Acute Myeloid Leukemia,” Blood

106:4086-4092), but not only by normal hematopoietic stem cells

<p dir="rtl">5 -CD34+/CD38. CD123 is genetically expressed by basophils and mast cells</p>

Plasma-form stem cells and some genetic expression by monocytes, macrophages, and eosinophils, and there is low or no genetic expression by neutrophils and megakaryocytes. Some non-blood-forming tissues (placenta, Leydig cells of the testis, certain elements of brain cells and some endothelial cells) express

<p dir="rtl">10 CD123 is hereditary; However, expression is mostly cytoplasmic.</p>

It is reported that CD123 is expressed congenitally by metastatic leukemia cells and stem cells

Jordan, CT et al. (2000) “The Interleukin-3 (LSC) Leukemia Receptor Alpha Chain Is A Unique Marker For Human Acute Myelogenous

“Leukemia Stem Cells,” Leukemia 14:1777-1784; Jin, W. et al. (2009) “Regulation Of Th17 Cell Differentiation And EAE Induction By MAP3K 15

NIK 113:6603-6610 (Figure 1). In human normal progenitor populations, CD123 is expressed congenitally by a subset of hematopoietic progenitor cells.

CD123 is also genetically expressed by HPC (hematopoietic progenitor cells) but not by normal hematopoietic stem cells (HSC).

<p dir="rtl">20 By plasmacytoid dendritic cells (pDC) and cells</p>

Lopez, A. F. et al. (1989) Reciprocal Inhibition Of Binding Between Interleukin 3 And Granulocyte

Macrophage Colony-Stimulating Factor To Human Eosinophils,” Proc.

Natl. Acad. Sci. (USA) 86:7022-7026; Sun, Q. et al. (1996) “Monoclonal Antibody 7G3 Recognizes The N-Terminal Domain Of The 25”

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Human Interleukin-3 (IL-3) Receptor Alpha Chain And Functions As A Specific IL-3 Receptor Antagonist,” Blood 87:83-92; Muñoz, L. et al.

<p>(2001) “Interleukin-3 Receptor Alpha Chain (CD123) Is Widely Expressed</p>

In Hematologic Malignancies,” Haematologica 86(12):1261-1269;

Masten, B.J. et al. (2006) “Characterization Of Myeloid And Plasmacytoid 5 Dendritic Cells In Human Lung,” J. Immunol. 177:7784-7793;

Korpelainen, E.I. et al. (1995) “Interferon-Gamma Upregulations

Interleukin-3 (IL-3) Receptor Expression In Human Endothelial Cells And Synergizes With IL-3 In Stimulating Major Histocompatibility Complex

“Class II Expression And Cytokine Production,” Blood 86:176-182 10.

CD123 is reported to be genetically overexpressed on malignant cells or tumors in a wide range of hematological malignancies including acute myeloid leukemia.

myelodysplastic syndrome and myeloid leukemia (AML) Muñoz, L. et al. (2001) “Interleukin-3 Receptor Alpha Chain (MDS)

(CD123) Is Widely Expressed In Hematologic Malignancies,” 15

1261-1269:(12)86 Haematologica. Genetic overexpression of CD123 is associated with

Tettamanti, M. S. et al. (2013) “Targeting Of AML With Poorer Prognosis In Acute Myeloid Leukaemia By Cytokine-Induced Killer Cells Redirected

With A Novel CD123-Specific Chimeric Antigen Receptor,” Br. J.

.)Haematol. 161:389-401 20

AML and MDS are thought to arise and be populated by a small population of leukemic stem cells (LSCs), which are generally quiescent (specifically, cells that are dividing rapidly) and therefore resistant to apoptotic cell death and conventional chemotherapeutic agents. LSCs are characterized by high levels of CD123 expression

<p dir="rtl">25 Hereditary, not found in the corresponding normal hematopoietic stem cell populations in the marrow</p>

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Jin, W. et al. (2009) ( normal human bone marrow A Unique Marker For Human Acute

<p dir="rtl">5 14:1777-1784 “Myelogenous Leukemia Stem Cells.”</p>

Genetic CD123 expression is expressed in 45%-95% of hairy cell leukemia (HCL), and 40% of acute B lymphoblastic leukemia (B-ALL). Genetic CD123 expression has also been linked to malignancies/ or various other pre-malignant leukemias: chronic myeloid leukemia (CML 10) progenitor cells (including CML); Reed-Sternberg cells

Hodgkin's lymphoma (Sternberg's RS); transformed non-Hodgkin's lymphoma (NHL); some chronic lymphocytic leukemia (CLL); a subset of T-ALL T acute T lymphoblastic leukemia (15 (16%), mostly immature, mostly mature), malignant plasmacytoid dendritic cell tumors

pDC (DC2) plasmacytoid dendritic cell syndrome and CD34+/CD38- myelodysplastic myelodysplastic syndrome (MDS).

AML is a monoclonal disease characterized by the proliferation and accumulation of transformed myeloid progenitor cells in the bone marrow, leading largely to failure of hematopoiesis. The incidence of AML increases with age

Robak, T. et (20) Older patients typically have worse treatment outcomes than younger patients al. (2009) “Current And Emerging Therapies For Acute Myeloid

2:2349-2370.Leukemia,” Clin. Ther(. Unfortunately, currently, most adults with AML die from their disease.

Treatment for AML focuses primarily on induction therapy. Once 25% disease relief has been achieved, treatment shifts to focus on tightening this relief (post-dilution or stabilization therapy).

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In some cases, a treatment to maintain the status quo. The standard induction model for AML is chemotherapy with anthracycline/cytarabine combination, followed by either consolidation chemotherapy (always with higher doses of the same drugs used during the induction period) or a human stem cell transplant, depending on the patient's ability to achieve remission. Withstand treatment

<p dir="rtl">5 Intensive and potential cure with chemotherapy alone (see, for example, Roboz, GJ</p>

<p>(2012) “Current Treatment Of Acute Myeloid Leukemia,” Curr. Opin.</p>

.)Oncol. 24:711-719

Agents frequently used in induction therapy include cytarabine and anthracycline compounds. Ceta-arpin, also known as AraC, kills cancer cells

<p dir="rtl">10 (and other normal, rapidly dividing cells) by interfering with DNA synthesis. Side effects associated with AraC treatment include decreased resistance to infection, due to low production of white blood cells; bleeding, due to low production of platelets; and anemia, due to low production of white blood cells. Possible in red blood cells. Other side effects include nausea and vomiting, including anthracycline compounds (for example, daunorubicin,</p>

<p dir="rtl">15 Doxorubicin and idarubicin have multiple modes of action including inhibition of DNA and RNA synthesis, disruption of higher-order DNA structures, and production of cell damage by oxygen-free radicals. The most significant adverse effect of anthrecycline compounds is myocardial damage, which significantly restricts Its dose given over life and to some extent its benefit.</p>

Hence, unfortunately, despite significant progress in the treatment of newly diagnosed AML, it is not being achieved

<p dir="rtl">20 Relief occurs in 20% to 40% of patients with standard induction chemotherapy, and 50% to 70% of patients are expected to enter a first complete remission state that will relapse within 3 years. The optimal strategy for times of relapse, or for patients with resistant disease, remains uncertain. Stem cell transplantation has been established as the most effective form of anti-leukemia therapy in affected patients</p>

Roboz, GJ (2012) “Current Treatment in case of first or subsequent remission of AML with

.)Of Acute Myeloid Leukemia,” Curr. Opin. Oncol. 24:711-719 25

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CD3.II

Wucherpfennig, (2010) “Structural Biology Of The T-Cell Receptor: Insights

“Into Receptor Assembly, Ligand Recognition, And Initiation Of Signaling,”

In mammals, Cold Spring Harb. Perspect. Biol. 2(4):a005140; pages 1-14 5

The complex contains a CD3γ chain, a CD3δ chain, and two CD3ε chains. These chains bind to a molecule known as the T cell receptor in order to generate an activation signal in T lymphocytes. In the absence of CD3, TCRs do not properly assemble and be degraded. (Thomas

S. et al. (2010) “Molecular Immunology Lessons From Therapeutic T-Cell

<p dir="rtl">10 129 Receptor Gene Transfer,” Immunology(2):177–170. CD3 found.</p>

It is associated with the membranes of all adult T cells, and is not apparently associated with any other cell type (see,

Janeway, CA et al. (2005) In: IMMUNOBIOLOGY: THE IMMUNE SYSTEM IN HEALTH AND DISEASE,” 6th ed. Garland Science

Publishing, NY, pp. 214- 216; Sun, Z. J. et al. (2001) “Mechanisms Contributing To T Cell Receptor Signaling And Assembly Revealed By 15

“The Solution Structure Of An Ectodomain Fragment Of The CD3ε:γ Heterodimer,” Cell 105(7):913-923; Kuhns, M.S. et al. (2006) “Deconstructing The Form And Function Of The TCR/CD3 Complex,” Immunity. 2006 Feb;24(2):133-139

<p dir="rtl">20 III. Bispecific objects</p>

The ability of an unmodified antibody (for example, IgG) to bind an antigen epitope depends on the presence of variable domains on the immunoglobulin light and heavy chains (specifically, the VL and VH domains, respectively). A binary body on a fragment created by a single Fv string

25 scFv( chain Fv construct( )See, for example, Holliger et al (1993).

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“'Diabodies': Small Bivalent And Bispecific Antibody Fragments,” Proc.

0058400/2004; US Patent No. Natl. Acad. Sci. (USA) 90:6444-6448

Hollinger et al (; US Patent No. 0220388/2004). Mertens et al (; Alt et

al. (1999) FEBS Lett. 454(1-2):90-94; Lu, D. et al. (2005) “A Fully

Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal 5 Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For

“Enhanced Antitumor Activity,” J. Biol. Chem. 280(20):19665-19672

Olafsen, T. et al. (Mertens et al.) (2004) 02781/02 International Application No. “Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific

Conjugation And Radiolabeling For Tumor Targeting Applications,” Protein 10 Eng. Des. Sel. 17(1):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) “A Diabody For Cancer Immunotherapy And Its

Functional Enhancement By Fusion Of Human Fc Domain,” Abstract 3P- 15

<p>683, J. Biochem. 76(8):992; Takemura, S. et al. (2000) “Construction Of</p>

A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding

System,” Protein Eng. 13(8):583-588; Baeuerle, P. A. et al. (2009) “Bispecific T-Cell Engaging Antibodies For Cancer Therapy,” Cancer Res.

.)69(12):4941-4944 20

The interaction between the antibody light chain and the antibody heavy chain, specifically, the interaction of the VL and VH domains constitutes one of the epitope binding sites of the antibody. Otherwise, the scFv construct comprises an antibody VL and VH domain contained in a 25 single polypeptide chain where the domains are separated by a ligand

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Flexible has sufficient length to allow self-assembly of two domains to obtain a functional epitope binding position. Since self-assembly is impossible because the linker length is insufficient (less than about 12 amino acid residues), two scFv constructs interact with each other to form a divalent molecule, in which the VL of one chain combines with the VH of one chain.

<p dir="rtl">5 On the other hand (referenced in “Recombinant Approaches To” (2005). Marvin et al.</p>

<p>Sin Acta Pharmacol, 26:649-658. “IgG-Like Bispecific Antibodies.”</p>

Natural antibodies are able to bind to only one type of epitope (namely, mono-specific), although they are able to bind to multiple copies of that type (i.e., appearing bivalent or multivalent). The art has previously indicated their ability to produce Binary bodies differ

<p dir="rtl">10 Reported natural antibodies are capable of binding two or more different types of epitope (namely, bispecific or polyspecific as well as bivalent or multivalent) (see, for example, Holliger et al. (1993) “'Diabodies': Small</p>

Bivalent And Bispecific Antibody Fragments,” Proc. Natl. Acad. Sci.

90:6444-6448 (.USA); US Patent No. 0058400/2004 (Hollinger et al.);

15 US Patent No. 00220388/2004 (Mertens et al.); Alt et al. (1999) FEBS

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

<p>; International Application No. “Antitumor Activity,” J. Biol. Chem. 280(20):19665-19672 Mertens, N. et al., “New Recombinant Bi- (Mertens et al.) 02781/02 20</p>

and Trispecific Antibody Derivatives,” In: NOVEL FRONTIERS IN THE

PRODUCTION OF COMPOUNDS FOR BIOMEDICAL USE, A.

VanBroekhoven et al. (Eds.), Kluwer Academic Publishers, Dordrecht, The Netherlands (2001), pages 195-208; Wu, A. et al. (2001) “Multimerization Of A Chimeric Anti-CD20 Single Chain Fv-Fv Fusion 25

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Protein Is Mediated Through Variable Domain Exchange,” Protein

Engineering 14(2):1025-1033; Asano et al. (2004) “A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of

Human Fc Domain,” Abstract 3P-683, J. Biochem. 76(8):992; Takemura,

<p>S. et al. (2000) “Construction Of A Diabody (Small Recombinant 5 Bispecific Antibody) Using A Refolding System,” Protein Eng. 13(8):583-588; Baeuerle, P. A. et al. (2009) “Bispecific T-Cell Engaging Antibodies</p>

.)For Cancer Therapy,” Cancer Res. 69(12):4941-4944

Providing non-univocal binary objects has a major benefit: the ability to

<p dir="rtl">10 Co-splicing and co-localization of cells that genotypedly express different adhesin epitopes. Bivalent antibodies have a wide range of applications including immunotherapy and diagnosis. Bivalency allows great flexibility in the design and conduct of genetic manipulation of bivalent antibodies in many applications, while providing enhanced affinity for multi-subunit antigens, cross-linking of different antigens and targeted targeting of specific cell types dependent on the presence of both.</p>

<p dir="rtl">15 Target antigens. As a result of their increased valency, low separation rates and rapid clearance from circulation (for bivalent antibodies of small size, about 50 kDa), known bivalent antibody molecules in the art also show specific use in the field of tumor imaging (1997). TargetingBy Fitzgerald et al.</p>

Disulphide Stabilized Diabodies Expressed In Pichia pastoris, “Protein

<p dir="rtl">20 10:1221.Eng). The synaptic connectivity of different cells is important, for example</p>

Crosslinking of cytotoxic T cells with tumor cells (Staerz et al., 1985).

Antibodies Can Target Sites For Attack By T Cells,” Nature 314:628-631,

<p>(1996) “Specific Killing Of Lymphoma Cells By .and Holliger et al. Cytotoxic T-Cells Mediated By A Bispecific Diabody,” Protein Eng</p>

.)9:299-305 25

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Diabody epitope-binding domains can also be directed to the specific surface of any immune effector cell such as CD3, CD32, CD16 or CD64, which are expressed on T lymphocytes and natural killer (NK) cells. natural killer) or other mononuclear cells 5. In several studies, it has also been found that the determinants of the bivalent antibody bound

In the neurotransmitter cell, for example, Fcγ receptors, activate the neurotransmitter cell (Holliger et al. 1996).

“By Cytotoxic T-Cells Mediated By A Bispecific Diabody,” Protein Eng

<p>(1999) “Carcinoembryonic Antigen (CEA)-9:299-305; Holliger et al</p>

Specific T-cell Activation In Colon Carcinoma Induced By Anti-CD3 x 10

Anti-CEA Bispecific Diabodies And B7 x Anti-CEA Bispecific Fusion

Cancer Res 59:2909-2916. International Patent Application No.

113665/2006; 157379/2008; 080538/2010; 018687/2012;

162068/2012). Normally, activation of the neurotransmitter cell is carried out by attaching a body

<p dir="rtl">15 antigen bound to the neurotransmitter cell via Fc-FcγR interaction; Hence, in this regard, the bivalent antibody molecules of the invention can exhibit Ig-like function regardless of whether they comprise an Fc domain {for example, as assayed in any neurotransmitter function assay known in the art or demonstrated Example in this document {For example, (ADCC test). By cross-linking the tumor and neurotransmitter cells, the body does not</p>

<p dir="rtl">20 The bivalent antagonist not only brings the transmitter cell close to the tumor cells but leads to effective killing of the tumor (see, for example, “Bispecific Cao et al., 2003).</p>

55:171- .Rev .Deliv .Drug .Antibody Conjugates In Therapeutics, “Adv.

.)197

However, the aforementioned benefits require a significant cost. The formation of the 25 specific non-monomeric dimers requires the appropriate assembly of two or more distinct and different polypeptides, i.e., it requires

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The mentioned formation is that dimers are formed by heterogeneous polymerization of different types of polypeptide chains. This fact is in contrast to monospecific dimers, which are formed by homodimerization of identical polypeptide chains. Because at least two polypeptides must be provided, that is, two types of polypeptides

<p dir="rtl">5 Peptides in order to form a non-monospecific dimer, and because the homopolymerization of polypeptides</p>

Takemura, S. et al. (2000) (The aforementioned leads to the formation of inactive molecules “Construction Of A Diabody (Small Recombinant Bispecific Antibody))

583-588:(8)13. Using A Refolding System,” Protein Eng). The production of these polypeptides must be accomplished in such a way as to prevent covalent bonding between the

<p dir="rtl">10 Peptides from specifically the same samples, to prevent homodimerization</p>

Takemura, S. et al. (2000) “Construction Of A Diabody (Small ( Recombinant Bispecific Antibody) Using A Refolding System,” Protein

583-588:(8)13.Eng). The prior art therefore explains the non-covalent attachment of these polypeptides (see, e.g., Olafsen et al. (2004) “Covalent Disulfide-Linked Anti

CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For 15

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 Fc 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 20 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

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However, the art has previously recognized that monovalent dimers composed of non-covalently linked polypeptides are unstable and rapidly dissociate into non-functional monomers

Lu, D. et al. (2005) “A Fully Human Recombinant, see for example IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor

Receptor And The Insulin-Like Growth Factor Receptor For Enhanced 5.)Antitumor Activity,” J. Biol. Chem. 280(20):19665-19672

To address this challenge, the art has succeeded in developing stable, covalently linked heterodimeric heterodimers (see, for example, International Applications No. 113665/2006; 157379/2008; 080538/2010; 018687/2012;

Johnson, S. et al. (2010) “Effect Cell Recruitment With 10 162068/2012; Novel Fv-Based Dual-Affinity Re-Targeting Protein Leads To Potent

Tumor Cytolysis And In Vivo B-Cell Depletion,” J. Molec. Biol.

399(3):436-449; Veri, M. C. et al. (2010) “Therapeutic Control Of B Cell

Activation Via Recruitment Of Fcgamma Receptor IIb (CD32B) Inhibitory

Function With A Novel Bispecific Antibody Scaffold,” Arthritis Rheum. 15 62(7):1933-1943; Moore, P. A. et al. (2011) “Application Of Dual Affinity

Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-

4542-4551:(117 Cell Lymphoma,” Blood). The mentioned curricula include engineering

One or more cysteine residues in each polypeptide used. for example

<p dir="rtl">20 For example, the addition of a cysteine residue to the C terminus of the constructs has been demonstrated to allow the formation of disulfide bonds between the polypeptide chains, stabilizing the resulting heterodimer without interfering with the binding properties of the divalent molecule.</p>

Despite this success, the production of stable dimeric, functional heterodimers, and non-monospecific dimers can also be improved by taking into account cysteine residues and their placement.

<p dir="rtl">25 In one or more polypeptide chains used. Objects can be produced</p>

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These enhanced dualities are more active and more active than unimproved dualities. Therefore, the present invention is directed to solve the crisis of providing polypeptides specifically designed and optimized to form heterodimers. The invention solves this problem by providing improved modular CD123 x CD3 antibodies.

<p dir="rtl">5 General description of the invention</p>

The present invention relates to CD123 x CD3 bispecific antibodies capable of immediately binding to CD123 and CD3, and to uses of these molecules in the treatment of diseases, particularly hematologic malignancies.

The CD123 x CD3 bi-specific diabodies of the invention 10 comprise at least two different polypeptide chains linked together in a manner

Heterodimer to form one epitope-specific binding site for CD123 and one epitope-specific binding site for CD3. The CD123 x CD3 dimer of the invention is also monovalent and can therefore bind only one copy of the CD123 epitope and only one copy of the CD3 epitope. Individual polypeptide chains are linked to dimers

<p dir="rtl">15 Covalently linked to each other, for example through disulfide bonds to cysteine residues</p>

Located within the polypeptide chain. In certain embodiments, the dimers of the present invention also have an immunoglobulin Fc domain or an albumin binding domain

Albumin-Binding Domain to extend the half-life in the organism.

In detail, the invention also provides monovalent CD123 x CD3 bispecific antibodies

<p dir="rtl">20 It can specifically bind to the CD123 epitope and to the CD3 epitope, wherein the secondary body comprises a first polypeptide chain and a second polypeptide chain, covalently linked to each other, wherein:</p>

<p dir="rtl">a. The first polypeptide chain, from the N-terminus to the C-terminus, includes:</p>

<p dir="rtl">1. Scope 1, includes:</p>

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<p dir="rtl">(1) Sub-Domain (1A), which includes the VL domain of a monoclonal antibody</p>

monoclonal antibody can bind to CD3 (VLCD3) (sequence ID number: 21); and

<p dir="rtl">(2) subdomain (1B), including the VH domain of a monoclonal antibody that can bind CD123 (VHCD123) (sequence ID number: 26);</p>

<p dir="rtl">5 Where subdomains 1A and 1B are separated from each other by a peptide linker (sequence ID number: 29);</p>

<p dir="rtl">2. band 2, wherein band 2 is an E-coil domain (sequence ID number: 34) or a K-coil domain (sequence ID number: 35), wherein band 2 is separated from band 1 by a peptide linkage (sequence ID number: 30); And</p>

<p dir="rtl">10 B. The second polypeptide chain includes, from the N-terminus to the C-terminus:</p>

<p dir="rtl">1. Scope 1, includes:</p>

<p dir="rtl">(1) sub-domain 1A, which includes the VL domain of a monoclonal antibody that can bind CD123 (VLCD123) (sequence ID #: 25); and</p>

<p dir="rtl">(2) subdomain (1B), including the VH domain of a monoclonal antibody that can bind to CD3 15 (VHCD3) (sequence ID number: 22);</p>

Where subdomains 1A and 1B are separated from each other by a peptide linkage (sequence ID number: 29);

<p dir="rtl">2. Band 2, wherein band 2 is a K-coil domain (sequence ID: 35) or an E-coil domain (sequence ID: 34), wherein band 2 is separated from band 1 by a peptide linkage</p>

<p dir="rtl">20 (Sequence ID No.: 30); and wherein band 2 of the first and second polypeptide chains are neither E-coil domains nor K-coil domains;</p>

And where:

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(a) said VL domain of said first polypeptide chain and said VH domain of said second polypeptide chain form an antigen-binding domain capable of specifically binding to an epitope of CD3; and

(b) said VL domain of said second polypeptide chain and said VH domain of said second polypeptide chain

<p dir="rtl">5 The first polypeptide mentioned forms an antigen-binding domain capable of binding specifically to the epitope of L</p>

.CD123

The invention also provides a non-sequence-enhanced monovalent CD123 x CD3 bispecific body that can bind specifically to a CD123 epitope and a CD3 epitope, wherein the secondary body comprises a first polypeptide chain and a second polypeptide chain, covalently linked

<p dir="rtl">10 with each other, where:</p>

<p dir="rtl">a. The first polypeptide chain includes, from the N-terminus to the C-terminus:</p>

<p dir="rtl">1. Scope 1, includes:</p>

(1) a subdomain (A1), which includes the VL domain of a monoclonal antibody that can bind to CD3 (VLCD3) (sequence ID #: 23); and

<p dir="rtl">15 (2) A subdomain (B1), which includes the VH domain of a monoclonal antibody that can bind to</p>

CD123) VHCD123 (sequence ID number: 28);

Where subdomains 1A and 1B are separated from each other by a peptide link (sequence ID number: 29);

<p dir="rtl">2. Range 2, where Range 2 is a file range E (sequence ID number: 34) or a range</p>

20 two K files (sequence ID: 35), where band 2 is separated from band 1 by a peptide linker (sequence ID: 30); and

B. The second polypeptide chain includes, from the N-terminus to the C-terminus:

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<p dir="rtl">1. Scope 1, includes:</p>

<p dir="rtl">(1) a subdomain (A1), which includes the VL domain of a monoclonal antibody that can bind CD123 (VLCD123) (sequence ID number: 27); and</p>

<p dir="rtl">(2) Subdomain (B1), which includes the VH domain of a monoclonal antibody that can bind to CD3</p>

5 (VHCD3) (sequential ID number: 24);

Where subdomains 1A and 1B are separated from each other by a peptide link (sequence ID number: 29);

<p dir="rtl">2. Band 2, wherein band 2 is a K-coil domain (sequence ID: 35) or an E-coil domain (sequence ID: 34), wherein band 2 is separated from band 1 by a peptide linkage 10 (sequence ID: 30). ; wherein band 2 of the first and second polypeptide chains are not bands</p>

Two E coils or two K coils

And where:

<p dir="rtl">(a) said VL domain of said first polypeptide chain and said VH domain of said second polypeptide chain form an antigen-binding domain capable of specifically binding to the epitope of CD3 15; and</p>

<p dir="rtl">(b) said VL domain of said second polypeptide chain and said VH domain of said first polypeptide chain form an antigen-binding domain capable of specifically binding to an epitope of</p>

.CD123

The invention further provides a model for the described specific bivalent bivalent objects

<p dir="rtl">20 above, wherein the first or second polypeptide chain additionally comprises an albumin-binding domain (sequence ID number: 36) attached, at the C terminus to domain 2 or</p>

At the N-terminus of band 1, by linking a peptide (sequence ID number: 31).

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The invention further provides an embodiment of the above-described bi-specific monovalent bodies wherein the first or second polypeptide chain further comprises a band 3 comprising a CH2 and CH3 domain of an IgG Fc immunoglobulin domain (sequence ID number: 37), wherein band 3 is linked , at the N-terminus, of band 1A by linking a peptide (sequence ID number: 33).

<p dir="rtl">5 The invention further provides an embodiment of the above-described bi-specific monovalent bodies wherein the first or second polypeptide chain further comprises a 3 domain including a CH2 and CH3 domain of an IgG Fc immunoglobulin domain (sequence ID number: 37), wherein the domain is ligated 3, at the C terminus, in band 2 by linking a peptide (sequence ID number: 32).</p>

<p dir="rtl">10 The invention further provides an embodiment for any of the above-described specific monovalent dimers wherein domain 2 of the first polypeptide chain is a K-coil domain (sequence ID number: 35) and domain 2 of the second polypeptide chain is an E-coil domain (sequence ID number: 34).</p>

The invention further provides a model for any of the specific bivalent bivalent objects

<p dir="rtl">15 described above, where domain 2 of the first polypeptide chain is an E-coil domain (sequence ID number: 34) and domain 2 of the second polypeptide chain is a K-coil domain (sequence ID number: 35).</p>

The invention further provides an embodiment of a monovalent bi-specific body that can bind specifically to a CD123 epitope and a CD3 epitope, wherein the secondary body comprises 20 first polypeptide chains and a second polypeptide chain, covalently linked to each other, wherein: The dual type mentioned on:

<p dir="rtl">a. A first polypeptide chain containing the amino acid sequence of sequence ID number: 1; And</p>

<p dir="rtl">B. A second polypeptide chain containing the amino acid sequence of sequence ID number: 3;</p>

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The first and second polypeptide chains are covalently linked to each other by disulfide bonds.

The binary objects of the invention unexpectedly exhibit enhanced functional activities as further described below.

<p dir="rtl">5 The dimers of the invention would preferably be capable of cross-linking with CD123 and CD3 proteins from both humans and primates, preferably with CD123 and CD3 proteins from the baboons.</p>

It is preferable that the binary objects of the invention are capable of depleting, in an in vitro cell-based test, plasmacytoid dendritic cells.

<p dir="rtl">10 (pDC) from a primary culture of PBMCs with an IC50 of about 1 ng/ml or less, about 0.8 ng/ml or less, about 0.6 ng/ml or less, about 0.4 ng/ml or less, about 0.2 ng /ml or less, about 0.1 nanog/ml or less, about 0.05 nanog/ml or less, about 0.04 nanog/ml or less, about 0.03 nanog/ml or less, about 0.02 nanog/ml or less or About 0.01 nanog/ml or less and preferably an IC50 of about 0.01 nano</p>

<p dir="rtl">15 g/ml or less. In the test described above, the primary culture of PBMCs can be from a baboon monkey and in such a case said depletion of plasmacytoid dendritic cells (pDC) is from a baboon monkey. The binary objects of the invention can optionally be capable of depleting pDCs. (from a primary culture of PBMCs as described above where the test is performed by or in accordance with the protocol of Example 14, as described herein in</p>

<p dir="rtl">20 This request, or by modifying this test as understood by those of ordinary skill, or by other means known to those of ordinary skill.</p>

The dimers of the invention preferentially exhibit cytotoxicity in the in vitro 3-Kasumi test with an EC50 of approximately 0.05 ng/mL or less. Ideally, the EC50 is about 0.04 ng/ml or less, about 0.03 ng/ml or less, about 0.02 ng/ml or less, or

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About 0.01 nanog/ml or less. The dimers of the invention may optionally exhibit cytotoxicity as described above where the test is performed by or in accordance with the protocol of Example 3 as described herein, or by a modification of this test as understandable to those of ordinary skill, Or by other means known to those of ordinary skill.

<p dir="rtl">5 The dimers of the invention preferentially exhibit cytotoxicity in the 13-Molm in vitro assay with an EC50 of approximately 5 ng/mL or less. Ideally, the EC50 is about 3 ng/ml or less, about 2 ng/ml or less, about 1 ng/ml or less, about 0.75 ng/ml or less, or about 0.2 ng/ml or less. The binary objects of the invention may optionally exhibit cytotoxicity as described above where the test is performed by or according to</p>

<p dir="rtl">10 of the protocol for Example 3 as described herein, or by a modification of this test as understood by those of ordinary skill, or by other means known to those of ordinary skill.</p>

The dimers of the invention would ideally be able to inhibit the growth of a 13-MOLM tumor xenograft in a mouse. The invention may preferably be the binary objects

<p dir="rtl">15 Able to inhibit the growth of 13-MOLM tumor xenografts in a mouse at a concentration of at least about 20 μg/kg, at least about 4 μg/kg, at least about 0.8 μg/kg, at least about 0.6 μg/ kg or at least about 0.4 µg/kg. The preferred antibody of the invention inhibits the growth of a 13-MOLM tumor xenograft in a mouse by at least 25%, but the inhibition rate is likely to be at least about 40% or</p>

<p dir="rtl">20 more, or at least about 50% or more, or at least about 60% or more, or at least about 70% or more, or at least about 80% or more, or at least about 90% or more, or so that Complete inhibition of 13-MOLM tumor growth after a certain period of time or due to tumor relapse or disappearance. This inhibition will occur for at least the NSG strain. The dual objects of the invention may optionally be capable of inhibiting the growth of a tumor xenograft -MOLM</p>

<p dir="rtl">25 13 in a mouse by the method described above or according to the protocol of Example 6 as described</p>

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Here in this application, or by modifying this test as understood by those with ordinary skill, or by other means known to those with ordinary skill.

The invention's dimers would ideally be able to inhibit the growth of an xenograft of an RS4-11 tumor in a mouse. The binary objects of the invention may preferably be capable of

<p dir="rtl">5 On inhibiting the growth of 11-RS4 tumor xenografts in a mouse at a concentration of at least about 0.5 mg/kg, at least about 0.2 mg/kg, at least about 0.1 mg/kg, at least about 0.02 mg/kg or at least about 0.004 mg/kg. The preferred antibody of the invention inhibits the growth of an xenograft of an RS4-11 tumor in a mouse by at least about 25%, but the inhibition rate is likely to be at least about 40%, at least about</p>

<p dir="rtl">10 50%, at least about 60%, at least about 70%, at least about 80%, at least</p>

About 90%, or even complete inhibition of RS4-11 tumor growth occurs after a certain period of time or due to tumor recurrence or disappearance. This inhibition will occur for at least the NSG strain. The dimers of the invention may optionally be capable of inhibiting the growth of an RS4-11 tumor xenograft in a mouse by the method described above or according to the protocol of Example 6 as described above.

<p dir="rtl">15 Here in this application, or by modifying this test as understood by those with ordinary skill, or by other means known to those with ordinary skill.</p>

The dimers of the invention would ideally be able to deplete metastatic leukemia cells in vitro in a primary culture of AML bone marrow cells. The binary objects of the invention may preferably be able to deplete in vitro proliferation leukemia cells in culture

<p dir="rtl">20 Majority of AML bone marrow cells in Turkey is at least about 0.01 ng/ml, at least about 0.02 ng/ml, at least about 0.04 ng/ml, at least about 0.06 ng/ml, at least about 0.08 nanog/ml or at least about 0.1 nanog/ml. Preferably, the dimers of the invention may preferably be able to deplete metastasized leukemia cells in vitro in a primary culture of AML bone marrow cells into</p>

<p dir="rtl">25 Less than 20% of the total colony of primary proliferation leukemia cells when a dual antibody is detected</p>

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At least about 0.01 ng/ml, at least about 0.02 ng/ml, at least about 0.04 ng/ml, at least about 0.06 ng/ml, at least about 0.08 ng/ml or at least about 0.1 nano g/ml, optionally following incubation of the main culture with the binary for approximately 120 h. Proliferation leukemia cells are best depleted in the laboratory

<p dir="rtl">5 Primary culture of AML bone marrow cells reduced to less than 20% of the total primary proliferation cell colony at approximately 0.01 ng/ml or 0.1 ng/ml following incubation of the primary culture with the binary antibody for approximately 120 hours.</p>

Ideally, the invention's dimers would be able to induce T cell colony expansion in vitro in a primary culture of AML bone marrow cells. This expansion is preferable

<p dir="rtl">10 About 70% or more of the maximum T cell colony that can be expanded in the test. The binary bodies of the invention may preferably be capable of inducing in vitro T cell colony expansion in a primary culture of AML bone marrow cells to about 70% or more of the maximum T cell colony expandable in the test when a binary antibody is formed. At least about 0.01 nanog/ml, at least about 0.02 nanog/ml, on</p>

<p dir="rtl">15 At least about 0.04 ng/ml, at least about 0.06 ng/ml, at least about 0.08 ng/ml or at least about 0.1 ng/ml, optionally following incubation of the master culture with the binary for about 120 hours. The in vitro T cell colony in a primary culture of AML bone marrow cells should ideally be expanded to about 70% or more of the maximum T cell colony that can be expanded in the test when a binary antibody concentration of about</p>

<p dir="rtl">20 0.01 ng/ml or 0.1 ng/ml following incubation of the main culture with the binary antibody for</p>

<p dir="rtl">120 About an hour.</p>

Ideally, the binary objects of the invention would be able to induce T cell colony activation in vitro in a primary culture of AML bone marrow cells. This activation can occur at dimer concentrations of at least about 0.01 ng/ml, at least about 0.02-25 ng/ml, at least about 0.04 ng/ml, at least about 0.06 ng/ml,

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At least about 0.08 ng/ml or at least about 0.1 ng/ml, optionally following incubation of the main culture with the binary for about 72 hours. This activation can be measured by genotype expression of a T cell activation marker such as CD25. Preferably, T cell colony activation can occur in vitro in a primary culture of AML bone marrow cells as measured by genotypic expression of CD25 at a binary antibody concentration of approximately 0.01 ng/ml or

<p dir="rtl">0.1 ng/ml following incubation of the main culture with the binary antibody for approximately 72 hours.</p>

The dimers of the invention would ideally be able to deplete leukemia proliferation cells in vitro in a primary culture of AML bone marrow cells to less than 20% of the total primary leukemia proliferation cell colony and at the same time induce T cell colony expansion in

<p dir="rtl">10 The laboratory in the primary culture of AML bone marrow cells to 80% or more of the maximum T cell colony can be expanded in the test at a binary antibody concentration of at least about 0.01 ng/ml, at least about 0.02 ng/ml, at least about 0.04 ng/ml, at least about 0.06 ng/ml, at least about 0.08 ng/ml or at least about 0.1 ng/ml, optionally following incubation of the main culture with the binary for 120</p>

<p dir="rtl">15 About an hour. It is best to have a concentration of approximately 0.01 ng/ml or 0.1 ng/ml and incubate the main culture with the dimer for approximately 120 hours.</p>

The binary objects of the invention may preferably be able to deplete leukemic proliferation cells in vitro in a primary culture of AML bone marrow cells and/or induce T cell colony expansion in vitro in a primary culture of AML bone marrow cells and/or induce Colony

<p dir="rtl">20 T cells in vitro in a primary culture of AML bone marrow cells by the method described above or in accordance with the protocol of Example 8 as described herein herein, or by a modification of this test as understood by those of ordinary skill, or by other means known to those of ordinary skill. Normal skill.</p>

For the avoidance of doubt, the binary objects of the invention may exhibit one, two, 25, three, or more than three or all of the functional features described herein. and then,

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The binary objects of the invention may exhibit any combination of the functional features described herein.

The binary objects of the invention may be for use in a pharmaceutical substance. Binomials are best used in the treatment of a disease or condition associated with or characterized by the hereditary expression of

<p dir="rtl">5 CD123. The invention also relates to the use of the binary objects of the invention in the manufacture of a composition</p>

Pharmaceutical, preferably for the treatment of a disease or condition associated with or characterized by hereditary expression of CD123 as also defined herein.

A disease or condition associated with or characterized by hereditary expression of CD123 may be cancer. For example, cancer can be selected from the group consisting of: Leukemia

<p dir="rtl">10 Acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), including CML, the Abelson oncogene associated with CML (Bcr-ABL translocation), and myelodysplastic syndrome (MDS). B-ALL (acute lymphoblastic leukemia), chronic B lymphoblastic leukemia (B-ALL).</p>

<p dir="rtl">15 CLL (lymphocytic leukemia), including Richter's syndrome or Richter's transformation of CLL, hairy cell leukemia (HCL), hairy cell leukemia</p>

blastic plasmacytoid dendritic cell neoplasm diffuse plasmacytoid cell neoplasm

Non-Hodgkin lymphomas (BPDCN), including mantel cell leukemia (NHL), including mantel cell leukemia

<p dir="rtl">20 SLL (small lymphocytic lymphoma), Hodgkin's lymphoma, systemic mastocytosis, and Burkitt's lymphoma</p>

Burkitt's lymphoma

A disease or condition associated with or characterized by hereditary expression of CD123 may be an inflammatory condition. For example, the inflammatory condition can be selected from the group consisting of: lupus

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Autoimmune Lupus (SLE), allergies, asthma, and rheumatoid arthritis.

The invention further provides a pharmaceutical composition comprising any of the above-described dimers and a physiologically acceptable carrier.

<p dir="rtl">5 The invention further provides for the use of the pharmaceutical composition described above in the treatment of a disease or condition associated with or characterized by hereditary expression of CD123.</p>

The invention is particularly directed to an embodiment of this use, where the disease or condition associated with or characterized by hereditary expression of CD123 is a cancer (particularly a cancer selected from the group consisting of: acute myeloid leukemia

<p dir="rtl">10 (AML), chronic myelogenous leukemia (CML), including CML proliferation crisis and the CML-associated Abelson oncogene (Bcr-ABL translocation), myelodysplastic syndrome (MDS), and B acute lymphoblastic leukemia. -ALL (acute B lymphoblastic leukemia), chronic lymphocytic leukemia (CLL), including Richter syndrome or Richter transformation of CLL,</p>

<p dir="rtl">15 Hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin lymphomas (NHL), including mantel cell leukemia, Small lymphocytic lymphoma (SLL), Hodgkin's lymphoma, frequent</p>

<p dir="rtl">20 systemic mastocytosis, and Burkitt's lymphoma</p>

.)lymphoma

The invention is particularly directed to an embodiment of this use, wherein the disease or condition associated with or characterized by hereditary expression of CD123 is an inflammatory condition (particularly an inflammatory state selected from the group consisting of: Autoimmune Lupus

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(SLE), allergy, asthma, and rheumatoid arthritis

.)rheumatoid arthritis

Terms such as “about” should be taken to mean within 10%, and most preferably 5%, of the specified value, unless the context requires otherwise.

<p dir="rtl">5 Brief explanation of the drawings</p>

Figure 1 shows that CD123 is known to be expressed congenitally on leukemia stem cells.

Figure 2 shows the structures of the first and second polypeptide chains of the CD123 x CD3 monovalent antibody chains of the present invention.

<p dir="rtl">10 Figures 3a and 3b show the structures of two copies of the first, second, and third polypeptide chains of three monovalent CD123 Version 2, Figure 3b).</p>

Figure 4 shows the ability of different CD123 x CD3 dimers to be lethal to target cells redirected with T cells showing a variable amount of CD123. Provides shape

<p dir="rtl">15 Dose-response curves showing that sequence-enhanced CD123 or non-sequence-enhanced CD123 x CD3 dual-specific antibody (“DART-B”) in target cell types: RS4-11 (panel A); Panel D); and 1-THP</p>

<p dir="rtl">20 (Panel E) with a T:E (effector:target) ratio of 10:1.</p>

Figure 5 (Panels A-D) shows the potential of the sequence-enhanced CD123 Specificity CD123 x CD3 enhanced

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sequences containing the immunoglobulin DART-A domain (IgG Fc with w/Fc "Fc") are likely to cause activation of T cells during the killing of redirected target cells. The figure represents dose-response curves illustrating the cytotoxicity induced by DART-A, and DART-A w/ABD and DART-A w/Fc in 3-Kasumi (panel A), 1-THP (panel B) and CD8 T cells

<p dir="rtl">5 purified at a T:E (effector:target) ratio of 10:1 (incubation for 18 h). Panels C and D show dose-response curves for T cell activation with the CD25 marker on CD8 T cells in the presence (panel D) and absence (panel C) for target cells.</p>

Figure 6 (panels A-B) shows the levels of granzyme B and perforin in CD4 and CD8 T cells after treatment with the dual-specific CD123 x CD3 sequence-enhanced antibody (DART-A).

<p dir="rtl">10 (Panel A) or a comparison dual-specific antibody (Control DART) (Panel B) in the presence of 3-Kasumi target cells and keeping T cells at a T:E ratio of 10:1.</p>

Figure 7 (Panels A-B) shows the anti-tumor activity of the CD123 × dual-specificity antibody (DART-A)-enhanced sequence with nanogarms at each kilogram dose levels. 13-MOLM cells were co-mixed (expression CD123 medium) with T cells and...

<p dir="rtl">15 Subcutaneous implantation (1:1 E:T) in the NSG group. The treatment was administered intravenously once a day for eight (QDx8) intervals at implantation. Different concentrations of DART A were compared with double-dose Panel B showed that multiple doses of DART-A had an effect on tumor volume even at time periods greater than 30 days. Panel B showed the effect of multiple doses of DART-A on tumor size (DART comparison sample).</p>

<p dir="rtl">20 Increased doses of DART-A on tumor size were seen in NSG mice that received 13-MOLM cells and 1:1 E:T(T) cells. Panel B showed that the effect of increased doses of DART-A on tumor size was seen in NSG mice that received Received 13-MOLM cells and (1:1 E:T) cells for a period of 0-18 days.</p>

Figure 8 shows the in vivo antitumor activity of the bispecific

<p dir="rtl">25 CD123</p>

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Monocytes. The cells were co-mixed with T cells and transplanted into the skin (E:T).

<p dir="rtl">1:1) in the NSG group. The treatment is a single four-day intravenous treatment (QDx4) starting at implantation. Multiple concentrations of DART-A were compared with the second type of antibody from the control sample (DART for the control sample).</p>

<p dir="rtl">5 Figure 9 (Panels A-B) shows CD123+ attack in bone mononuclear cells (BM MNCs), peripheral blood megakaryocytes (PBMCs), and Patient 1 AML (Panel A) compared to the Kasumi-3 AML cell lineage (Panel A). B</p>

Figure 10 (Panels A-C) shows the ability of a dual-specificity CD123 x CD3 antibody enhanced with the DART-A sequence to induce a reduction in AML proliferation first 10 at 120 hours (Panel A), T cell expansion in AML at 120 h (panel B) and reactivation induction

T cell in AML at 48 h and 72 h (panel c).

Figure 11 (Panels A-H) shows the identification of the CD123+ proliferation population in the primary cell sample from ALL PBMCs. Panels A, E showed the side scatter of the input populations from normal PBMC (Panel A) and ALL PBMCs (Panel E). , and that the 15 selection of a lymphocyte population consists of primary B cells (panel B) and leukemic metastatic cells (panel F). Panels C, G showed a population of lymphocytes that are CD123+. Panels D, H showed a selection of lymphocytes CD19+ and CD123+.

Figure 12 (Panels A-B) shows the determination of CD4 and CD8 T cell populations in an initial sample of ALL PBMCs. Panel A showed the means of forward and side scatter from the input ALL 20 PBMCs. Panel B showed the CD4 or CD8 T cell population which It was displayed

In the samples. Figures showed that CD4 T cells represented approximately 0.5% of the total cells and CD8 T cells represented approximately 0.4% of the total cells present in the ALL PBMC sample.

Figure 13 (Panel A-H) shows the ability of dual-specific CD123 x CD3 antibody-mediated depletion-mediated (DART-A) depletion of ALL with autologous CTL.

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Panels a,e show the means of lateral forward scatter from the input pool of normal PBMCs (panel a) and ALL PBMCs (panel e). PBMCs were not treated (panel b,f) and were treated with the double-specificity antibody from the control sample (DART from control sample) (panels c, g) or treated with DART-A (panels d, h) and incubated

<p dir="rtl">5 For 7 days followed by staining for CD34 and CD19.</p>

Figure 14 (Panels A-L) shows the power of the binary object of binary quality

CD123 Panels a-f) and ALL PBMC (panels g-l). Cells were untreated (panels a,d,g,j) or

<p dir="rtl">10 Treated with dual-specific antibody (DART comparison sample) (panels b, e, h, k) or DART-A (panels c, f, i, l) for seven days.</p>

Figure 15 (Panels A-C) shows the identification of the AML proliferation population and T cells in the primary AML sample. Panel A shows the forward and side scattering of input AML PBMCs. Panel B shows the identification of the AML proliferation population in the AML sample. Panel C shows the identification of the AML proliferation population and T cells in the primary AML sample.

<p dir="rtl">15 T cell population from an AML sample.</p>

Figure 16 (panels A-C) shows the ability of the dual-specificity CD123 x CD3 sequence optimization (DART-A) to predict AML with autologous CTL and T cell expansion. Primary AML PBMCs from the patient were incubated 2 Using PBS with a comparison sample from a binary body with a specific binary quality for the comparison sample (DART comparison sample) or

<p dir="rtl">20 DART-A for 144 hours. Attacking cells (panel A), CD4 T cells (panel B) and CD8 T cells (panel C) were counted.</p>

Figure 17 (Panels A-D) shows the ability of a dual-specific CD123 x CD3 sequence-enhancing antibody (DART-A) to mediate cell activation in AML. CD25 (Panel A) and 67-Ki (Panel B) were identified. Regarding CD4 and CD8 T cells from patient 2 AML follows

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This was done by incubation with a dual-specific antibody from a comparison sample (DART comparison sample) or DART-A with independent PBMCs. Perforin (panel C) and corenzyme B (panel D) levels were determined in relation to CD4 and CD8 T cells from two patients after Incubation with the comparison sample of DART or DART-A with autologous PBMCs.

<p dir="rtl">5 Figure 18 (Panels A-D) shows that the second type DART-enhanced dimer (CD123 x CD3) is able to cross-link with both CD123 and CD3 proteins. The panels show that small portions of the BIACORETM plot Analysis of the analysis of the ability of DART-A to bind to human CD3 (panels A, C) and non-human primate proteins (panels B and D) and CD3 (panels A,</p>

<p dir="rtl">10 b) and CD123 (panels c, d). KD values are indicated.</p>

Figure 19 (Panels A-B) shows the ability of a CD123 Dose–response of DART-A-mediated cytotoxicity with human PBMCs (panel A)

<p dir="rtl">15 Or PBMCs from baboons (panel B).</p>

Figure 20 (Panels A-N) shows the ability to optimize the CD3 × CD123 double-specific antibody (DART-A) sequence to induce pDC-specific depletion in a baboon without systemic cytokine induction. D: The results of the comparison sample obtained at 4 o’clock and on the 4th day with a carrier substance and a carrier substance. Panels 20 of E-H show the results of the comparison sample obtained at 4 o’clock and on the 4th day with.

Bispecific body (comparison sample DART). Panels t-n show the results obtained at hour 4 and day 4 with 10 nmol/kg/day and at day 4 with 30 nanograms/kg/day of DART. -A.

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Figure 21 (Panels A-D) shows the power of the body-to-body optimization sequence

Bispecific CD123 x CD3 (DART-A) to cause depletion depends on

On the dosage of pDC from baboons. The baboons were given a dose of DART-A at 0.1,

<p dir="rtl">1, 10, 30, 100, 300 or 1000 nm/kg. PBMCs were evaluated at the indicated time points</p>

<p dir="rtl">5 In total B cells (panel A), monocytes (panel B), NK cells (panel C) and pDC counted (panel D).</p>

Figure 22 (Panels A-D) shows the power of the optimized sequence from the two-body

Dual specificity DART-A (CD123 x CD3) for intermittent modulation of T cells in monkeys

The baboons. Baboons were dosed with DART-A at 0.1, 1, 10, 30, 100, 300.

<p dir="rtl">10 Or 1000 nanogarm/kg. PBMCs were evaluated at the indicated time points and total B cells were counted</p>

(panel a), total T cells (panel a), CD4 T cells (panel b), CD69 cells (panel c),

CD8 T cells (panel D).

Figure 23 shows SDS-PAGE analysis of purified DART-A protein under reducing conditions (left) and non-reducing conditions (right).

<p dir="rtl">15 Figures 24a-24b show the physiochemical properties of DART-A. Figure 24a shows: SEC plot of DART-A protein on an equilibrated TSK G3000SWxL column. Figure 24b shows the mass spectrum of the DART-A protein.</p>

Figure 25A-25D shows SPR analysis of DART-A binding to human CD123 and CD3.

Fixed or from baboons. The dashed lines showed the general configuration at 1:1 of the model

<p dir="rtl">20 Langmuir of the experiment correlation curves when DART-A ranges from zero, 6.25, 12.5,</p>

25, 50 or 100 nM (solid lines). Data are illustrative of three

m independent experiments.

Figures 26a-26e show that DART-A is able to specifically bind to CD3.

And CD123. Figures 26a-26b provide results for the bifunctional ELISA and identify the cross-linking

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It is induced by both DART-A target antigens. ELISA dishes were coated with CD123 (Figure 26A) or CD123 from baboons (Figure 26B). DART-A titration and DART concentrations were followed by detection of human CD3 biotin. Figures 26C-26E showed cell surface binding of DART-A to the target cell 13 -CD123+ Molm) Figure No

<p dir="rtl">5 26C), human T cells (Figure 26D), T cells from baboons (Cells 26D), and T cells from</p>

baboons (Figure 26e). Binding was detected by FACS analysis and using a monoclonal antibody specific for the E. coli region and the K-coil region of DART-A or the DART molecule of the control sample.

Figures 27A-27H illustrate the ability of DART-A to mediate target cell killing

<p dir="rtl">10 Through neurotransmitter cells from humans or monkeys versus leukemia cell lines from</p>

3-CD123+ Kasumi, demonstrating the ability of the molecules to bind to leukocyte subsets, which include pDCs and monocytes, demonstrating the ability of the molecules to deplete high pDC cells (CD14-CD123 and basophils) without affecting monocytes (CD14+ cells). Figure 27a shows the relative binding positions of CD123-PE on U937

<p dir="rtl">15 and Kasumi leukemia type 3 cell lineages as determined by QFACS analysis. Figure 27B shows the lower relative cytotoxicity of DART-A or DART on an AML cell line (U937 cells), which, as shown in Figure 27A, have relatively few CD123 binding sites). Figure 27C shows the percentage For toxicity mediated by DART-A or DART comparator in the presence of cells</p>

<p dir="rtl">20 Purified human T cells (neurotransmitter cells) are based on AML cell lineages (Kasumi-3 cells) on which, as shown in Figure 27A, a number of CD123 binding sites are localized. In Figures 27B-27C, the T:E ratio is 10 1. Figure 27D shows the percentage toxicity mediated by DART-A or the control sample DART in the presence of purified baboon PBMCs (in the form of neurotransmitter cells) on Kasumi-3 cells (T:E ratio of 15:</p>

<p dir="rtl">25 1) It has been shown that DART-A can bind T cells from baboons. Figure 27E shows</p>

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The relative binding positions of CD123-PE on 3-Kasumi cells, human monocytes, human plasmacytoid dendritic cells (“pDC”), baboons and baboons, as determined by QFACS analysis, are shown in Fig. 27 The specific ability of DART-A to deplete CD14–CD123lo cells is demonstrated

<p dir="rtl">5 Figure 27g Ability of DART-A to deplete human CD14–CD123Hi cells. Figure 27h shows the ability of DART-A to deplete CD14–CD123Hi cells. Cytotoxicity was determined by release with LDH with EC50 values determined through GraphPad PRISM® software.</p>

Figure 28 shows the use of a two-compartment model to estimate kinematic variables

<p dir="rtl">10 Pharmacokinetics of DART-A. Data showed the end of DART-A serum concentrate infusion (EOI) in baboons after receiving a 96-hour infusion at a dose of 100 nanograms/kg/day, 300 nanograms/kg/day, and 600 nanograms/kg/day. today and 1000 nanograms/kg/day.</p>

Figures 29A-29C show the effect of DART-A infusions on the production of the cytokine, IL-6. Serum IL-6 levels (mean ± SEM) in infusion-dose monkeys are shown

<p dir="rtl">15 With DART-A using pretreatment dose. The control baboons were treated with a control sample of the vector on day 1 followed by a 4-week infusion of either the vector (Group 1) (Figure 29A) or DART-A which was given as a four-day infusion starting at Days 8, 15, 22 and 29 (Groups 2-5) (Figure 29B) or seven-day infusion doses for four weeks starting on Day 8 (Group 6) (Figure 29C).</p>

<p dir="rtl">20 Illustration of treatment times through filled gray bars.</p>

Figures 30a-30f show the effect of DART-A infusions on the depletion of CD14+/CD123+ cells (Figures 30a-30c) and CD303+ cells (Figures 30d-30f). Mean ± SEM of circulating levels of CD14+ are shown. CD123-/ (Figures 30A-30C) or CD303+ (Figures 30D-30F) from today's study and the combination shown.

<p dir="rtl">25 Baboons were treated with a control sample of the carrier on day 1 followed by a duration infusion</p>

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<p dir="rtl">4 weeks of either the vector (Group 1) (Figures 30A and 30D) or DART-A given as 4-day infusions on days 8, 15, 22, and 29 (Groups 2-5) (Figures 30A and 30E) or as Infusion at 7 days/week for four weeks starting on day 8 (Group 6) (Figures 30C and 30F). Treatment intervals are indicated by 5 filled gray bars.</p>

Figures 31a-31i show the changes observed in the T cell populations (Figures 31a-31c), CD4+ cell populations (Figures 31d-31f), and CD8+ cell populations (Figures 31g-31i) that receive DART. -A given in four-day infusions starting on days 8, 15, 22 and 29. Figure legend: CD25+ (gray triangles), PD1+

<p dir="rtl">10 (white triangles); +3-Tim (white squares). T cells were annotated with CD4 and CD8 markers, other than cone CD3 in order to eliminate the possibility of interference with DART-A. Baboons were treated with a comparator sample of vector on the day 1 This is followed by a four-day infusion of the vector (Group 1) or DART-A given as a four-day infusion weekly beginning on days 8, 15, 22, and 29 (Group 5) or as a 7-day infusion therapy</p>

<p dir="rtl">15 days/week for 4 weeks beginning on day 8 (Group 6). Treatment intervals are indicated by filled gray bars. The mean ± SEM of the number of total circulating T cells required by study day and group is shown (Figures The percentage values (mean percentage ± SEM) of CD25+, CD69+, 1-PD+, 3-Tim, and CD4+ (Figures 31D-31E) or CD8 T cells (Figures 31F-31H) are shown before</p>

<p dir="rtl">20 On the day of the study and in the group shown.</p>

Figures 32a-32f show the changes observed in CD4+ T cell populations (Figures 32a-32c) and CD8+ T cell populations (Figures 32d-32f) during and after a 7-day continuous infusion of DART-A. The average percentage From ± SEM of CD25+, CD69+, 1-PD+, and 3-Tim+ on CD4 (Figures 32A-32C) or CD8 (Figures

<p dir="rtl">25 32d-32f) of T cells by study day for groups 2, 3 and 4. Intervals are indicated</p>

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Processing through filling bars. Figure legend: +CD25 (gray triangles), +1-PD (white triangles); +3-Tim (white squares).

Figures 33a-33f show the changes observed in CD4+ T cell populations (Figures 33a-33c) and CD8+ T-cell populations (Figures 33d-33f) during and after a 5-7 day continuous infusion of DART-A. The percentages are the mean ± SEM of CD95+/CD28+ (CD4), CD95+/CD28+ (CMT) and EMT (CD95+/CD28-) T cells in the CD4+ group (Figures 33a-33c) or CD8 group (Figures 33d-33f) on study day for groups 2 , 3 and 4 shown. Baboons were treated with a control sample of vector on day 1 of a 4-week infusion compared to DART-A, which was given four 10-day infusions per week beginning on days 8, 15, 22 and 29 (groups 2–4). Treatment times are shown by filled gray bars. Figure legend: untreated sample (white triangles); CMT (black triangles), EMT (gray triangles).

Figure 34 shows the cytotoxicity mediated by DART-A versus 3-Kasumi cells with PBMCs from either untreated or treated monkeys.

<p dir="rtl">15 Multiple infusion of DART-A.</p>

Figures 35a-35f show that DART-A exposure increased the relative frequency of central memory CD4+ cells and neurotransmitter memory CD8+ cells at the expense of the corresponding progenitor T cell populations. The mean ± SEM of the percentage of native CD4+ (CMT+, CD95+/CD28+, CD95+/CD28+), and EMT (CD95+/CD28-) T cells are shown in 20 CD4+ populations (Figures 35a-35c) or in CD8+ (Figures 35a-35f) by day of study and by group.

Rabahi monkeys were treated with the comparator with the vector on day 1, followed by 4 weekly infusions of either vector (Group 1) or DART-A was given as 4 daily infusions weekly starting on Days 8, 15, 22, and 29 (Group 5). Or as an infusion 7 days/week for 4 weeks starting on day 8 (group 6). Treatment intervals are indicated in filled gray bars. Figure 25 Key: Original (white triangles); CMT (black triangles), EMT (gray squares).

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Figures 35a-35f show the effect of DART-A on red cell parameters in monkeys that received infusions of the molecules. Circled RBCs (Figures 36A-36C) or reticulocyte levels (Figures 36A-36F) are shown (mean ± SEM) in samples collected at the indicated time points from DART-A-treated monkeys.

<p dir="rtl">5 Figures 37a-37b show that the frequency (mean percentage ± SEM) of CD123+ cells (Figure 37a) or HSC (CD34+/CD38-/CD45-/CD90+ cells) (Figure 37b) in the range of Lin-cell populations in brain samples Bone collected at the time points shown from DART-A-treated monkeys Rabahi monkeys were treated with the comparator on day 1, followed by 4 weekly infusions of either vector (group 1) or DART-A given as 4 infusions.</p>

<p dir="rtl">10 per day weekly starting on days 8, 15, 22, and 29 (groups 2-5) or as an infusion 7 days/week for 4 weeks starting on day 8 (group 6).</p>

Detailed description:

The present invention is directed to sequence-enhanced CD123 x CD3 monovalent bi-specific antibodies that can bind immediately to CD123 and CD3, and to uses of these molecules in

<p dir="rtl">15 Treatment of hematological malignancies. However, non-optimized CD123 x CD3 dimers are fully functional and correspond to the improvements in gene expression obtained during codon optimization (see, for example, Grosjean, H. et al (1982).</p>

“Preferential Codon Usage In Prokaryotic Genes: The Optimal CodonAnticodon Interaction Energy And The Selective Codon Usage In

<p dir="rtl">20 199-209:(3)18 Efficiently Expressed Genes” Gene), and it is also possible to improve</p>

Stability and/or function of CD123 x CD3 dimers through modification or refinement of their sequences.

The preferred CD123 x CD3 dimers of the present invention are formed from at least two polypeptide chains linked together to form

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One specific binding site for the epitope of CD123 and one specific binding site for the CD3 epitope (Figure 2). The individual dimeric polypeptide chains are linked to each other covalently, for example by disulfide bonding to the cysteine residues within each polypeptide chain. Peptide. Each polypeptide chain contains a light-chain variable domain 5 antigen-binding domain, a heavy-chain variable domain antigen-binding domain, and a heterodimer domain.

An intervening binding peptide (ligand 1) separates the light-chain variable domain-binding domain antigen from the heavy-chain variable domain-binding domain antigen. The light-chain variable domain-binding domain antigen of the first polypeptide interacts with the heavy-chain variable domain-binding domain antigen of the first polypeptide. 10 The second polypeptide in order to form a first functional antigen-specific binding site for the first antigen (namely, that of CD123 or CD3). Similarly, the antigen-binding domain of the first antigen interacts with

The light-chain variant of the second polypeptide chain combines with an antigen-binding domain of the heavy-chain variable domain of the first polypeptide chain in order to form a second functional antigen-binding site specific for the second antigen (specifically, either CD123 or CD3, depending on the identification of the first antigen). Therefore, the antigen selection coordinates for the light-chain variable domain binding domain and antigen 15 for the heavy-chain variable domain binding domain of the first and second polypeptide chains are set such that

The two polypeptide chains collectively contain antigen-binding domains of light- and heavy-chain variable domains capable of binding to CD123 and CD3.

The formation of heterodimers of the first and second polypeptide chains can be accomplished by the heterodimers. The domains mentioned include GVEPKSC (sequential ID number: 54) (or 20 VEPKSC; Sequence ID No.: 55) on a single polypeptide chain and GFNRGEC (sequence ID: 55) on a single polypeptide chain and GFNRGEC

No.: 56 or FNRGEC; sequence ID No.: 57) on the other polypeptide chain (US Patent No. 0004909/2007). Alternatively, said domains may be engineered to contain coils of opposite charges. The heterodimer domain of one of the polypeptide chains comprises a sequence of at least six, or at least seven or at least eight of the 25 positively charged amino acids, the heterodimer domain of the polypeptide chains comprising a sequence of six

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Least, at least seven or at least eight negatively charged amino acids. For example, the first or second heterologous domain can comprise a sequence including eight positively charged amino acids and the other heterologous domains can comprise a sequence including eight negatively charged amino acids. An amino acid can be positively charged

<p dir="rtl">5 Lysine, arginine, histidine, etc. And/or the negatively charged amino acid could be glutamic acid, aspartic acid, etc. The positively charged amino acid is preferably a lysine and/or the negatively charged amino acid is preferably a glutamic acid.</p>

The CD123 x CD3 specific dimers of the present invention are engineered such that said first and second polypeptide chains are covalently linked to each other along their length via 10 cysteine residues. These cysteine residues can be inserted into the intervening linker that separates

VL and VH domains of polypeptides. Alternatively, and more preferably, a second peptide (linker 2) is inserted into each polypeptide chain, for example, at the amino-terminal ends of the polypeptide chains or at a site that places linker 2 between the heterodimer domain and the antigen-binding domain of the polypeptide chain. Light-chain variable or heavy-chain variable range.

<p dir="rtl">15 In certain embodiments, CD123 x CD3 dimers enhanced with the monovalent sequence of the present invention also comprise an immunoglobulin Fc domain or an albumin binding domain to extend the half-life in the organism.</p>

The monovalent CD123 x CD3 dimers comprise the monovalent CD123 x CD3 dimers of the present invention comprising the Fc domain of immunoglobulin (i.e., monovalent CD123 x CD3 dimers).

<p dir="rtl">20 The polypeptide chain is bi-specific (of a first polypeptide chain, a second polypeptide chain and a third polypeptide chain. The first and second polypeptide chains are linked to each other to form one binding position specific to the epitope of CD123 and one binding position specific to the epitope of CD3. The polypeptide chain is linked to The first and third polypeptide chains join together to form the Fc domain of immunoglobulin (Figure 3A and Figure 3B). The first and second polypeptide chains of the anti-Fc dimer are monomeric</p>

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Divalents are covalently linked to each other, for example by disulfide bonding to cysteine residues located within each polypeptide chain.

The first and third polypeptide chains are covalently linked to each other, for example by disulfide bonding to the cysteine residues within each polypeptide chain. Contains all

<p dir="rtl">5 One of the first and second polypeptide chains contains an antigen-binding domain with a light chain variable domain, an antigen-binding domain with a heavy chain variable domain, and a heterodimer domain. An interference binding peptide (ligand 1) separates the antigen-binding domain of the light-chain variable domain from the antigen-binding domain of the heavy-chain variable domain. The antigen-binding domain of the light-chain variable domain of the first polypeptide interacts with the antigen-binding domain</p>

<p dir="rtl">10 The antigen binds to the heavy-chain variable domain of the second polypeptide chain to form a binding site for the first functional antigen that is specific for the first antigen (i.e., either CD123 or CD3). Likewise, the antigen-binding domain of the light-chain variable domain of the second polypeptide chain interacts with The antigen-binding domain of the heavy-chain variable domain of the first polypeptide chain to form a binding site for a second functional antigen that is specific for the second antigen (i.e., either CD3 or</p>

<p dir="rtl">15 CD123, depending on the identity of the first antigen). Thus, selection of the antigen-binding domain is coordinated</p>

The antigen-binding domain of the light-chain variable domain and the antigen-binding domain of the heavy-chain variable domain of the first and second polypeptide chains, wherein the two polypeptide chains collectively comprise antigen-binding domains of light- and heavy-chain variable domains that can bind CD123 and CD3. The first and third polypeptide chains each contain some or all of the CH2 domain

<p dir="rtl">20 and/or some or all of the CH3 domain of the Fc domain of a whole immunoglobulin and a cysteine-containing peptide. Some or all of the CH2 domain and/or some or all of the CH3 domain are linked to form the Fc domain of the monovalent bispecific immunoglobulin antibody of the present invention. The first and third polypeptide chains of the monovalent bispecific anti-Fc antibodies of the present invention are covalently linked to each other, e.g.</p>

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Disulfide binding of cysteine residues within a cysteine-containing polypeptide to polypeptide chains.

<p dir="rtl">I. Sequence-enhanced CD123 x CD3 bispecific antibody, “DART-A”</p>

The invention offers a series-enhanced dual-specificity binary body that can bind at the same time and simultaneously

<p dir="rtl">5 Identified by the CD123 epitope and on the epitope of CD123 Non-sequence-enhanced CD3 is of similar composition, and thus a CD123 x CD3 bispecific antibody is called “sequence-enhanced”.</p>

<p dir="rtl">10 The sequence-enhanced CD123 Light Chain Variable Domain (VL domain) of a monoclonal antibody that can bind CD3 (VLCD3), cross-linking peptide (bind 1), heavy chain variable domain</p>

<p dir="rtl">15 (VH domain) of a monoclonal antibody that can bind to CD123 (VHCD123), and its C terminus.</p>

The preferred sequence of the aforementioned VLCD3 domain is sequence ID number: 21:

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGL

IGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWV

FGGGTKLTVLG

<p dir="rtl">20 The CDR1 VLCD3 antigen-binding domain includes a sequence with identity number:</p>

38:CDR2,RSSTGAVTTSNYAN, sequence with ID number: 39:GTNKRAP, and CDR3, sequence with ID number:40:ALWYSNLWV.

The preferred sequence for the mentioned linkage 1 is a sequence with identity number: 29: GGGSGGGG. The preferred sequence of the aforementioned VHCD123 band is sequence ID number: 26:

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EVQLVQSGAELKKPGASVKVSCKASGYTFTDYYMKWVRQAPGQGLEWI

GDIIPSNGATFYNQKFKGRVTITVDKSTSTAYMELSSLRSEDTAVYYCARS

HLLRASWFAYWGQGTLVTVSS

The antigen-binding domain of VHCD123 includes a CDR1 sequence with ID number:

<p dir="rtl">5 47:CDR2, DYYMK, sequence with ID number: 48: DIIPSNGATFYNQKFKG, and CDR3</p>

Sequence ID No.: 49: SHLLRAS.

A second polypeptide chain includes, in an N-terminal to C-terminal direction, the N-terminus, the VL domain of a monoclonal antibody that can bind CD123 (VLCD123), a binding peptide for interference (e.g., bind 1), the VH domain of a monoclonal antibody The clone can bind to CD3

<p dir="rtl">10 (VHCD3), and terminal C. The preferred sequence for the aforementioned VLCD123 band shall be sequence ID number:</p>

:25

DFVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQ

PPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDY

SYPYTFGQGTKLEIK

<p dir="rtl">15 The antigen-binding domain of VLCD123 includes the CDR1 sequence ID: 44: KSSQSLLNSGNQKNYLT, sequence ID: 45: of WASTRES CDR2, and sequence ID: 46: of QNDYSYPYT CDR3.</p>

The preferred sequence for the aforementioned VHCD3 domain is sequence ID number: 22:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWV

GRIRSKYNNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYC 20

VRHGNFGNSYVSWFAYWGQGTLVTVSS

The antigen-binding domain of VHCD3 includes the CDR1 of sequence ID No. 41: TYAMN, the CDR2 of sequence ID No. 42: RIRSKYNNYATYYADSVKD, and the CDR3 of sequence ID No. 41: TYAMN.

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No.: 43: HGNFGNSYVSWFAY.

The sequence-enhanced CD123 x CD3 dimers of the present invention are genetically processed such that said first and second polypeptides are covalently linked to each other via cysteine residues along their length. The mentioned building blocks of cysteine can be introduced

<p dir="rtl">5 In cross-linking (e.g., linking 1) the VL and VH domain of the polypeptides separates. Alternatively, and more preferably, a second peptide (linking 2) is inserted into each polypeptide chain, e.g., at the N-terminal position to The VL domain or C-terminal to the VH domain of said polypeptide chain is the preferred sequence of said ligand 2 is sequence ID number: 30: GGCGGG.</p>

The formation of heterodimers can be directed by additional genetic engineering

<p dir="rtl">10 of said polypeptide chains such that they contain polypeptide coils of corresponding charge. Thus, in a preferred embodiment, one of the polypeptide chains will be genetically engineered to contain an “E-coil” domain (sequence ID number: 34: EVAALEKEVAALEKEVAALEKEVAALEK) whose residues will form a negative charge at pH 7, while the other will be engineered from The two polypeptide chains are hereditary and contain a “K-coil” domain (sequence ID number: 35):</p>

<p dir="rtl">15 KVAALKEKVAALKEKVAALKEKVAALKE) whose residues will form a positive charge at pH 7. The presence of such charged domains enhances the union between the first and second polypeptides, and thus enhances the heterodimerization.</p>

It is of no importance which coil is presented to the first or second polypeptide chain. However, the preferred CD123 x CD3 bispecific antibody is enhanced by the sequence of the present invention

<p dir="rtl">20 (“DART-A”) a first polypeptide chain having the sequence (sequence ID number: 1):</p>

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGL

IGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWV

FGGGTKLTVLGGGGGSGGGGEVQLVQSGAELKKPGASVKVSCKASGYTFT

DYYMKWVRQAPGQGLEWIGDIIPSNGATFYNQKFKGRVTITVDKSTSTAY

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MELSSLRSEDTAVYYCARSHLLRASWFAYWGQGTLVTVSSGGCGGGEV

AALEKEVAALEKEVAALEKEVAALEK

The DART-A 1 sequence consists of: Sequence ID: 21 ─ Sequence ID: 29 ─ Sequence ID: 26 ─ Sequence ID: 30 ─ Sequence ID: 34. The DART-A 5 1 sequence encoding the polynucleotide is Sequence with identity number: 2:

caggctgtggtgactcaggagccttcactgaccgtgtccccaggcggaactgtgaccctgacatgcaga tccagcacaggcgcagtgaccacatctaactacgccaattgggtgcagcagaagccaggacaggca

ccaaggggcctgatcggggggtacaaacaaaagggctccctggacccctgcacggttttctggaagtctg ctgggcggaaaggccgctctctgactattaccggggcacaggccgaggacgaagccgattactattgtgct

ctgtggtatagcaatctgtgggtgttcgggggtggcacaaaactgactgtgctgggagggggtggatccg 10 gcggcggaggcgaggtgcagctggtgcagtccggggctgagctgaagaaacccggagcttccgtga aggtgtcttgcaaagccagtggctacaccttcacagactactatatgaagtgggtca ggcaggctccag gacagggactggaatggatcggcgatatcattccttccaacggggccactttctacaatcagaagtttaa aggcagggtgactattaccgtggacaaatcaacaagcactgcttatatggagctgagctccctgcgctct gaagatacagccgtgtactattgtgctcggtcacacctgctgagagccagctggtttg cttattggggaca 15 gggcaccctggtgacagtgtcttccggaggatgtggcggtggagaagtggccgcactggagaaagag

gttgctgctttggagaaggaggtcgctgcacttgaaaaaggaggtcgcagccctggagaaa

The second polypeptide chain of the DART-A sequence (sequence ID number: 3) has:

DFVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQ

PPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDY 20 SYPYTFGQGTKLEIKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASG

FTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRD DSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTV SSGGCGGGKVAALKEKVAALKEKVAALKEKVAALKE

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The DART-A 2 sequence consists of: sequence ID number: 25 - sequence ID number: 29 - sequence ID number: 22 - sequence ID number: 30 - sequence ID number: 35. The DART-2 sequence that encodes the polynucleotide is a sequence With ID number: 4:

5

ggccagcccccctaaactgctgatctattgggcttccaccagggaatctggcgtgcccgacagattcagcg gcagcggcagcggcacagattttaccctgacaatttctagtctgcaggccgaggacgtggctgtgtactat tgtcagaatgattacagctatccctacactttcggccaggggaccaagctggaaattaaaggaggcgga tccggcggcggaggcgaggtgcagctggtggagtctgggggaggcttggtccagcctggagggtccct

gagactctcctgtgcagcctctctggattcaccttcagcacatacgctatgaattgggtccgccaggctccag 10 ggaaggggctggagtgggttggaaggatcaggtccaagtacaacaattatgcaacctactatgccgact

ctgtgaaggatagattcaccatctcaagagatgattcaaagaactcactgtatctgcaaatgaacagcct gaaaaccgaggacacggccgtgtattactgtgtgagacacggtaacttcggcaattcttacgtgtcttggtt

tgcttattggggacaggggacactggtgactgtgtcttccggaggatgtggcggtggaaaagtggccgc actgaaggagaaagttgctgctttgaaagagaaggtcgccgcacttaaggaaaaaggtcgcagccctga 15

aagag

As discussed below, the sequence-enhanced CD123 x CD3 dual-specific antibody (DART-A) has been shown to be able to simultaneously bind CD123 and CD3 as regulated in human and monkey cells. Providing DART-A has been shown to cause cell activation. T, to mediate

<p dir="rtl">20 To reduce blasts, to direct T cell expansion, to induce T cell activation and to effect redirected killing of target cancer cells.</p>

2- The non-sequence-enhanced CD123 x CD3 di-specific dimer, “DART-B.” DART-B represents an unsequence-enhanced CD123 x CD3 di-specific dimer with an overall structure similar to that of DART-A. The first polypeptide chain includes DART-B, in the direction from the N-terminus

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To the C terminus, at the N terminus, the VL domain of a monoclonal antibody that can bind to CD3 (VLCD3), the cross-linking peptide (Lg1), the VH domain of a monoclonal antibody that can bind to CD123 (VHCD123), the cross-linking 2, and the E, and C-terminus. The VLCD3 domain of the first polypeptide chain of the body DART-B (sequence ID number: 23) has:

DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYD 5

TSKVASGVPYRFSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFG

AGTKLELK

The VHCD123 domain of the body's first polypeptide chain DART-B sequence (sequence ID number: 28):

QVQLVQSGAELKKPGASVKVSCKASGYTFTDYYMKWVRQAPGQGLEWI 10

GDIIPSNGATFYNQKFKGRVTITVDKSTSTAYMELSSLRSEDTAVYYCARS

HLLRASWFAYWGQGTLVTVSS

Thus, the DART-B chain 1 consists of: Sequence ID: 23 - Sequence ID: 29 - Sequence ID: 28 - Sequence ID: 30 - Sequence ID: 34. The sequence of the first 15 polypeptide chain of the B-body is DART is (sequence with identity number: 5):

DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYD

TSKVASGVPYRFSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFG

AGTKLELKGGGSGGGGQVQLVQSGAELKKPGASVKVSCKASGYTFTDYY

MKWVRQAPGQGLEWIGDIIPSNGATFYNQKFKGRVTITVDKSTSTAYMEL

SSLRSEDTAVYYCARSHLLRASWFAYWGQGTLVTVSSGGCGGGEVAALE 20

KEVAALEKEVAALEKEVAALEKThe 1 DART-B chain encoding the polynucleotide is a sequence with identity number: 6:

gacattcagctgacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgc agagccagttcaagtgtaagttacatgaactggtaccagcagaagtcaggcacctcccccaaaagatg

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gatttatgacacatccaaagtggcttctggagtcccttatcgcttcagtggcagtgggtctgggacctcatac tctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccaacagtggagtagtaacc cgctcacgttcggtgctgggaccaagctggagctgaaaggaggcggatccggcggcggaggcca gg tgcagctggtgcagtccggggctgagctgaagaaacccggagcttccgtgaaggtgtcttgcaaagcc

agtggctacaccttcacagactactatatgaagtgggtcaggcaggctccaggacagggactggaatg 5 gatcggcgatatcattccttccaacggggccactttctacaatcagaagtttaaaaggcagggtgactatta

ccgtggacaaatcaacaagcactgcttatatggagctgagctccctgcgctctgaagatacagccgtgta ctattgtgctcggtcacacctgctgagagccagctggtttgcttattggggacagggcaccctggtgacagt

gtcttccggaggatgtggcggtggagaagtggccgcactggagaaagaggttgctgctttggagaagg aggtcgctgcacttgaaaaaggaggtcgcagccctggagaaa 10

The second polypeptide chain of DART-B, in an N-terminal to C-terminal direction, includes the N-terminal, VL domain of a monoclonal antibody that can bind to CD123.

(VLCD123), a cross-linking peptide (ligand 1), the VH domain of a monoclonal antibody that can bind to CD3 (VHCD3), cross-linking 2, a K-coil domain, and a C terminus.

<p dir="rtl">15 The VLCD123 domain of the body's second polypeptide chain DART-B sequence (sequence ID: 27) has:</p>

DFVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQ PPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDY

SYPYTFGQGTKLEIK

<p dir="rtl">20 The VHCD3 domain of the body's second polypeptide chain DART-B sequence (sequence ID number:</p>

:)24

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIG YINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARY

YDDHYCLDYWGQGTTLTVSS

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Thus, the DART-B chain 2 consists of: Sequence ID No: 27 - Sequence ID No: 29 - Sequence ID No: 24 - Sequence ID No: 30 - Sequence ID No: 35. The sequence of the second polypeptide chain of the body is B-DART. They are (consecutive with ID number: 7):

DFVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQ PPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDY 5 SYPYTFGQGTKLEIKGGGSGGGGDIKLQQSGAELARPGASVKMSCKTSG YTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQ KFKDKATLTTDKS SSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGCG

GGKVAALKEKVAALKEKVAALKEKVAALKE

<p dir="rtl">10 The 2 DART-B sequence encoding the polynucleotide is sequence ID number: 8:</p>

gacttcgtgatgacacagtctcctgatagtctggccgtgagtctgggggagcgggtgactatgtcttgcaa gagctcccagtcactgctgaacagcggaaatcagaaaaactatctgacctggtaccagcagaagcca ggccagcccccctaaactgctgatctattggggcttccaccagggaatctggcgtgcccgacagatt cagcg gcagcggcagcggcacagattttaccctgacaatttctagtctgcaggccgaggacgtggctgtgtactat tgtcagaatgattacagctatccctacactttcggccaggggaccaagctggaattaaaggaggcgga 15 tccggcggcggaggcgatatcaaactgcagcagtcaggggctgaactggcaagacctgggg cctcag tgaagatgtcctgcaagacttctggctacacctttactaggtacacgatgcactgggtaaaacagaggcc tggacagggtctggaatggattggatacattaatcctagccgtggttatactaattacaatcagaagttcaa ggacaaggccacattgactacagacaaatcctccagcacagcctacatgcaactgagcagcctgaca tctgaggactctgcagtctattactgtgcaagatattatgatgatcattactgccttgactactggggccaag 20 gcaccactct cacagtctcctccggaggatgtggcggtggaaaagtggccgcactgaaggagaaagtt

gctgctttgaaagagaaggtcgccgcacttaaggaaaaaggtcgcagccctgaaagag

<p dir="rtl">3. Modified heterochromatin images of the sequence-enhanced CD123 x CD3 diploid</p>

(DART-A)

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<p dir="rtl">a. Sequence-enhanced CD123 x CD3 dual-specific antibody with an albumin binding domain (DART-A).</p>

)"w/ABD" ABD has

In a second embodiment of the invention, the sequence-enhanced CD123 x CD3 dual-specific antibody (DART-A) comprises one or more albumin-binding domain (“ABD”) (DART-A).

<p dir="rtl">5 ABD (w/ABD) contains one or both of the polypeptide chains of the dimer.</p>

As disclosed in International Application No. 2012/018687, to improve the in vivo pharmacokinetic properties of the binary bodies, the binary bodies may be modified to contain a polypeptide portion of a serum binding protein at one or more edges of the binary body. Ideally, the polypeptide portion of the serum-binding protein would be anchored to the C terminus of the dimer. It is a polycarbonate part

<p dir="rtl">10 A particularly preferred peptide of the serum binding protein for this purpose is the binding domain</p>

For albumin ABD (Albumin-Binding Domain) from the protein related to Streptococcus bacteria^

<p dir="rtl">G streptococcal protein. The Albumin-Binding Domain (ABD3) of the G protein of the Streptococcus bacterium G148 strain</p>

Streptococcus strain is particularly preferred.

<p dir="rtl">15 The albumin-binding domain 3 (ABD3) of the G protein of Streptococcus strain G148 consists of 46 amino acid residues forming a stable triple helix bundle.</p>

Johansson, M. U. et al. (It has a broad three-helix bundle (2002) “Structure, Specificity, And Mode Of Interaction For Bacterial

Albumin-Binding Modules,” J. Biol. Chem. 277(10):8114-8120

<p dir="rtl">20 Albumin is the most abundant protein in plasma and has a half-life of 19 days in humans. Albumin contains several small molecule binding sites that allow it to bind non-covalently to other proteins and thus prolong their serum half-life.</p>

Thus, the first polypeptide chain of such a dimeric antibody contains CD123 x

The sequence-enhanced CD3 has an albumin-binding domain on a third ligand (link 3), separating the E coil (or

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(K file) of the said polypeptide chain from the albumin binding domain. The preferred sequence of the said binding 3 shall be sequence with identity number: 31: GGGS. The preferred albumin binding domain shall be - Albumin

ABD(Binding Domain) sequence (sequence with ID number: 36):

LAEAKVLANRELDKYGVSDYYKNLIDNAKSAEGVKALIDEILAALP.

<p dir="rtl">5 Thus, the first preferred sequence of the CD123</p>

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGL IGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWV

FGGGTKLTVLGGGGGSGGGGEVQLVQSGAELKKPGASVKVSCKASGYTFT

DYYMKWVRQAPGQGLEWIGDIIPSNGATFYNQKFKGRVTITVDKSTSTAY 10 MELSSLRSEDTAVYYCARSHLLRASWFAYWGQGTLVTVSSGGCGGGEV

AALEKEVAALEKEVAALEKEVAALEKGGGSLAEAKVLANRELDKYGVSDY

YKNLIDNAKSAEGVKALIDEILAALP

The sequence-enhanced CD123 31 - Sequence ID No.: 36. The polynucleotide encoding such a CD123

caggctgtggtgactcaggagccttcactgaccgtgtccccaggcggaactgtgaccctgacatgcaga tccagcacaggcgcagtgaccacatctaactacgccaattgggtgcagcagaagccaggacaggca 20

ccaaggggcctgatcggggggtacaaacaaaagggctccctggacccctgcacggttttctggaagtctg ctgggcggaaaaggccgctctgactattaccggggcacaggccgaggacgaagccgattactattgtgct ctgtggtatagcaatctgtgggtgttcgggggtggcacaaaactgactgtgctgggaggggt ggatccg gcggcggaggcgaggtgcagctggtgcagtccggggctgagctgaagaaacccggagcttccgtga

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aggtgtcttgcaaagccagtggctacaccttcacagactactatatgaagtgggtcaggcaggctccag gacagggactggaatggatcggcgatatcattccttccaacggggccacttctacaatcagaagtttaa aggcagggtgactattaccgtggacaaatcaacaagcactgcttatatggagctgagctccctgcgctct ga agatacagccgtgtactattgtgctcggtcacacctgctgagagccagctggtttgcttattggggaca gggcaccctggtgacagtgtcttccggaggatgtggcggtggagaagtggccgcactggagaaagag 5 gttgctgctttggagaaggaggtcgctgcacttgaaaaggaggtcgcagccctggagaaagg cggcgg

gtctctggccgaagcaaaagtgctggccaaccgcgaactggataaatatggcgtgagcgattattataa gaacctgattgacaacgcaaaatccgcggaaggcgtgaaagcactgattgatgaaattctggccgccc tgcct

<p dir="rtl">10 The second polypeptide chain of such a sequence-enhanced CD123</p>

B. Sequence-enhanced CD123 x CD3 bispecific antibodies that have an IgG Fc domain

("Fc" w/Fc with DART-A)

<p dir="rtl">15 In a third embodiment, the invention provides a sequence-enhanced CD123 x CD3 bispecific antibody consisting of three polypeptide chains and characterized by an IgG Fc domain (DART-A) having Fc “w/Fc” copy 1 and copy 2 (Figures 3a-3b).</p>

To form the aforementioned IgG Fc domain, the first and third polypeptide chains of dimers contain, in the direction from the N-terminus to the C-terminus, a cysteine-containing peptide (preferably, peptide 1 of

<p dir="rtl">20 Amino acid sequence (Sequence ID No.: 55): DKTHTCPPCP), or some or all of the CH2 domain and/or some or all of the CH3 domain of the Fc domain of a complete immunoglobulin, and the C terminus. Some or all of the CH2 domain and/or some of Of or all of the CH3 domain to form the Fc domain of the immunoglobulin di-bodies containing the monovalent bi-specific Fc domain of the present invention the first and second polypeptide chains of the bi-dimers of the Fc di-bodies</p>

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The bivalent dimers of the present invention are covalently linked to each other, for example by disulfide bonding of cysteine residues contained within a cysteine-containing peptide of polypeptide chains.

The CH2 and/or CH3 domain of the first and third polypeptides does not have to be identical, and is...

<p dir="rtl">5 They are specifically modified to enhance the complexation between the two polypeptides. For example, an amino acid substitution (preferably a replacement using an amino acid with a bulky side group that forms a “cluster” (e.g., tryptophan)) can be introduced into the CH2 or CH3 domain such as a gap-like overlap that prevents interaction with the mutant domains in a similar way that will make the mutant domain In pair form with the mutant range in which the complementary or localized mutant is processed genetically, i.e., “hole” (e.g.</p>

<p dir="rtl">10 For example, replacement using glycine. The aforementioned mutant groups can be genetically processed into any pair of polypeptides containing the bivalent antibody molecule, and also genetically processed into any part of the polypeptide chains of the aforementioned pair. The methods of protein processing are genetically Through heterodimers compared to homodimers are known in the scope, specifically in relation to the processing of immunoglobulin-like molecules which are included herein</p>

15 (See, for example, 'Ridgway et al. (1996) “'Knobs-Into-Holes

Engineering Of Antibody CH3 Domains For Heavy 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 20 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 “block” is processed in the CH2-CH3 bands of the first polypeptide chain and the “hole” is processed in the CH2-CH3 bands of the third polypeptide chain. Thus, the “block” helps prevent

<p dir="rtl">25 The first polypeptide chain of the homodimer via two special CH2 and/or CH3 domains</p>

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With it. Glasses Because the third polypeptide chain preferably contains the “hole” substitution, it forms a heterodimer with the first polypeptide chain as well as forming a homodimer with itself. A preferred cluster is formed by modifying a native IgG Fc domain so that it contains the T366W modification. A preferred hole is formed by modifying a native IgG Fc domain such that it contains the L368A, T366S modification

<p dir="rtl">5 And Y407V. To assist in the purification of the homodimer of the third polypeptide chain from the end-specific monovalent Fc dimer comprising the first, second and third polypeptide chains, it is preferable to mutagen the protein A binding site of the CH2 and CH3 domains of the third polypeptide chain by substituting the amino acid at position 435. (H435R). Thus, the homodimer of the third polypeptide chain will not bind to protein A, whereas the anti-Fc dimer will not bind to protein A.</p>

<p dir="rtl">10 The monovalent bispecific will retain its ability to bind protein A via the protein A binding position on the first polypeptide chain.</p>

The preferred sequence for the CH2 and CH3 domains of the Fc domain of the antibody present in the first polypeptide chain is (sequence ID number: 56):

APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP 15

APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVE

WESNGQPENNYKTTPPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGK

The preferred sequence for the CH2 and CH3 domains of the Fc domain of the antibody present in the third polypeptide chain 20 is (sequence ID number: 11):

APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP

APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVE

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WESNGQPENNYKTTPPPVLDSDGSFFLVLVSKLTVDKSRWQQGNVFSCSVM

HEALHNRYTQKSLSLSPGK

C. Version 1 DART-A w/Fc architecture

To illustrate the aforementioned Fc binary objects, the invention provides a DART-A w/Fc version 1 structure. Includes

<p dir="rtl">5 The first polypeptide of the body DART-A w/Fc copy 1 structure, in an N-terminal to N-terminal direction</p>

<p dir="rtl">C, on the N terminus, is the VL domain of a monoclonal antibody that can bind to CD123</p>

(VLCD123), an interference binding peptide (ligand 1), and the VH domain of a monoclonal antibody that can

Binding to CD3 (VHCD3, bind2, E-coil domain, bind5, polypeptide 1, polypeptide containing

on the CH2 and CH3 domains of the Fc domain and the C terminus. The preferred 5′ linkage has the sequence (sequence

<p dir="rtl">10 Identity No.: 32(: GGG). The preferred polypeptide containing the CH2 and CH3 domains is</p>

For the sequence Fc domain (sequence ID number: 37):

APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP

APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVE

WESNGQPENNYKTTPPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM 15

HEALHNHYTQKSLSLSPGK

Thus, the first polypeptide of the aforementioned DART-A w/Fc body copy 1 structure consists of: Sequence ID No: 25 - Sequence ID No: 29 - Sequence ID No: 22 - Sequence ID No: 30 - Sequence ID No: 34 - Sequence ID No: 34 ID number: 32 - consecutive ID number: 55 - consecutive ID number: 37.

<p dir="rtl">20 The preferred sequence of the first polypeptide of clone 1 has the DART-A w/Fc structure mentioned</p>

Sequential (sequential with ID number: 13):

DFVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQ

PPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDY

SYPYTFGQGTKLEIKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASG

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FTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRD DSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTV

SSGGCGGGEVAALEKEVAALEKEVAALEKEVAALEKGGGDKTHTCPPCP

APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP 5

APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGK

The preferred polynucleotide encoding such a polypeptide is (sequence ID number: 14):

gacttcgtgatgacacagtctcctgatagtctggccgtgagtctctgggggagcgggtgactatgtcttgcaa 10 gagctcccagtcactgctgaacagcggaaatcagaaaaactatctgacctggtaccagcagaagcca

ggccagccccctaaactgctgatctattgggcttccaccagggaatctggcgtgcccgacagattcagcg gcagcggcagcggcacagattttaccctgacaatttctagtctgcaggccgaggacgtggctgtgtactat tgtcagaatgattacagctatccctacactttcggccaggggaccaagctggaaattaaaggaggcgga

tccggcggcggaggcgaggtgcagctggtggagtctgggggaggcttggtccagcctggagggtccct 15 gagactctcctgtgcagcctctctggattcaccttcagcacatacgctatgaattgggtccgccaggctccag ggaaggggctggagtggggttggaaggatcaggtccaagtacaacaattatgcaacctactatg ccgact

ctgtgaaggatagattcaccatctcaagagatgattcaaagaactcactgtatctgcaaatgaacagcct gaaaaccgaggacacggccgtgtattactgtgtgagacacggtaacttcggcaattcttacgtgtcttggtt

tgcttattggggacaggggacactggtgactgtgtcttccggaggatgtggcggtggagaagtggccgc 20 actggagaaagaggttgctgctttggagaaggaggtcgctgcacttgaaaaggaggtcgcagccctgg agaaaggcggcggggacaaaactcacacatgcccaccgtgcccagcacctgaagccgc gggggg accgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcaca tgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtgga

ggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgt 25

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cctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagcc ctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtac accctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgtggtgcctggtcaaaggctt ctat cccagcgacatcgccgtggagtggggagagcaatggggcagccggagaacaactacaagaccac gcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtgg 5 cagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagag cctctccctg tctccgggtaaa

The DART-A w/Fc second sequence of copy 1 structure includes, in an N-terminal to C-terminal direction, the N-terminal, VL domain of a monoclonal antibody that can bind to CD3

<p dir="rtl">10 (VLCD3), an interference binding peptide (ligand 1), the VH domain of a monoclonal antibody that can bind CD123 (VHCD123), binder 2, a K-coil domain, and a C terminus. Thus, the second polypeptide consists of the aforementioned DART-A w/Fc Copy 1 structure of: Sequence ID No: 21 ─ Sequence ID No: 29 ─ Sequence ID No: 26 ─ Sequence ID No: 30 ─ Sequence ID No: 35. Such a polypeptide has the sequence (Sequence ID No: 15):</p>

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGL 15 IGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWV

FGGGTKLTVLGGGGGSGGGGEVQLVQSGAELKKPGASVKVSCKASGYTFT DYYMKWVRQAPGQGLEWIGDIIPSNGATFYNQKFKGRVTITVDKSTSTAY MELSSLRSEDTAVYYCARSHLLRASWFAYWGQGTLVTVSSGGCGGGKV

AALKEKVAALKEKVAALKEKVAALKE 20

The preferred polynucleotide encoding said polypeptide shall have the sequence (sequence ID number:

:)16

caggctgtggtgactcaggagccttcactgaccgtgtccccaggcggaactgtgaccctgacatgcaga tccagcacaggcgcagtgaccacatctaactacgccaattgggtgcagcagaagccaggacaggca

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ccaaggggcctgatcggggggtacaaacaaaagggctccctggacccctgcacggttttctggaagtctg ctgggcggaaaaggccgctctgactattaccggggcacaggccgaggacgaagccgattactattgtgct ctgtggtatagcaatctgtgggtgttcgggggtggcacaaaactgactgtgctgggaggggt ggatccg gcggcggaggcgaggtgcagctggtgcagtccggggctgagctgaagaaacccggagcttccgtga aggtgtcttgcaaagccagtggctacaccttcacagactactatatgaagtgggtcaggcaggctccag 5 gacagggactggaatggatcggcgatatcattccttccaacggggccactttctacaatcagaag tttaa aggcagggtgactattaccgtggacaaatcaacaagcactgcttatatggagctgagctccctgcgctct gaagatacagccgtgtactattgtgctcggtcacacctgctgagagccagctggtttgcttattggggaca gggcaccctggtgacagtgtcttccggaggatgtggcggtggaaaaagtggccgcactgaaggagaaa

gttgctgctttgaaagagaaggtcgccgcacttaaggaaaaaggtcgcagccctgaaagag 10

The third polypeptide chain of the body DART-A w/Fc mentioned copy 1 includes the CH2 and CH3 domains of the IgG Fc domain. A preferred polypeptide consisting of peptide 1 (sequence ID number: 55) and the CH2 and CH3 domains of an Fc domain (sequence ID number: 11) has a sequence to sequence identity number: 54:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED 15 PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQ GNVFSCSVMHEALHNRYTQKSLSLSPGK

<p dir="rtl">20 The preferred polynucleotide encoding said polypeptide shall have the sequence (sequence ID number:</p>

:)12

Gacaaaactcacacatgcccaccgtgcccagcacctgaagccgcggggggacgtcagtcttcctctt ccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacg tgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgcca ag

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acaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcacc aggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcga gaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccg gga ggagatgaccaagaaccaggtcagcctgagttgcgcagtcaaaggcttctatcccagcgacatcg

ccgtggagtggggagagcaatggggcagccggagaacaactacaagaccacgcctcccgtgctggact 5 ccgacggctccttcttcctcgtcagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtc ttctcatgctccgtgatgcatgaggctctgcacaaccgctacacgcagaagagcctctccctg tctccggg

taaa

<p dir="rtl">Dr.. Body Structure DART-A w/Fc Version 2</p>

<p dir="rtl">10 As a second example of said DART-A w/Fc binary body, the invention provides a three-chain binary body, a “DART-A w/Fc version 2 binary body” (Figure 3b).</p>

The structure of the first polypeptide of the body DART-A w/Fc includes copy 2, towards the end

N to the C terminus, at the N terminus, a linking peptide (peptide 1), and a polypeptide containing the CH2 and CH3 domains of the Fc domain linked (via linkage 4) to the VL domain of a monoclonal antibody that can

<p dir="rtl">15 Binding to CD123 (VLCD123), an interference binding peptide (binding 1), the VH domain of a monoclonal antibody that can bind to CD3 (VHCD3), binding 2, a K-coil domain, and a C terminus.</p>

The polypeptide containing the CH2 and CH3 domains of the Fc domain is preferred

Continuous (Continuous with ID No.: 37):

APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP 20 APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVE

WESNGQPENNYKTTPPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGK

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It is preferable that “link 4” include the amino acid sequence (sequence ID number: 57): APSSS.

“Binder 4” has the sequence (Sequence ID No.: 33): APSSSPME. Thus, the first polypeptide of the aforementioned DART-A w/Fc structure, copy 2, consists of: Sequence ID No.: 55 - Sequence ID No.: 37 - Sequence ID No.: 37 : 33 - Continuous with ID No.: 25 - Continuous with ID No.: 29 - Continuous with ID No.:

<p dir="rtl">5 Number: 22 - Sequence with identity number: 30 - Sequence with identity number: 35. A polypeptide that has such a sequence (sequence with identity number: 17):</p>

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLV

KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ 10

QGNVFSCSVMHEALHNHYTQKSLSLSPGKAPSSSPMEDFVMTQSPDSLA VSLGERVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRE

SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLE

IKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVR QAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSL 15

KTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGCGGGKVAAL

KEKVAALKEKVAALKEKVAALKE

The preferred polynucleotide encoding said polypeptide shall have the sequence (sequence ID number:

:)18

gacaaaactcacacatgcccaccgtgcccagcacctgaagccgcggggggacgtcagtcttcctctt 20 ccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacg tgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcata atgccaag acaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcacc aggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcga

gaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccg 25

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ggaggagatgaccaagaaccaggtcagcctgtggtgcctggtcaaaggcttctatcccagcgacatcg ccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggact ccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtc ttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccggg taaagccccttccagctcccctatggaagacttcgtgatgacacagtctcctgatagtctggccgtgagtct 5 gggggagcgggtgactatgtcttgcaagagctcccagtcactgctgaacagcggaaat cagaaaaact atctgacctggtaccagcagaagccaggccagccccctaaactgctgatctattggggcttccaccaggg aatctggcgtgcccgacagattcagcggcagcggcagcggcacagattttaccctgacaatttctagtct gcaggccgaggacgtggctgtgtactattgtcagaatgattacagctatccctacactttcggccagggg

accaagctggaaattaaaggaggcggatccggcggcggaggcgaggtgcagctggtggagtctctggg 10 ggaggcttggtccagcctggagggtccctgagactctcctgtgcagcctctggattcaccttcagcacata cgctatgaattgggtccgccaggctccagggaaggggctggagtggggtttggaaggatcaggtc caagt acaacaattatgcaacctactatgccgactctgtgaaggatagattcaccatctcaagagatgattcaaa gaactcactgtatctgcaaatgaacagcctgaaaaccgaggacacggccgtgtattactgtgtgagaca

cggtaacttcggcaattcttacgtgtcttggtttgcttattggggacaggggacactggtgactgtgtcttccg 15 gaggatgtggcggtggaaaaagtggccgcactgaaggagaaagttgctgctttgaaagagaaggtcgc

cgcacttaaggaaaaaggtcgcagccctgaaagag

The second polypeptide chain of the mentioned DART-A w/Fc body structure includes transcript 2, in an N-terminal to C-terminal direction, and the VL domain of a monoclonal antibody that can bind to CD3.

<p dir="rtl">20 (VLCD3), an interference binding peptide (ligand 1) and the VH domain of a monoclonal antibody that can bind to CD123 (VHCD123). The molecule is linked (via binder 2) to the E-coil domain. Thus, the third polypeptide of the DART-A body structure is formed /Fc mentioned is copy 2 of: Sequence ID No.: 21 - Sequence ID No.: 29 - Sequence ID No.: 26 - Sequence ID No.: 30 - Sequence ID No.: 34. The polypeptide that has such a sequence is (sequence ID No.: 1), preferably poly-encoded</p>

<p dir="rtl">25 A nucleotide has sequence identity number: 2.</p>

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The third polypeptide chain includes the CH2 and CH3 domains of the IgG Fc domain. The preferred polypeptide consists of peptide 1 (sequence ID number: 55), which has the CH2 and CH3 domains of the Fc domain (sequence ID number: 11), and has sequence ID number: 54.

To evaluate the activity of the previously mentioned bispecific antibodies (DART-A (CD123 x CD3),

<p dir="rtl">5 A bispecific dimer (DART conjugate, DART-A w/Fc, DART-B w/ABD) is produced. The DART conjugate can simultaneously bind to FITC and CD3. The two polypeptide chains have the following corresponding sequences :</p>

Comparative DART series 1 (sequence with ID number: 19):

DVVMTQTPFSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSP

KVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVP 10

WTFGGGTKLEIKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFT

FNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDS

KNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

GGCGGGEVAALEKEVAALEKEVAALEKEVAALEK

<p dir="rtl">15 Comparative DART series 2 (sequence with ID number: 20):</p>

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGL

IGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWV

FGGGTKLTVLGGGGGSGGGGEVKLDETGGGLVQPGRPMKLSCVASGFTF

SDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSK

SSVYLQMNNLLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSGGCGGGKV 20

AALKEKVAALKEKVAALKEKVAALKE

<p dir="rtl">4 - Pharmaceutical compositions: The compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions, for example, impure or non-sterile compositions, and pharmaceutical compositions (i.e. compositions suitable for administration to a subject or patient), which can</p>

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Use them in preparing dosage unit images. Such compositions comprise a therapeutically prophylactic effective amount or of the therapeutic and/or prophylactic agent disclosed herein or a combination of the sequence-enhanced CD123 x CD3 bispecific antibodies of the present invention, or a combination of such agents

<p dir="rtl">5 The pharmaceutically acceptable carrier substance. Preferably, the compositions of the invention comprise a prophylactically or therapeutically effective amount of the sequence-enhanced CD123 x CD3 bispecific antibody of the invention and a pharmaceutically acceptable carrier.</p>

The invention also includes pharmaceutical compositions comprising CD123 x CD3 specificity dimers enhanced with the sequence of the invention, and a second therapeutic antibody, for example, an antibody

<p dir="rtl">10 A tumor specific monoclonal antibody that is specific to a specific cancer antigen and a pharmaceutically acceptable carrier.</p>

In a specific embodiment, “pharmaceutical accepted” means any approved by a federal or state regulatory agency or listed in the American Pharmacopoeia or other generally recognized medical encyclopedia for use in animals, more specifically in humans. The term “material” refers to

<p dir="rtl">15 "carrier" refers to the diluent, the adjuvant (eg, the complete or incomplete excipient, the excipient, or the carrier through which the therapeutic agent is administered. Said pharmaceutical carriers can be sterile liquids, such as water, oils Which includes petroleum, animal, vegetable or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like, and water</p>

<p dir="rtl">20 A preferred carrier when administering a pharmaceutical formulation via intravenous injection. Saline solutions, aqueous dextrose, and glycerol solutions can also be used as liquid carriers, specifically injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, and rice.</p>

<p dir="rtl">25 And flour, chalk, silica gel, and sodium stearate.</p>

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stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition may also include, if Demand, small amounts of wetting 5, emulsifying agents, or pH buffering agents

agents. These formulations can be in the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like.

<p dir="rtl">10 In general, the components of the compositions of the invention are supplied either separately or mixed together as a unit</p>

A dose, for example, in the form of a dry lyophilized powder, or a water-free concentration product in a hygienically sealed container such as an ampoule or ziplock with instructions on the amount of active ingredient. When the formulation is given by infusion, it may be dispersed with a dispensing bottle containing water or sterile pharmaceutical grade saline. When the composition is given by

<p dir="rtl">15 Injection: An ampoule of sterile water for injection may be provided or saline may be provided so that the components can be mixed before administration.</p>

The compositions of the invention may be formed in a neutral form or in a salt form. Pharmaceutical acceptable salts include, but are not limited to, those formed with anions such as those derived from hydrochloric, phosphoric, acetic, and oxalic.

<p dir="rtl">20 oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, and 2-ethyl 2-ethylamino ethanol, histidine, procaine, etc.</p>

25

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The invention also provides a pharmaceutical kit or package comprising one or more containers filled with sequence-enhanced CD123 x CD3 dimers of the invention alone or in combination with said pharmaceutically acceptable carrier. Additionally, one or more other prophylactic or therapeutic agents useful for treating a disease may also be included in a pharmaceutical package or package. He provides

<p dir="rtl">5 The invention also invents a pharmaceutical set or package comprising one or more containers filled with one or</p>

More than the ingredients of patented pharmaceutical formulations. Optionally in relation to the relevant container(s) there may be a note in a form prescribed by the government agent regulating the manufacture, for use or sale of pharmaceutical substances or biological products, and such note reflecting the approval of the agency of manufacture, use or sale for administration to humans.

<p dir="rtl">10 The present invention provides kits that can be used in the above methods. The kit may include the sequence-enhanced CD123 x CD3 dual-specific antibodies of the invention. The kit may also include one or more other prophylactic and/or therapeutic agents useful in 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 related antigens.</p>

<p dir="rtl">15 With cancer. In certain embodiments, the other prophylactic or therapeutic agent is chemotherapy. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic agent.</p>

<p dir="rtl">5- Methods of administration</p>

Compositions of the present invention may be provided to treat, prevent, or improve one or more symptoms associated with a disease, disorder, or infection by administering to the subject an effective amount of the fusion protein or

<p dir="rtl">20 The conjugated molecule of the invention, or a pharmaceutical composition comprising the fusion protein or conjugated molecule of the invention. In one preferred aspect, said compositions are substantially purified (i.e., substantially free of substances that restrict their action or produce unwanted side effects). In a specific embodiment, the subject is an animal, preferably a mammal such as a non-primate e.g., bovine, equine, feline, dog, rodent, etc. or primate (e.g., ape-e.g., baboon, human,</p>

<p dir="rtl">25 (etc.). In a preferred embodiment, the subject is a human being.</p>

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Many delivery systems are known and can be used to administer the compositions of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of transgenic expression of the antibody or fusion protein, receptor-mediated endocytosis (See, for example, Wu et al. (1987) “Receptor-Mediated In Vitro Gene

“Transformation By A Soluble DNA Carrier System,” J. Biol. Chem. 262 5

4429-4432), creating nucleic acid as part of a retrovirus or other vector, etc.

Routes of administration of the inventive molecule include, but are not limited to, non-gastrointestinal (e.g., transdermal, intramuscular, intraperitoneal, intravenous and subcutaneous) administration, epidural, and intramucosal (e.g. transnasal and oral routes) In a specific embodiment, the 10 sequence-enhanced CD123 x CD3 bispecific antibodies of the invention are administered intramuscularly, intravenously, or intravenously.

Under the skin. The compositions may be administered by any of the appropriate routes, e.g., by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucus, rectal and intestinal mucus, etc.) and may be administered in combination with other biologically active agents. Administration can be systemic or topical. In addition, transpulmonary administration can be used, for example, using an inhalation or nebulization method, and an agent formulation.

Aerosol. See, for example, US Patent Nos. 6,019,968; 55,985, 320; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; International Application No. 19244/92 was published; International Application No. 32572/97; 44013/97; 31346/98; and 66903/99, each of which is used by reference in its entirety herein.

<p dir="rtl">20 The invention also demonstrates that the sequence-enhanced CD123 x CD3 dimers of the invention are packaged in a hygienic sealed container such as an ampoule or a zipper bag indicating the quantity of those molecules. In one embodiment, the sequence-enhanced CD123 x CD3 dimers of the invention are supplied as a lyophilized powder or water-free concentrate product in a hygienic sealed container and can be reconstituted, e.g., with water or saline to a concentration appropriate for administration. To submissive. Preferably, the supply is done with binary specific objects</p>

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Sequence-enhanced CD123 At least one microgram, at least 100 micrograms, or at least 200 micrograms.

<p dir="rtl">5 Sequence-enhanced CD123 Within 3 hours, or within 1 hour after reshaping. In an alternative embodiment, the sequence-enhanced CD123 x CD3 bispecific dimers of the invention are supplied in liquid form in</p>

<p dir="rtl">10 A hygienic sealed container indicating the quantity and concentration of the molecule, fusion protein, or attached molecule. Preferably, the liquid form of the sequence-enhanced CD123 x CD3 dimers of the invention is supplied in a hygienic sealed container in which the molecules are present at a concentration of at least 1 μg/ml, preferably at least 2.5 μg/ml, At least 5 mcg/ml, at least 10 mcg/ml, at least 50 mcg/ml, or 100 mcg/ml</p>

<p dir="rtl">15 At least garm/ml.</p>

The amount of the composition of the invention that is effective in treating, preventing or alleviating one or more symptoms associated with the disease can be determined by standard clinical techniques. The specific dose to be used in the formula also depends on the method of administration and the seriousness of the condition, and must be decided in accordance with the instructions of the practitioner and the circumstances of each patient. Effective doses can be estimated inductively from curves

<p dir="rtl">20 Dose response derived from in vitro and animal model test systems.</p>

For the sequence-enhanced CD123 0.9 nano ARM/kg per day, from at least about 1 nano ARM/kg per day to about 25 3 nano ARM/kg per day, from at least about 3 nano ARM/kg per day to about

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<p dir="rtl">9 Nanograms/kg per day, from at least about 10 nanograms/kg per day to about 30 nanograms/kg per day, from at least about 30 nanograms/kg per day to about 90 nanograms/kg Kg per day, from at least about 100 nanograms/kg per day to about 300 nano</p>

nm/kg per day, from at least about 200 nm/kg per day to about 600 nm/kg

<p dir="rtl">5 nm/kg per day, from at least about 300 nm/kg per day to about 900 nm/kg</p>

nm/kg per day, from at least about 400 nm/kg per day to about 800 nm/kg

Garm/kg per day, from at least about 500 nanogarm/kg per day to about 1000 nanograms

nm/kg per day, from at least about 600 nm/kg per day to about 1000 nm/kg

nm/kg per day, from at least about 700 nm/kg per day to about 1000 nm/kg

<p dir="rtl">10 Nanograms/kg per day, from at least about 800 nanograms/kg per day to about 1,000 nanograms/kg per day, from at least about 900 nanograms/kg per day to about 1,000 nanograms/kg kg per day, or at least about 1,000 nanograms/kg per day.</p>

In another embodiment, the patient is administered a treatment regimen comprising one or more doses of such a prophylactically or therapeutically effective amount of the enhanced CD123 x CD3 bispecific antibody.

<p dir="rtl">15 In the sequence included in the invention, the treatment regimen is given over 2 days, 3 days, 4 days, 5 days, 6 days or 7 days. In certain embodiments, the treatment regimen comprises intermittent dosing of a prophylactically or therapeutically effective amount of CD123 4 of a specific week and not giving doses of the effective preventive or therapeutic amount of</p>

<p dir="rtl">20 Bispecific CD123 system or a different system.</p>

In another embodiment, the administered dose escalates over the first quarter, first half, first two-thirds, or three-quarters of the regimen(s) (e.g., over the first, second, or

<p dir="rtl">25 The first two-thirds of treatment with four courses) until the effective amount is achieved preventively or therapeutically daily</p>

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of the CD123 x CD3 dual-specific antibodies enhanced by the sequence included in the invention.

Table 1 provides 5 examples of the different dosing regimens described above for a typical course of treatment.

<tr><td colspan="7"><p dir="rtl">Table 1</p></td></tr><tr><td colspan="5"><p dir="rtl">Dosages from the body double (body double in nanograms per kg of the subject's weight per day)</p></td><td><p dir="rtl">today</p></td><td><p dir="rtl">the system</p></td></tr><tr><td><p>100</p></td><td><p>100</p></td><td><p>100</p></td><td><p>100</p></td><td><p>100</p></td><td><p>,3 ,2 ,1</p><p>4</p></td><td rowspan="2"><p>1</p></td></tr><tr><td><p dir="rtl">nothing</p></td><td><p dir="rtl">nothing</p></td><td><p dir="rtl">nothing</p></td><td><p dir="rtl">nothing</p></td><td><p dir="rtl">nothing</p></td><td><p>7 ,6 ,5</p></td></tr><tr><td><p>1000</p></td><td><p>900</p></td><td><p>700</p></td><td><p>500</p></td><td><p>300</p></td><td><p>,3 ,2 ,1</p><p>4</p></td><td rowspan="2"><p>2</p></td></tr><tr><td><p dir="rtl">nothing</p></td><td><p dir="rtl">nothing</p></td><td><p dir="rtl">nothing</p></td><td><p dir="rtl">nothing</p></td><td><p dir="rtl">nothing</p></td><td><p>7 ,6 ,5</p></td></tr><tr><td><p>1000</p></td><td><p>900</p></td><td><p>700</p></td><td><p>500</p></td><td><p>300</p></td><td><p>,3 ,2 ,1</p><p>4</p></td><td rowspan="2"><p>3</p></td></tr><tr><td><p dir="rtl">nothing</p></td><td><p dir="rtl">nothing</p></td><td><p dir="rtl">nothing</p></td><td><p dir="rtl">nothing</p></td><td><p dir="rtl">nothing</p></td><td><p>7 ,6 ,5</p></td></tr><tr><td><p>1000</p></td><td><p>900</p></td><td><p>700</p></td><td><p>500</p></td><td><p>300</p></td><td><p>,3 ,2 ,1</p><p>4</p></td><td rowspan="2"><p>4</p></td></tr><tr><td><p dir="rtl">nothing</p></td><td><p dir="rtl">nothing</p></td><td><p dir="rtl">nothing</p></td><td><p dir="rtl">nothing</p></td><td><p dir="rtl">nothing</p></td><td><p>7 ,6 ,5</p></td></tr>

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The dose and frequency of administration of the sequence-enhanced CD123 x CD3 dual-specific antibodies of the invention can be reduced or changed by improving the absorption and tissue penetration of the sequence-enhanced CD123 x CD3 dual-specific antibodies by modifications, for example, conversion to lipids.

<p dir="rtl">5 The doses of the sequence-enhanced CD123 x CD3 bispecific antibodies can be calculated from the invention given to the patient for use as single-agent therapy. Alternatively, the sequence-enhanced CD123 x CD3 dimers of the invention are used in combination with other therapeutic compositions and the dose administered to the patient is lower than when said molecules are used as a single therapeutic agent.</p>

<p dir="rtl">10 The pharmaceutical compositions of the invention can be administered topically to the area requiring treatment; This can be achieved, for example, but not limited to, by local infusion, by injection, or by implantation, and the aforementioned implant may be in the form of a porous, non-porous, or gelatinous material, including membranes, such as silicone rubber membranes, or fibers. Preferably, when administering a molecule of the invention, care should be taken in using substances that are not absorbed by the molecule.</p>

<p dir="rtl">15 The compositions of the invention may be delivered in vesicle form, specifically in the form of a liposome</p>

Langer (1990) “New Methods Of Drug Delivery,” Science 249:1527- see (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); Lopez-Berestein, ibid., pp. 3 17-327; see generally ibid.

<p dir="rtl">20 The compositions of the invention may be delivered in a controlled or sustained release system. Any technology known to a person skilled in the art may be used to produce extended-release dosage formulations comprising one or more sequence-enhanced CD123 x CD3 bispecific antibodies of the invention. See, for example, US Patent No. 4,526,938, International Application No. PCT 91/05548 publication, International Application No. 96/05548 PCT publication, and Ning</p>

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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

<p dir="rtl">5 Science &amp; Technology 50:372-397, Cleek et al. (1997) “Biodegradable</p>

Polymeric Carriers For A bFGF Antibody For Cardiovascular Application,”

Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854; and Lam et al.

<p>(1997) “Microencapsulation Of Recombinant Humanized Monoclonal Antibody For Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact.</p>

<p dir="rtl">10 24:759-760.Mater, each of which is used by reference throughout this document. In one</p>

Models, a pump may be used in controlled release systems (see Langer, supra

Sefton, (1987) “Implantable Pumps,” CRC Crit. Rev. Biomed. Eng.

Buchwald et al. (1980) “Long-Term, Continuous 240-14:201, and

Intravenous Heparin Administration By An Implantable Infusion Pump In Ambulatory Patients With Recurrent Venous Thrombosis,” Surgery 15

88:507-516, and Saudek et al. (1989) “A 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 achieve

Medical Applications of Controlled Release, controlled release of molecules (see

And, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974) 20

Controlled Drug Bioavailability, Drug Product Design and Performance,

Levy et al. (1985) and Smolen and Ball (eds.), Wiley, New York (1984) “Inhibition Of Calcification Of Bioprosthetic Heart Valves By Local

Controlled-Release Diphosphonate,” Science 228:190-192; During et al.

<p>(1989) “Controlled Release Of Dopamine From A Polymeric Brain Implant: 25</p>

Howard et al. And, “In Vivo Characterization,” Ann. Neurol. 25:351-356

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<p>(1989) “Intracerebral Drug Delivery In Rats With Lesion-Induced Memory.”</p>

(105-112:(1)7 .Deficits,” J. Neurosurg, US Patent No. 5,679,377, 5,916,597, 5,912,015, 5,989,463, and 5,128,326, and Application Publication No. 99/15154. PCT Publication No. 99/20253. Examples include:

<p dir="rtl">5 Polyart used in extended-release formulations, including, but not limited to, poly</p>

<p dir="rtl">(2-hydroxy ethyl methacrylate) (poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethyl-co-vinyl acetate) (ethylene-co-vinyl (acetate), poly(methacrylic acid), polyacrylamide</p>

<p dir="rtl">10 polyanhydrides, poly(ethylene glycol), polylactides</p>

(poly(lactide-co-glycolides) (PLA), poly(lactide-co-glycolides) (PLGA), polyorthoesters. In yet another embodiment, the controlled release system may be located near The therapeutic target such as lungs, which

Goodson, in Medical Applications of Requires a portion of the systemic dose (see

In another embodiment, Controlled Release, supra, vol. 2, pp. 115-138 (1984) 15

Use of useful polymeric formulations as controlled release primers according to Dunn et al (see US Patent No. 5,945,155). The specific method is based on the therapeutic effect in the form of controlled release in situ of the bioactive substance from the polymer system. Implantation in general anywhere in the patient's body that needs treatment

<p dir="rtl">20 Using a non-polymeric extended-release delivery system, where a non-polymeric implant is used in the subject’s body as a drug delivery system. Upon implantation, the organic solvent of the implant diffuses, disperses, or seeps from the composition into the surrounding tissue fluid, and the nonpolymeric material gradually coagulates or precipitates to form a solid or microporous material (see US Patent No. 5,888,533).</p>

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Langer (1990, “New Methods of Controlled Release System” is described in Ref

(249:1527-1533 Drug Delivery,”Science. Any technique known to those skilled in the art may be used to prepare sustained-release formulations comprising one or more therapeutic agents of the invention. See, for example, US Patent No. 4,526,938, and International Publications

<p dir="rtl">5 Nos. 05548/91, 20698/96, and Ning et al. (1996) “Intratumoral</p>

“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

<p dir="rtl">10 50:372-397, and Cleek et al. (1997) “Biodegradable Polymeric Carriers</p>

“For A bFGF Antibody For Cardiovascular Application,” Pro. Int'l. Symp.

Lam et al. (1997) and,Control. Rel. Bioact. Mater. 24:853-854 “Microencapsulation Of Recombinant Humanized Monoclonal Antibody For

“Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater.

<p dir="rtl">15 24:759-760, each of which is incorporated by reference in its entirety herein.</p>

When the composition of the invention is DNA encoding a CD123 x CD3 di-specific body enhanced by the sequence of the invention, the DNA can be administered into the organism to enhance the genetic expression of the CD123 x CD3 di-specific body enhanced with its coding sequence, by constructing it as a fragment. of the appropriate DNA gene expression vector and administering it so that 20 are present inside the cell, i.e. using a retroviral vector (see US Patent No. 4,980,286), or

By direct injection, by microparticle bombardment (e.g., gene drive, Biolistic, Dupont), by encapsulation with lipids, cell surface receptors, or transfectants, or by co-administration with a homologous box-shaped peptide known to enter the nucleus (See, for example, Joliot et al. (1991) “Antennapedia Homeobox

Peptide Regulates Neural Morphogenesis,” Proc. Natl. Acad. Sci. USA 25

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88:1864-1868), etc. Alternatively, DNA can be introduced into cells and incorporated into the DNA of the host cell for genetic expression with the product of homozygous recombination.

Treatment of the subject using a therapeutically or prophylactically effective amount of the sequence-enhanced CD123

<p dir="rtl">5 It includes a series of treatments. In a preferred example, the subject is treated with the sequence-enhanced CD123 And more preferably also for a period of about 4, 5, or 6 weeks. In other embodiments, the pharmaceutical compositions of the invention may be administered once a day, or twice a day</p>

<p dir="rtl">10 a day, or three marts a day. Alternatively, pharmaceutical formulations may be given once a week, twice a week, once every two weeks, once a month, once every six months, once every two months, twice a year or once a year. It can also be realized that the effective dose of molecules used in treatment can be increased or decreased in the specified treatment period.</p>

<p dir="rtl">6- Uses of the compositions of the invention The CD123 x CD3 binary antibodies are bispecific</p>

<p dir="rtl">15 Enhanced sequences of the present invention have the ability to treat any disease or condition associated with or characterized by the expression of CD123. Thus, without limitation, such molecules may be used in the diagnosis or treatment of acute myeloid leukemia (AML) and chronic myelogenous leukemia (CML), including metastatic crisis CML (chronic myelogenous leukemia). (And the associated Abelson oncogene</p>

<p dir="rtl">20 Chronic myelogenous leukemia (Bcr-ABL translocation), myelodysplastic syndrome (MDS), B-ALL, chronic lymphocytic leukemia (CLL). , including Richter's syndrome or Richter's transformation into chronic lymphocytic leukemia.</p>

<p dir="rtl">25 CLL (hairy cell leukemia) HCL (hairy cell leukemia)</p>

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blastic plasmacytoid dendritic cell neoplasm diffuse plasmacytoid cell neoplasm

5 Systemic mastocytosis (BPDCN), non-Hodgkin lymphomas (NHL), mantel cell leukemia (MCL), small lymphocytic lymphoma (SLL). Burkitt's lymphoma (see example 2); small lymphocytic lymphoma (SLL), allergy, asthma, and rheumatoid arthritis.

rheumatoid arthritis. The bispecific dimers of the present invention can be further used in the manufacture of drugs to treat the conditions described above.

<p dir="rtl">10 Now that the invention has been described in general terms, it will be more easily understood by cross-referencing the following examples, which are provided by way of illustration and are not intended to be limiting to the present invention unless otherwise specified.</p>

Example 1

Construction of CD123 x CD3 bispecific antibodies and comparative protein

<p dir="rtl">15 Table 2 contains a list of expressed and purified bispecific dimers. The sequence-enhanced CD123 x CD3 dual-specific antibody (DART-A) and the non-sequence-enhanced CD123 x CD3 dual-specific antibody (DART-B) can bind simultaneously to CD123 and CD3. The dual-specific antibody (DART conjugate) can simultaneously bind to FITC and CD3 are heterodimeric</p>

<p dir="rtl">20 Homologous to the aforementioned amino acid sequences. Methods for forming bispecific bi-objects are presented in International Application No. 2006/113665, International Application No. 2008/157379, International Application No. 2010/080538, International Application No. 2012/018687, International Application No. 2012/162068 and International Application No. 2012/162067.</p>

Table 2:

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<tr><td colspan="3"><p dir="rtl">Table 2</p></td></tr><tr><td><p dir="rtl">Sequences that encode DNA</p></td><td><p dir="rtl">Amino acid sequences of a polypeptide chain</p></td><td><p dir="rtl">Binary objects are binary</p></td></tr><tr><td><p dir="rtl">Sequence with ID number:</p><p dir="rtl">2</p><p dir="rtl">Sequence with ID number:</p><p dir="rtl">4</p></td><td><p dir="rtl">Sequence with identity number: 1</p><p dir="rtl">Sequence with ID number:</p><p dir="rtl">3</p></td><td><p dir="rtl">Dual-specificity CD123 x CD3 sequence-enhanced (DART-A) antibody (binds to CD3 at epitope 1)</p></td></tr><tr><td><p dir="rtl">Sequence with ID number: 6</p><p dir="rtl">Sequence with ID number:</p><p dir="rtl">8</p></td><td><p dir="rtl">Sequence with identity number: 5</p><p dir="rtl">Sequence with ID number:</p><p dir="rtl">7</p></td><td><p dir="rtl">Unenhanced CD123 x CD3 bispecific antibody</p><p dir="rtl">With the DART-B sequence (binds to CD3 at epitope 2)</p></td></tr><tr><td><p dir="rtl">Sequence with ID number: 10</p><p dir="rtl">Sequence with ID number: 4</p></td><td><p dir="rtl">Sequence with ID number: 9</p><p dir="rtl">Sequence with ID number:</p><p dir="rtl">3</p></td><td><p dir="rtl">Bispecific CD123 x CD3 antibody enhanced by a sequence having an albumin-binding domain (DART-A)</p><p>w/ABD)</p><p dir="rtl">(Binds to CD3 at epitope 1)</p><p dir="rtl">It contains an albumin-binding domain (ABD) to extend its half-life in the organism</p></td></tr>

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<tr><td colspan="3"><p dir="rtl">Table 2</p></td></tr><tr><td><p dir="rtl">Sequences that encode DNA</p></td><td><p dir="rtl">Amino acid sequences of a polypeptide chain</p></td><td><p dir="rtl">Binary objects are binary</p></td></tr><tr><td><p dir="rtl">Sequence with ID number: 12</p><p dir="rtl">Sequence with ID number:</p><p dir="rtl">14</p><p dir="rtl">Sequential with ID number: 16</p></td><td><p dir="rtl">Sequential ID number: 54</p><p dir="rtl">Sequence with ID number:</p><p dir="rtl">13</p><p dir="rtl">Sequential with ID number: 15</p></td><td><p dir="rtl">The dual body is a dual quality</p><p dir="rtl">CD123 x CD3 sequence-enhanced</p><p dir="rtl">It has an IgG Fc copy 1 domain</p><p>(1 copy DART-A w/Fc)</p><p dir="rtl">(Binds to CD3 at epitope 1)</p><p dir="rtl">It contains an Fc domain to extend its half-life in the organism</p></td></tr><tr><td><p dir="rtl">Sequence with ID number: 12</p><p dir="rtl">Sequential ID number: 18</p><p dir="rtl">Sequence with ID number:</p><p dir="rtl">2</p></td><td><p dir="rtl">Sequential ID number: 54</p><p dir="rtl">Sequential ID number: 17</p><p dir="rtl">Sequence with identity number: 1</p></td><td><p dir="rtl">The dual body is a dual quality</p><p dir="rtl">CD123 x CD3 sequence-enhanced</p><p dir="rtl">It has an IgG Fc copy 2 domain</p><p>(2 DART-A w/Fc version)</p><p dir="rtl">(Binds to CD3 at epitope 1)</p><p dir="rtl">It contains an Fc domain to extend its half-life in the organism</p></td></tr><tr><td></td><td><p dir="rtl">Sequential ID number: 19</p></td><td><p dir="rtl">Comparative DART (or DART Comparative)</p></td></tr>

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<tr><td colspan="3"><p dir="rtl">Table 2</p></td></tr><tr><td><p dir="rtl">Sequences that encode DNA</p></td><td><p dir="rtl">Amino acid sequences of a polypeptide chain</p></td><td><p dir="rtl">Binary objects are binary</p></td></tr><tr><td></td><td><p dir="rtl">Sequence with ID number:</p><p dir="rtl">20</p></td><td><p dir="rtl">(Binds to CD3 at epitope 1)</p><p dir="rtl">(Linked to unrelated target FITC)</p></td></tr>

Example 2

Antibody Labeling Of Target Cells for FACS

(QFACS) Quantitative FACS

A total of 106 target cells were collected from the culture and resuspended in 10% serum AB

<p dir="rtl">5 Human cells in a buffer solution of 0.1% NaAzide + 1% BSA + PBS (FACS) and incubated for 5 minutes to block Fc receptors. The antibody was encoded from microspheres with different potencies.</p>

(Quantum™ Simply Cellular® (QSC), Bangs for binding to anti-PE antibody and target cells labeled with antibody Laboratories, Inc., Fishers, IN)

for CD123 from BD Biosciences according to the manufacturer's instructions. In short, a drop was added

<p dir="rtl">10 One of each QSC microsphere was transferred to a 5 ml polypropylene tube and PE-encoded anti-CD123 antibody at a concentration of 1 μg/ml was added to each target cell and microspheres. The tubes were incubated in the dark for 30 minutes at 4°C. Cells and microspheres were washed by adding 2 ml of FACS buffer and centrifuged at 2500 × G for 5 minutes. And it was added</p>

<p dir="rtl">15 One drop of fine ball set mold after washing. The microspheres were first analyzed on a flow cytometer to adjust the instrument settings specified by the test voltages and compensation values.</p>

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Using the same instrument settings, the geometric mean fluorescence values of the microspheres and target cells were recorded. A standard curve of the antibody binding sites on the microsphere clusters was constructed from the geometric mean of the fluorescence of the microsphere clusters. The antibody binding sites on the target cells were calculated based on the geometric mean of the cells' fluorescence

<p dir="rtl">5 Target using the microsphere standard curve in the QuickCal spreadsheet</p>

.(Bangs Laboratories)

To identify suitable target cell lines for evaluation of CD123 x bispecific antibodies

CD3, CD123 surface expression levels on target strains - Kasumi (TF-1 (Erythroleukemia), THP-1 (AML), Molm13 (AML), 3 (AML), and RS4 10 11 (ALL)) were evaluated by FACS) ) Quantitative. Absolute numbers of body binding positions were calculated

Anti-CD123 on the cell surface using a QFACS kit. As shown in Table 3, the absolute numbers of CD123 antibody binding sites on cell lineages were on the order of 3-Kasumi (high) greater than Molm13 (medium) greater than 1-THP (medium) greater than 1-TF (medium low). Greater than RS4-11 (low). The three highest expression cell lines were

15 MOLM13, Kasumi-3:AML, and 1-THP cell lines. and non-AML cell lineages: 1-TF and 11-RS4 had moderate-low/low expression of CD123, respectively.

<tr><td colspan="2"><p dir="rtl">Table 3</p></td></tr><tr><td><p dir="rtl">Cell lineage</p></td><td><p dir="rtl">Surface expression of CD123</p></td></tr><tr><td><p dir="rtl">Targeted</p></td><td><p dir="rtl">(antibody binding sites)</p></td></tr><tr><td><p>Kasumi-3</p></td><td><p>118620</p></td></tr><tr><td><p>Molm13</p></td><td><p>27311</p></td></tr><tr><td><p>THP-1</p></td><td><p>58316</p></td></tr><tr><td><p>TF-1</p></td><td><p>14163</p></td></tr>

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<tr><td><p>RS4-11</p></td><td><p>957</p></td></tr><tr><td><p>A498</p></td><td><p dir="rtl">negative</p></td></tr><tr><td><p>HT29</p></td><td><p dir="rtl">negative</p></td></tr>

Example 3

CTL cytotoxicity test (LDH release test)

Adherent target tumor cells were detached with 0.25% EDTA-trypsin solution and collected by centrifugation at 1000 rpm.

<p dir="rtl">5 For 5 minutes. Target cell lines were collected in suspension from the culture and washed with the test medium. Cell concentration and viability were measured by trypan blue exclusion using a Beckman Coulter Vi-Cell count. The target diluted cells were diluted to 4 x 105 cells/ml in diluted cell medium. 50 μL of diluted cell suspension was added to a 96-well cell culture dish (bottom cell culture U).</p>

(BD Falcon Cat#353077) 10

Three sets of comparators were prepared to measure maximum target release (MR) and antibody independent cytotoxicity.

spontaneous release and immediate release of the target cell (AICC) cellular cytotoxicity

SR((as follows:

<p dir="rtl">15 MR: 200 microliters of test medium without CD123 x bispecific antibodies</p>

CD3 and 50 μl target cells; The detergent was added at the end of the experiment to determine the maximum LDH release.

2) AICC: 50 microliters of test medium without CD123 x 50 microlitres, CD3 x 50 microliters of target cells and 100 microliters with T.

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3) SR: 150 μl of medium without CD123 x CD3 bispecific antibodies and 50 μl target cells.

Bispecific dimers (DART-A w/ABD, CD123 x CD3, and DART-A) and conjugates were initially diluted to a concentration of 4 μg/ml, and then 5 serial dilutions were prepared to a final concentration of 0.00004. nanograms/ml (i.e., 40 microns).

carm/ml). 50 μL of dilution solutions were added to the dish containing 50 μL target cells/eye.

Purified T cells were washed once with test medium and resuspended in test medium at a cell density of 2 × 106 cells/ml. 2 × 105 T cells in 100 μl were added to

<p dir="rtl">10 Each eye, for a final neurotransmitter-to-target cell (E:T) ratio of 10:1. The plates were incubated for approximately 18 hours at 37°C in 5% carbon dioxide.</p>

(CO2).

After following incubation, 25 μL of 10× Promega #G182A dissolution solution (or 1 mg/mL digitonin) was added to control eyes with

<p dir="rtl">15 For maximum release, they were mixed by pipetting 3 times and the plates were incubated for 10 minutes to completely lyse the target cell. The plates were centrifuged at 1200 rpm for 5 minutes and 50 μL of supernatant solution from each test plate was transferred to a flat-bottomed ELISA dish and 50 μL of Promega LDH substrate solution (#G1780) was transferred to each eye. Bake dishes for 10-20 minutes at room temperature</p>

<p dir="rtl">20 RT (room temperature) in the dark, after which 50 μL of stop solution was added. Optical density (OD) was measured at 490 nm within 1 hour on a Perkin Elmer plate reader (Victor2 Multilabel #1420-014). Percent cytotoxicity was calculated as described below and dose-response curves were generated using software</p>

.GraphPad PRISM5®

<p dir="rtl">25 The analysis of specific cells was calculated from the OD data using the following equation:</p>

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Cytotoxicity (%) = 100 × (OD of sample - OD of AICC) / (OD of MR - OD of SR)

Redirected killing of target cell lineages at different levels of CD123 surface levels

CD123

<p dir="rtl">5 mL, regardless of the determination of epitope binding for DART-A (CD3 vs. DART-B) in target cell lineages with high CD123 expression, Kasumi nanogar/mL (EC50=3-0.01) (Figure 4 Group D), Expression of CD123 on medium, Molm13 = 0.18 (EC50 nanograms/ml) and THP = 0.18 nanograms/ml (Figure 4, CandE group, respectively) and expression on medium is low or expression of CD123</p>

<p dir="rtl">10 Low, TF=0.46 (EC50 nG/mL) and RS4=0.5-11 (EC50 nG/mL) (Figure 4, group B and A, respectively). Similarly, killing was also observed The CD123 x CD3 bispecific molecules mediated redirection to multiple target cell lineages with T cells from different donors and no redirection killing efficacy was observed in cell lines that do not express CD123. The results are summarized in Table 4.</p>

<p dir="rtl">15 Table 4</p><table border="1"><tbody><tr><td colspan="4"><p dir="rtl">Table 4</p></td></tr><tr><td><p dir="rtl">% for maximum kill</p></td><td><p>EC50</p><p dir="rtl">For sequence-enhanced CD123 x CD3 bispecific antibodies</p><p dir="rtl">(nanoarms/ml)</p><p>10:1=E:T</p></td><td><p dir="rtl">Expression on the surface of CD123)</p><p dir="rtl">Antibody binding sites (</p></td><td><p dir="rtl">strain</p><p dir="rtl">target cell</p></td></tr></tbody></table>

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<tr><td><p>94</p></td><td><p>0.01</p></td><td><p>118620</p></td><td><p>Kasumi-</p><p>3</p></td></tr><tr><td><p>43</p></td><td><p>0.18</p></td><td><p>27311</p></td><td><p>Molm13</p></td></tr><tr><td><p>40</p></td><td><p>0.24</p></td><td><p>58316</p></td><td><p>THP-1</p></td></tr><tr><td><p>46</p></td><td><p>0.46</p></td><td><p>14163</p></td><td><p>TF-1</p></td></tr><tr><td><p>60</p></td><td><p>0.5</p></td><td><p>957</p></td><td><p>RS4-11</p></td></tr><tr><td><p dir="rtl">Without activity</p></td><td><p dir="rtl">Without activity</p></td><td><p dir="rtl">negative</p></td><td><p>A498</p></td></tr><tr><td><p dir="rtl">Without activity</p></td><td><p dir="rtl">Without activity</p></td><td><p dir="rtl">negative</p></td><td><p>HT29</p></td></tr>

If it were necessary to repeat this example, we would realize that a person skilled in the art could, within reasonable and acceptable limits, vary the protocol described above in a way appropriate to replicate the results described. Therefore, it is not intended to adhere to the specified protocol in a way that is fully compliant with it.

<p dir="rtl">5 Example 4: T cell activation during redirected killing by sequence-enhanced CD123 x CD3 dimers (DART-A w/ABD, DART-A and DART-A w/Fc)</p>

Sequence-optimized CD123 Expressive

<p dir="rtl">10 High CD123, 3-Kasumi, and middle, 1-THP, and expression of CD123 in the middle, (Figure 5,</p>

Groups A and B, respectively) To characterize T cell activation during redirected killing mediated by a sequence-enhanced CD123 As shown in Figure 5, CD25,D group was increased in a controlled manner in cells

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CD8 T cells in a dose-dependent manner suggesting that sequence-enhanced CD123 x CD3 dimers induce T cell activation in the process of redirected killing. Conversely, in the absence of target cells there is no activation of CD8 T cells (Figure 5, group C), indicating that CD123

<p dir="rtl">5 Consequently, T cells do not activate in the absence of target cells. Likewise, CD8 T cells are not activated when incubated with target cell and dual-specific antibody recombinant (DART conjugate) (Figure 5, group D), indicating that cross-linking of T cell and target cell with sequence-optimized dual-specificity CD123 x CD3 dimers is required. .</p>

Example 5: Intracellular staining of granzyme B and perforin

<p dir="rtl">10 To determine intracellular levels of granzyme B and perforin in T cells, a CTL assay was set up as described above. After approximately 18 hours, cells from the test dish were immunostained with anti-CD4 and anti-CD8 antibodies by incubating for 30 minutes at 4°C. After staining at the surface, cells were incubated in 100 μl of BD BioSciences fixation and permeabilization buffer solution for 20 min at 4°C. The cells were washed with buffer solution</p>

<p dir="rtl">15 Cells were then washed with 250 μl of perforin antibodies (prepared in 1X permeabilization/washing buffer) BD BioSciences. permeabilization/wash and were resuspended in FACS acquisition.</p>

<p dir="rtl">20 Controlled increase of L-arenzyme B and perforin by sequence-enhanced dual-specific CD123 x CD3 (DART-A) in T cells during redirected killing</p>

.Redirected Killing

To test the possible mechanism of CD123 x DART-A(CD3)-mediated cytotoxicity mediated by T cells, the levels of G-arenzyme B and perforin were measured.

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Intracellular T cells after redirected killing. A dose-dependent, controlled increase of corenzyme B and perforin levels was observed in both CD8 and CD4 T cells after incubation of T cells and 3-Kasumi cells with DART-A (Figure 6, panel A). Interestingly, the controlled increase was almost twice as much in CD8 T cells compared to CD4 T cells (Figure 6, 5, panel A). When the test was performed in the presence of the inhibitors of granzyme B and perforin, no cell killing was observed.

There was no controlled increase of G or perforin in CD8 or CD4 T cells when the T cells were incubated with 3-Kasumi target cells and the dual-specific antibody conjugate (DART conjugate) (Figure 5, panel B). These data indicate that DART-A-mediated killing of target cells may be mediated through the mechanisms of arenzyme B and perforin.

<p dir="rtl">10 Example 6: In vivo antitumor efficacy of the CD123 x CD3 sequence-enhanced Bi-Specific Diabody (DART-A).</p>

Isolation of PBMCs and T cells from human whole blood

PBMCs isolated from healthy human volunteers were isolated from whole blood using Ficoll gradient centrifugation. Briefly, whole blood was diluted 1:1 with sterile PBS. Fifteen layers of fifty-three milliliters of diluted blood were made on 15 milliliters of Ficoll-PaqueTM.

Plus in 50 ml tubes and the tubes were subjected to centrifugation at 1400 rpm for 20 minutes with the separator stopped. The interphase buffy coat was collected in a 50 ml tube and washed with 45 ml of PBS by centrifuging the tubes at 600× g (1620 rpm) for 5 min. The supernatant was discarded and the cell pellet was washed once 20 times with PBS and live cell counts were determined by Trypan Blue staining and resuspended

PBMCs to a final concentration of 2.5 × 106 cells/ml in complete medium (1640 RPMI,

<p dir="rtl">10 2% FBS, 10 mM Glutamine, 100 mM Glutamine, 100 mM HEPES / 100 mM Penicillin / Streptomycin P/S (Streptomycin).</p>

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T cell isolation: Unused T cells were isolated by negative selection from PBMCs from human whole blood using the Dynabeads Untouched Human T Cell Isolation Kit (Life Technologies) according to the manufacturer's instructions. After isolation, T cells were cultured overnight in RPMI medium with 10 %1%, FBS penicillin / Streptomycin

5 Tumor model: Human T cells and tumor cells (Molm13 or 11-RS4) were combined at a 1:5 ratio (1 × 106 and 5 × 106, respectively), resuspended in 200 μL of sterile saline, and injected subcutaneously (s.c. On study day zero (SD0). The sequence-enhanced CD123 microliter as

10 It is shown in Table 5 (MOLM13) and Table 6 (RS4-11).

<tr><td colspan="4"><p dir="rtl">Table 5</p><p dir="rtl">Study design according to the MOLM13 model</p></td></tr><tr><td><p dir="rtl">number</p><p dir="rtl">the animals</p></td><td><p dir="rtl">timetable</p></td><td><p dir="rtl">Dosage mg/kg</p></td><td><p dir="rtl">Treatment group</p></td></tr><tr><td><p>8</p></td><td><p>SD0, 1,</p><p>2, 3</p></td><td></td><td><p dir="rtl">Comparator (13-MOLM cells infused alone or + T cells)</p></td></tr><tr><td><p>8</p></td><td><p>SD0, 1,</p><p>2, 3</p></td><td><p>0.5</p></td><td><p>DART-A</p></td></tr><tr><td><p>8</p></td><td><p>SD0, 1,</p><p>2, 3</p></td><td><p>0.2</p></td><td><p>DART-A</p></td></tr>

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<tr><td><p>8</p></td><td><p>SD0, 1,</p><p>2, 3</p></td><td><p>0.1</p></td><td><p>DART-A</p></td></tr><tr><td><p>8</p></td><td><p>SD0, 1,</p><p>2, 3</p></td><td><p>0.02</p></td><td><p>DART-A</p></td></tr><tr><td><p>8</p></td><td><p>SD0, 1,</p><p>2, 3</p></td><td><p>0.004</p></td><td><p>DART-A</p></td></tr><tr><td><p>8</p></td><td><p>SD0, 1,</p><p>2, 3</p></td><td><p>0.0008</p></td><td><p>DART-A</p></td></tr><tr><td><p>8</p></td><td><p>SD0, 1,</p><p>2, 3</p></td><td><p>0.00016</p></td><td><p>DART-A</p></td></tr>

<tr><td colspan="4"><p dir="rtl">Table 5</p><p dir="rtl">Study design for Model 11-RS4</p></td></tr><tr><td><p dir="rtl">number</p><p dir="rtl">the animals</p></td><td><p dir="rtl">timetable</p></td><td><p dir="rtl">Dosage mg/kg</p></td><td><p dir="rtl">Treatment group</p></td></tr><tr><td><p>8</p></td><td><p>SD0, 1,</p><p>2, 3</p></td><td></td><td><p dir="rtl">Comparative carrier material</p><p dir="rtl">(RS4-11 cells cultured alone)</p></td></tr><tr><td><p>8</p></td><td><p>SD0, 1,</p><p>2, 3</p></td><td></td><td><p dir="rtl">Comparative carrier material</p><p dir="rtl">(Infused RS4-11+ T cells)</p></td></tr>

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<tr><td><p>8</p></td><td><p>SDO, 1,</p><p>2, 3</p></td><td><p>0).2</p></td><td><p dir="rtl">DART comparison</p></td></tr><tr><td><p>8</p></td><td><p>SDO, 1,</p><p>2, 3</p></td><td><p>0.5</p></td><td><p>DARTA</p></td></tr><tr><td><p>8</p></td><td><p>SDO, 1,</p><p>2, 3</p></td><td><p>0).2</p></td><td><p>DARTA</p></td></tr><tr><td><p>8</p></td><td><p>SDO, 1,</p><p>2, 3</p></td><td><p>0.1</p></td><td><p dir="rtl">M!L2</p></td></tr><tr><td><p>8</p></td><td><p>SDO, 1,</p><p>2, 3</p></td><td><p>0.02</p></td><td><p>Di-k</p></td></tr><tr><td><p>8</p></td><td><p>SDO, 1,</p><p>2, 3</p></td><td><p>0.004</p></td><td><p>DARTA</p></td></tr>

Data collection and statistical analysis:

Animal weights - The weights of individual animals were recorded twice a week until the end of the study, starting from the time of injection with tumor cells.

Mortality/Death - Animals were observed twice weekly for general moribund monitoring and daily

<p dir="rtl">5 To confirm that death occurred. Animal deaths were assessed as being drug-related or technical based on factors including total observation and weight loss: animal deaths were recorded daily.</p>

Tumor Volume - Individual tumor volumes were recorded twice weekly starting within 1 week of tumor implantation and continuing until study completion.

Length(mm)»Width2

Tumor size (mm) = 2

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Animals that experienced technical or drug-related deaths were censored from the data counts.

Tumor growth inhibition-tumor growth inhibition (A5)1 values were calculated for each group containing treated animals using the equation:

Average final tumor size (normalized) - Average primary tumor size (normalized) Average final tumor size (comparative) - Average primary tumor size (compared)

<p dir="rtl">5 Animals that experienced a partial or complete response, or animals that experienced technical or drug-related deaths were censored from TGI calculations. They were National Cancer Institute criteria</p>

Corbett et al. Anticancer (2004) greater than 58 TGI for compound efficacy (Drug Development Guide; Totowa, NJ: Humana 99-123).

Partial/total tumor response was classified - individual mice with tumors measuring &lt;1 mm3

<p dir="rtl">10 On day 1, it was determined that it had a partial response (PR), and the percentage of tumor regression (JR) was determined using the equation:</p>

Issal, 5/5100

Initial tumor size (HM)

Individual mice lacking palpable tumors were classified as undergoing a complete response (CR).

Statistical analyzes were performed for tumor volume between treated and comparison groups

<p dir="rtl">15 Tumor sizes. To conduct these analyses, two-way analyzes of variance followed by a Bonferroni post hoc test were used. All analyzes were performed using 855 GraphPad (version 5.02). Weight and tumor data from individual animals that experienced technical or drug-related deaths were excluded from the analysis. However, tumor data from animals in which partial or complete responses were reported were included in these calculations.</p>

<p dir="rtl">20 ΜΟΙΜ1 Results: The AML cell line from ΜΟΙΜ1 was premixed with activated 1 cells and infused subcutaneously into mice genetically induced with NOD/SCID (NSG gamma) (p:</p>

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<p dir="rtl">8/group) at SD0 as detailed above. The MOLM13 tumors in the vector-treated group (MOLM13 cells alone or in addition to T cells) showed relatively penetrant growth properties in vivo (Figure 7, groups A and B). At SD8, the average volume of tumors in the vector-treated group was 129.8 ± 29.5 mm3, and by SD15 the tumors reached an average</p>

<p dir="rtl">5 Volume 786.4 ± 156.7 mm3. By the end of the experiment at SD18, the tumors reached an average volume of 1398.8 ± 236.9 mm3.</p>

Treatment with DART-A was initiated on the same day that the tumor cell/T cell mixture [(SD0)] was infused and was then continued with daily infusions for an additional 7 days for a total of 8 injections per day. Animals were treated with DART-A on 9 Dosage levels (0.5, 0.2, 0.1, 0.02,

<p dir="rtl">10 And 0.004 mg/kg and 20, 4, 0.8 and 0.16 micrograms/kg). The results are shown in the figure</p>

<p dir="rtl">Figure 7, Group A (0.5, 0.2, 0.1, 0.02, and 0.004 mg/ kg) and Figure 7, Group B (20, 4, 0.8, and 0.16 μg/kg). By study day 11, growth of MOLM13 tumors was inhibited. With a statistically significant degree at 0.16, 0.5, 0.2, 0.1, 0.02, and 0.004 mg/kg dose levels (p &lt; 0.001). In addition to the above, treatment of mice carrying tumors resulted in</p>

<p dir="rtl">15 MOLM13 at 20 and 4 mcg/kg dose levels for 8/8 and 8/7 CRs, respectively. By the end of the trial at SD18, the average size of tumors treated with DART-A (0.8-20 μg/kg) ranged from 713.6.0 ± 267.4 to 0 mm3, and all were statistically significantly smaller than tumors in the control group. With the carrier material, the values of 100 TGI, 94, and 49% were for 20, 4, and 0.8 micrograms/kg for the dose groups, respectively</p>

<p dir="rtl">20 In the carrier-treated MOLM13 tumor cell group, the group that received DART-A at a concentration of 20 and 4 mcg/kg dose level reached statistical significance by SD15, while the group treated with 0.8 mcg/kg reached statistical significance at SD18.</p>

RS4-11 results

The RS4-11 ALL tumor cell line was pre-mixed with activated T cells and infused subcutaneously into 25 NOD/SCID gamma-mutated mice (n=8/group) at SD0 according to

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Detailed above. The RS4-11 ORM in the vector-treated group (RS4-11 cells alone or in addition to T cells) showed relatively penetrant growth properties in vivo (Figure 8).

Treatment with DART-A was initiated on the same day as the infusion of the tumor cell/T cell mixture [(SD0)] and was then continued with daily infusions for an additional 3 days for a total of 4 injections per day.

<p dir="rtl">5 Animals were treated with DART-A at 5 dose levels (0.5, 0.2, 0.1, 0.02, and 0.004 mg/kg). The results are shown in Figure 8.</p>

Sequence-enhanced CD123 or

<p dir="rtl">10 more. Based on criteria established by the National Cancer Institute, DART-A at a dose level of 0.1 mg/kg and higher (TGI &gt;58) was considered effective in the RS4-model 11 and DART-A doses of 0.004 mg/kg and higher were considered effective in the RS4 model. MOLM13 The lower DART-A doses that were associated with tumor growth inhibition in the MOLM13 model compared to the 11-RS4 model were consistent with in vitro data demonstrating that MOLM13 cells are highly differentiated.</p>

<p dir="rtl">15 Higher expression of CD123 from RS4-11 cells, which was associated with increased sensitivity to DART-A-mediated cytotoxicity in MOLM13 cells in vitro.</p>

If it is necessary to repeat this example, it should be recognized that someone skilled in the art can, within reasonable and acceptable limits, change the protocol described above in a way that is appropriate to replicate the results shown. As such, the example protocol is not intended to be adhered to inflexibly

<p dir="rtl">20 Accurately.</p>

Example 7

Surface expression of CD123 on blast cells and leukemia-related stem cells in

<p>1 AML number from a patient Stem Cells In Primary Tissue Sample</p>

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To define the expression pattern of CD123 in the primary samples of AML patient 1, cryopreserved primary bone marrow PBMC samples from the AML patient were evaluated for surface expression of CD123 on leukoblasts.

.leukemic blast cells

<p dir="rtl">5 Bone marrow sample of AML- clinical report</p>

Age: 42

Gender: Female

Subtype of M2: AML

Percentage of cancer cells based on composition: 67.5%

<p dir="rtl">10 Classification of bone marrow immunophenotypes:</p>

<p>CD45 = 81.8%, CD38 = 28.8%, CD33 = 98.5%, CD15 = 19%.</p>

<p>%0.53 = CD5, %1.8 = CD2, %17 = HLA-DR, %42.9 = CD117, %39.7</p>

%0.71 = CD22, %1.4 = CD20, %1.1 = CD19, %0.41 = CD10, %0.2 = CD7

%0.82 = CD34

<p dir="rtl">15 Expression of CD123 in leukemia-related myeloid mononuclear cells</p>

(BM MNC) Bone Marrow Mononucleocytes

A total of 0.5×106 bone marrow mononuclear cells (BM MNC Marrow Mononucleocytes) and peripheral blood mononuclear cells (PBMC) from AML patient 1 were evaluated for expression of CD123. The 3-Kasumi cell lineage was included as a sample

<p dir="rtl">20 comparison. Leukemia-related blast cells were identified using the myeloid marker</p>

CD33. As shown in Figure 9, panel A, 87% of cells from bone marrow in AML from patient 1 expressed CD123 and CD33. Expression levels of CD123 were lower

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The Kasumi-3 AML cell line highly expressed CD123 (Figure 9, panel B).

Example 8

Autologous CTL killing test using primary samples from an AML patient

<p dir="rtl">5 A primary cryopreserved AML sample was fused with bone marrow mononuclear cells (BMNC) and peripheral blood mononuclear cells.</p>

RPMI 1640 No. 1 in AML from a patient (PBMC) blood mononucleocytes

using 10% FBS and allowed to be recovered overnight at 37°C in 5% CO2. The cells were washed with test medium (1640 FBS + 10% RPMI) and the number of viable cells was determined by excluding

<p dir="rtl">10 Trypan Blue. CD123 , 0.01 ng/ml and 50 μL of each dilution was added to each eye (final volume = 200 μL). The double-specific body dilution was</p>

<p dir="rtl">15 The comparator (Control DART) was administered to 0.1 ng/ml and 50 μl of each dilution was administered to each eye (final volume = 200 μl). A separate test plate was prepared for each time point (48, 72, 120 and 144 h) and the plates were incubated at 37 h. m in 5% CO2 incubation. At each time point, cells were stained with CD33, CD45, CD25, CD8, CD4, and CD123 antibodies. Labeled cells were analyzed in a FACS Calibur flow cytometer equipped with the software</p>

<p dir="rtl">20 Cell acquisition Quest Pro, version 5.2.1 (BD Biosciences). Data were analyzed with Flowjo software version 9.3.3 (Treestar, Inc). T cell expansion was measured by gating on CD4+ and CD8+ complexes and activation was determined by measuring mean fluorescence intensity Fluorescent expression of MFI(CD25) on clusters stained by CD4+ and CD8+. The leukoplasty group was identified by CD45+CD33+ gating.</p>

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Autologous tumor cell exhaustion, T cell expansion and activation by dual-specificity CD123 x CD3 dual-specific antibody DART-A (CD123 x CD3) with orthotopic sequences in primary samples from AML patient 1

To determine the CD123 x CD3 double-specific activity

<p dir="rtl">5 (DART-A) with optimal sequences In AML patient 1, patient samples were incubated with 0.1 ng/ml or 0.01 ng/ml DART-A and the percentage of leukoblasts and T cells was measured at different time points after treatment. Identification of leukemia-related blast cells by CD33+ /D45 gating. Incubation of primary bone marrow samples from AML with DART-A led to depletion of the leukemia-related cell pool by passage</p>

<p dir="rtl">10 time (Figure 10, panel A), accompanied by a simultaneous expansion of lagging T cells (Figure 10, panel B) and the inclusion of signs of T cell activation (Figure 10, panel C). In samples treated with DART A, T cells expanded by About 7% to about 80% over 120 hours reached cell activation</p>

T measured by expression of CD25 on CD4 and CD8 cells peaked at 72 h and decreased by the 120 h time point.

<p dir="rtl">15 If it is necessary to repeat this example, it should be recognized that someone skilled in the art can, within reasonable and acceptable limits, change the protocol described above in a way that is appropriate to replicate the results shown. As such, the example protocol is not intended to be strictly adhered to inflexibly.</p>

Example 9

<p dir="rtl">20 Surface expression of CD123 on blast cells and leukemia-related stem cells in a primary tissue sample from an ALL patient</p>

To determine the expression pattern of CD123 in primary ALL patient samples, a primary cryopreserved PBMC sample from an ALL patient was evaluated for surface expression of CD123 on leukemia-related blast cells.

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Expression of CD123 in leukemia-related hematopoietic mononuclear cells

Peripheral Blood Mononucleocytes (PBMC).

A total of 0.5×106 peripheral blood mononuclear cells (PBMC) from a healthy donor and an ALL patient were evaluated for expression of CD123. As shown in Figure 11, panels e-5h, the overwhelming majority of cells from bone marrow expressed In ALL for CD123 and vice versa,

As expected, normal donor B cells are CD123 negative and pDCs and monocytes are CD123 positive (Figure 11, panel D).

The T cell population in the ALL patient sample was identified by staining of cells for CD4 and CD8. As shown in Figure 12, panel b, only a small proportion of the total PBMCs are in a sample

<p dir="rtl">10 ALL patients are T-cell-positive (about 0.5% are CD4 T cells and about 0.4% are CD8 T cells).</p>

Example 10

Autologous CTL killing test using primary ALL patient samples

The primary ALL specimen was cryopreserved with peripheral blood mononuclear cells (PBMC)

<p dir="rtl">15 at RPM I1640 using 10% FBS and allowed to be recovered overnight at 37°C in 5% CO2. Cells were washed with test medium (1640 FBS+10% RPMI) and the number of viable cells was determined by Trypan Blue exclusion. 150,000 cells/eye in 150 μl of test medium were added to a 96-well U-bottomed plate (BD Biosciences (.The CD123 x CD3 double-specific antibody was diluted</p>

<p dir="rtl">20 (DART-A) with optimized sequences was reduced to 10.1 ng/ml and 50 μL of each dilution was added to each eye (final volume = 200 μL). A separate test plate was prepared for each time point (48, 72, 120 and 144 hours). Plates were incubated at 37°C in 5% CO2 incubation and at each time point, cells were stained with antibodies to CD8, CD4, CD33, CD45, CD25, and CD123</p>

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FACS Calibur equipped with Quest Pro cell acquisition software, version 5.2.1 (BD Biosciences). Data were analyzed with Flowjo software, version 9.3.3 (Treestar, Inc). T cell expansion was measured by gating on CD4+ and CD8+ populations and activation was determined by measuring

CD25 MFI on groups opened by CD4+ and CD8+. A group has been identified

<p dir="rtl">5 Leukemia-related blast cells are characterized by CD45+CD33+</p>

Autologous tumor cell exhaustion, T cell expansion and activation with dual specificity (CD123 x CD3) DART-A with optimal sequences in primary samples from patients

ALL

To determine the CD123 x CD3 double-specific activity

<p dir="rtl">10 (DART-A) with optimal sequences in primary ALL patient samples. Patient samples were incubated with 1 ng/ml DART-A and the percentage of leukoblasts and T cells was measured at different time points after treatment. Leukemia with CD33+/CD45+ genotype Incubation of primary bone marrow samples in ALL with DART-A resulted in depletion of the leukemia-related cell pool over time compared with the sample.</p>

<p dir="rtl">15 The untreated comparison or the DART comparison (Figure 13, panel h versus panels f and g). When done</p>

Counting T cells (CD8 and CD4 staining) and testing for activation (CD25 staining), T cells expanded and were activated in the DART-A sample (Figure 14, long panels, respectively) compared to the untreated or control DART samples (Figure 14, h panels). , g, k and y, respectively (.

Example 11

<p dir="rtl">20 Surface expression of CD123 on blast cells and leukemia-related stem cells in a primary tissue sample from AML patient 2.</p>

To define the expression pattern of CD123 in primary samples from AML patient 2, cryopreserved primary samples of AML patient's bone marrow and PBMC were evaluated for surface expression of CD123 on leukemia-related blast cells.

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Expression of CD123 in leukemia-related myeloid mononuclear cells

(BMNC) bone marrow mononucleocytes

A total of 106 x 0.5 bone marrow mononuclear cells (BM MNC) and monocytes were evaluated.

AML from a patient with peripheral blood mononucleocytes (PBMC).

<p dir="rtl">5 No. 2 regarding the identification of leukemia-related blast cells. Leukemia-related blast cells were identified using myeloid markers CD33 and CD45. As shown in Figure 15, panel B, 94% of the cells in the bone marrow in AML are leukoblasts. The T cell population was identified by expression of CD3. As shown in Figure 15, panel c, the percentage of cells in the marrow sample was approximately 15%</p>

<p dir="rtl">10 Osteoblasts in AML and PBMC are T cells.</p>

Example 12

Autologous CTL killing assay using primary samples from AML patient #2

A cryopreserved primary AML sample (bone marrow mononuclear cells (mNC) and peripheral blood mononuclear cells) was fused

RPMI #2 in AML from a patient (PBMC)peripheral blood mononucleocytes 15

1640 using 10% FBS and allowed to be recovered overnight at 37°C in 5% CO2. Cells were washed with test medium (1640 FBS10%+RPMI) and the number of viable cells was determined by Trypan Blue exclusion. 150,000 cells/eye in 150 μl test medium were added to a 96-well U-bottomed plate (BD Biosciences) The body has been softened

20 Dual-specific CD123 x CD3 (DART-A) with ideal sequences and dual-specific antibody (DART comparator) were reduced to 0.1, and 0.01 ng/ml and 50 μl of each dilution were added to each eye (final volume = 200 μL). A separate test dish was prepared for each time point (48, 72, 120 and 144 h) and the plates were incubated at 37°C in a 5% CO2 incubator. At each time point, cells were stained with

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Antibodies CD33, CD45, CD25, CD8, CD4, and CD123. Labeled cells were analyzed in a FACS Calibur flow cytometer equipped with Quest Pro cell acquisition software, version 5.2.1 (BD Biosciences). Data were analyzed with Flowjo software, version 9.3.3 (Treestar, Inc). T cell expansion was measured by gating on CD4+ kits CD8+ was selected

<p dir="rtl">5 Activation measured by CD25 MFI on CD4+ and CD8+ opened groups. Leukemia-related blast cell populations were identified by CD45+CD33+ gating.</p>

Autologous tumor cell exhaustion, T cell expansion and activation in primary samples from AML patient 2

To determine the activity of the dual-specific CD123 x CD3 antibody (DART-A) with optimal sequences in primary samples from AML patient 2,

<p dir="rtl">10 Patient samples were treated with 0.1 or 0.01 ng/ml DART-A and the percentage of leukemia-related blast cells and T cells was measured at different time points after treatment. Incubation of primary bone marrow samples from AML with DART-A resulted in depletion of the leukemia-related cell pool over time (Figure 16, panel A), accompanied by a concurrent expansion of lagging T cells (both CD4 and CD8) (Figure 16, panel B and Figure 16, panel C, respectively). To determine what</p>

<p dir="rtl">15 If T cells were activated, the cells were stained for CD25 or 67-Ki, each representing a marker for T cell activation. As shown in Figure 17, panels a and b, incubation of primary bone marrow samples from AML with DART-A resulted in Activated T cells. These data represent the 144-hour time point.</p>

Intracellular staining of L-arenzyme B and perforin

<p dir="rtl">20 To determine the intracellular levels of corenzyme B and perforin in T cells, a CTL assay was set up. After approximately 18 hours, cells from the test dish were immunostained with anti-CD4 and anti-CD8 antibodies by incubating for 30 minutes at 4°C. After surface staining, cells were incubated in 100 μl of fixation and permeabilization buffer for 20 min at 4°C. Cells were washed with permeabilization/washing buffer and incubated in 50 μl of arenzyme B mixture.</p>

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and perforin antibody prepared in 1X buffer solution for permeabilization/washing at 4°C for 30 minutes. The cells were then washed with 250 μl of permeabilization/washing buffer and resuspended in permeabilization/washing buffer.

.FACS

<p dir="rtl">5 Increased G-arenzyme B and perforin are regulated by dual specificity CD123 DART-A (x CD3) with optimal sequences in T cells during redirected killing.</p>

To test the possible mechanism of cytotoxicity induced by the dual-specific antibody DART-A (CD123 x CD3) with ideal sequences by T cells, the levels of intracellular arrenzyme B and perforin were measured in T cells after redirected killing. There was no increase

<p dir="rtl">10 A significant increase in arrenzyme B and perforin levels was observed in both CD8 and CD4 T cells with the CD123 x DART conjugate. A) CD3 T cells with ideal sequences (Figure 17, panels c and d). Interestingly, the mitotic increase was approximately two times higher in CD8 T cells than in CD4 T cells.</p>

<p dir="rtl">15 (Figure 17, panel C and Figure 17, panel D). These data indicate that the killing of target cells by DART-A was due to the G-renzyme B and perforin pathway.</p>

Example 13

The heterospecific CD123 x CD3 diploid with ideal sequences interacts cross-reactively with CD123 and CD3 proteins in non-human primates.

<p dir="rtl">20 In order to quantify the extent of binding between the DART-A (CD123 x CD3) double-specific antibody with perfect sequences and CD3 in humans or baboons, BIACORETM analyzes were performed. BIACORETM analyzes measure the rate of dissociation arrest, kd. The binding affinity is (kd). KD) between an antibody and its target is a function of the kinetic constants of binding (on rate, ka) and dissociation (off rate, kd) according to the following equation: [KD = [kd]/[ka).</p>

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BIACORETM analyzes surface plasmon resonance to directly measure these kinetic variables. CD3 recombinant release from a human or a monkey was directly immobilized in a carrier.

Purified CD3 from human or baboons was captured and immobilized with a carrier. The time span of separation was measured and bivalent fit of the data was performed. Binding and affinity constants were obtained

<p dir="rtl">5 Using 1:1 link compatibility. The results of BIACORETM analyzes comparing binding to human CD123 and CD3 from baboons are shown in Figure 18. The binding affinity values for CD123 (Figure 18d) and CD3 (Figure 18b) from baboons are close to those for CD123 (Figure 18c). ) and CD3 (Figure 18a) in humans.</p>

Example 14

<p dir="rtl">10 Depletion of autologous monocytes in vitro with human PBMCs and from baboons</p>

PBMCs from human or baboons whole blood samples were added to U-bottomed dishes at a cell density of 200,000 cells/eye in 150 μl test medium. Dilutions of CD123 x DART-A (CD3 or ABD/DART-A w) duplexes with ideal sequences were prepared in the test medium.

<p dir="rtl">15 Add 50 µl of each dilution of DART-A or DART-A w/ABD to the dish containing PBMCs in paired eyes. Plates were incubated for ~18–24 h at 37°C.</p>

Supernatants were used to determine cytotoxicity as described above. As shown in Figure 19 (Panels A and B), depletion of pDCs was observed in both human PBMCs (Figure 19, Panel A) and from the baboons (Figure 19, Panel B). These results show that

<p dir="rtl">20 Use of pDC as a pharmacokinetic marker in preclinical toxicity studies in baboons.</p>

If it is necessary to repeat this example, it should be recognized that someone skilled in the art can, within reasonable and acceptable limits, change the protocol described above in a way that is appropriate to replicate the results shown. As such, the example protocol is not intended to be strictly adhered to inflexibly.

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Example 15

Depletion of plasmacytoid dendritic cells in baboons treated with dual specificity CD123 x CD3 antibody with optimal sequences (DART-A).

As part of a toxicological study to determine the dose range, baboons were given a body double

<p dir="rtl">5 Dual specificity of DART-A (CD123 x CD3) with optimal sequences as 4-day infusions at doses of 0.1, 1, 10, 30, 100, 300, or 1000 ng/kg. Comparator DART was administered at 100 ng/kg. To identify pDCs populations Monocytes in baboose monkey PBMCs, cells were labeled with the CD14-FITC antibody, monocytes were identified as the CD14 + group and pDCs were identified as the CD14 - group</p>

<p dir="rtl">10 +CD123. As shown in Figure 20 panels k and l, pDCs were exhausted after a while</p>

as early as 4 days after a low-infusion of 10 ng/kg DART-A. No pDC depletion was observed in monkeys treated with the comparator DART or vector+vector-treated monkeys at the 4-day time point (Figure 20, panels g, h, c and d, respectively). Determination of cytokine levels of interferon

<p dir="rtl">15 – Gamma, TNF-alpha, IL4, IL5, IL6 and IL2 4 hours after infusion. There was a rise</p>

There was little or no increase in cytokine levels in animals treated with DART A compared to animals treated with the comparator DART or the vector.

Figures 21 and Figure 22 show the results of FACS analysis for CD20+ B cells (Figure 21, panel A), monocytes (CD14+) (Figure 21, panel B), and CD159+CD16+ NK cells (Figure

<p dir="rtl">20 21, panel c), pDC (CD14-, CD123HI) (Figure 21, panel d), and T cells (overall,</p>

<p dir="rtl">CD4+ and CD8+ (Figure 22, panel A, Figure 22, panel B, and Figure 22, panel D, respectively).</p>

Treatment of monkeys with comparator DART had no significant effects on T or B lymphocytes, NK cells, monocytes, and pDCs. Treating monkeys with DART-A at doses of 10

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ng/kg/day or higher abolished pDCs (Figure 21, panel D). pDC depletion was complete and persistent, with a return to pre-dosing levels several weeks after dosing was completed. Circulating levels of T lymphocytes decreased upon DART-A administration , but it returned to the previous level on doses at the end of each weekly cycle, which suggests changes in passage and not real exhaustion. Both CD4 and CD8 T lymphocytes followed the same pattern, which was a sign of activation

CD69 T lymphocytes (Figure 22, panel C) were only marginally positive among DART-A cells and did not follow the effect of DART-A doses. B lymphocytes, monocytes, and NK cells fluctuated over the course of DART-A doses, with significant variation observed between monkeys. A tendency to increase DAR levels in B lymphocytes and monocytes in monkeys was observed at the highest doses.

<p dir="rtl">10 Summary of the above, the previous results demonstrate the therapeutic effect of the dual-specificity antibody (DART-A) (CD123 x CD3) with optimal sequences. The dual-specificity antibody (DART-A) (CD123 x CD3) with optimal sequences can be used as a therapeutic agent for the treatment of diseases. and multiple conditions, including: pDCL, MDS, CLL, ABL, AML, cortical cell leukemia, hairy cell leukemia, Ricter transformation of CLL, blastic attack of</p>

<p dir="rtl">15 BLL, CML (subtype is CD123+) (see example 2); autoimmune lupus (SLE), allergies (basophils are CD123+), asthma, etc.</p>

Example 16

Comparative characteristics of the DART-CD123 x CD3 A dimer with ideal sequences and the CD123 x CD3 A-specific dimer

<p dir="rtl">20 (DART-B) Non-ideal sequences</p>

Unexpected advantage and characteristic of CD123 x CD3 duplexes with perfect sequences

As described above, DART-A and DART-B are similarly designed and the first polypeptide of both constructs includes, in the N-terminal to C-terminal direction, an N-terminal, VL domain

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From a monoclonal antibody that can bind to CD3 (VLCD3), an overlapping binding peptide (Bind 1), a VH domain from a monoclonal antibody that can bind to CD123 (VHCD123), Bind 2, an E-coil domain, and a C terminus. Likewise, A second polypeptide of both structures comprises, in the N-terminal to C-terminal direction, the N-terminal, VL domain of a monoclonal antibody that can

<p dir="rtl">5 Binding to CD123 (VLCD123), overlapping binding peptide (ligand 1), VH domain of a monoclonal antibody that can bind to CD3 (VHCD3), binding 2, K-coil domain and C terminus.</p>

As shown in Example 1, it has been found that both types of CD123 x CD3 dimers can concomitantly bind to CD3 and CD123. In addition, as shown in Example 3 and Figure 4, Panels C and D, both body doubles show

<p dir="rtl">10 The dual specificity nature of CD123 x CD3 has a great ability to kill. Redirected in Turkish: AZT</p>

This is required to achieve 50% of maximum activity (EC50s) in the sub-ng/ml range, regardless of the specific nature of CD3 epitope binding of DART-A (vs. DART-B) in target cell lines with a high degree of CD123 expression. Syntactically, slight variations in the specific sequences of the CD123 x duplexes do not result in

<p dir="rtl">15 CD3 completely abolishes biological activity.</p>

However, in all cell lines tested, DART-A was found to be more active and more potent in redirected killing than DART-B (see, for example, Figure 4, panels A, C, and D). As such, DART-B showed An unexpected advantage compared to similar DART-B.

Example 17

<p dir="rtl">20 Pharmacological properties in non-human primates of DART-A in the treatment of malignant aneurysms</p>

The alpha chain of the interleukin-3 receptor (CD123-3), IL, is overexpressed on cells.

Munoz, L. et al. (2001) Malignancy in a wide variety of malignant aneurysms “Interleukin-3 Receptor Alpha Chain (CD123) Is Widely Expressed In

Hematologic Malignancies,” Haematologica 86:1261-1269; Testa, U. et

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al. (2014) “CD123 Is A Membrane Biomarker And A Therapeutic Target and Is Associated with Poor Prognosis (In Hematologic Malignancies,” Biomark. Res. 2:4 Vergez, F. et al. (2011) “High Levels Of CD34+CD38low/-CD123+ ( Blasts Are Predictive Of An Adverse Outcome In Acute Myeloid Leukemia:

A group of people with money and Maladies to sing 5

96:1792-1798 (GOELAMS Study,” Haematologica). Furthermore, it is reported that CD123 is expressed by leukemia stem cells (LSC).

Jordan, CT et al. (2000) “The Interleukin-3 Receptor Alpha Chain Is A ( Unique Marker For Human Acute Myelogenous Leukemia Stem Cells,” Leukemia 14:1777-1784; Jin, L. et al. (2009) “Monoclonal Antibody- 10 Mediated Targeting Of CD123, IL-3 Receptor Alpha Chain, Eliminates “Human Acute Myeloid Leukemic Stem Cells,” Cell Stem Cell 5:31-42

This represents an attractive feature that allows targeting the basic cause of this mother land. Consistent with this finding, CD123 is also involved in the autocrine complex that maintains leukemia, 15 as shown by the ability of a CD123-blocking monoclonal antibody to reduce engraftment of leukemic stem cells and improve survival in a murine model of acute myelogenous leukemia.

Jin, L. et al. (2009) “Monoclonal Antibody-Mediated Targeting Of (AML) CD123, IL-3 Receptor Alpha Chain, Eliminates Human Acute Myeloid

5:31-42 Leukemic Stem Cells,” “Cell Stem Cell.” However, in a phase 1 study 20 in patients with high risk of AML, the monoclonal antibody did not show activity

Roberts, A. W. et al. (2010) “A Phase I Study Of Anti-Leukemia CD123 Monoclonal Antibody (mAb) CSL360 Targeting Leukemia Stem and As such, of Cells (LSC) In AML,” J. Clin. Oncol. 28(Suppl):e13012

Alternative methods for targeting CD123, including depletion strategies, are desirable. 25 Although CD123 is expressed by a subset of normal hematopoietic progenitor cells (HPC), expression of CD123 by hematopoietic stem cells (HSC) is minimal.

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Jordan, CT et al. (2000) “The Interleukin-3 Receptor Alpha Chain Is A Unique Marker For Human Acute Myelogenous Leukemia

Stem Cells,” Leukemia 14:1777-1784; Jin, W. et al. (2009) “Regulation

Of Th17 Cell Differentiation And EAE Induction By MAP3K NIK,” Blood

<p dir="rtl">5 113:6603-6610), which indicates that CD123 cell depletion strategies allow the restoration of</p>

Formation through normal hematopoiesis.

Enabling a patient's T lymphocytes to target leukemia-related cells represents a promising immunotherapeutic strategy for the treatment of malignant hematology. The therapeutic potential of this method has been tested using blinatumomab, BiTE (blinatumomab), based on an antibody of a specific nature.

<p dir="rtl">10 Dual specificity: It can bind CD19 antigen to CD3 and B cells in patients with tumor</p>

Klinger, ( B and acute lymphoblastic leukemia in cells producing B cells lymphocytes

<p>M. et al. (2012) “Immunopharmacologic Response Of Patients With B-Lineage Acute Lymphoblastic Leukemia To Continuous Infusion Of T Cell</p>

Engaging CD19/CD3-Bispecific BiTE Antibody Blinatumomab,” Blood

119:6226-6233; Topp, M. S. et al. (2012) “Long-Term Follow-Up Of 15

Hematologic Relapse-Free Survival In A Phase 2 Study Of Blinatumomab

“In Patients With MRD In B-Lineage ALL,” Blood 120:5185-5187; Topp,

MS et al. (2011) “Targeted Therapy With The T-Cell-Engaging Antibody Blinatumomab Of Chemotherapy-Refractory Minimal Residual Disease In

B-Lineage Acute Lymphoblastic Leukemia Patients Results In High 20

Response Rate And Prolonged Leukemia-Free Survival,” J. Clin. Oncol.

.29:2493-2498

The CD123 x CD3 dual specificity molecules of the present invention, such as DART-A, comprise a specificity antibody-based pattern

Johnson, S. et al. (2010) (Double 25 provides improved stability and more consistent manufacturing properties

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“Effector Cell Recruitment With Novel Fv-Based Dual-Affinity ReTargeting Protein Leads To Potent Tumor Cytolysis And In Vivo B-Cell

Depletion,” J. Mol. Biol. 399:436-449; Moore, P. A. et al. (2011) “Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell Lymphoma,” Blood 117:4542-4551 5

To demonstrate the superiority and effectiveness of the dual-specific CD123 x CD3 molecules of the present invention, the bioactivity of DART-A described above was confirmed in vitro and its pharmacokinetic, pharmacokinetic, pharmacodynamic and safety-related pharmacokinetic properties were evaluated in preclinical models of leukemia. Macaque monkeys (Macaca).

<p dir="rtl">10 fascicularis) for the comparator DART shown above (dual specificity for CD3 and fluorescein or “2-Control DART” was dual specificity for CD123 and fluorescein).</p>

Amino acid sequence of the first polypeptide chain of the “2-DART conjugate” (CD123VL-

The Connect ─ 4-4420VH ─ The Connect -E-coil; Links are underlined (Sequence No.: 58):

DFVMTQSPDS LAVSLGERVT MSCKSSQSLL NSGNQKNYLT 15

WYKPGQPP

KLLIYWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA

VYYCQNDYSY

PYTFGQGTKL EIKGGGSGGG GEVKLDETGG GLVQPGRPMK

LSCVASGFTF 20

SDYWMNWVRQ SPEKGLEWVA QIRNKPYNYE TYYSDSVKGR

FTISRDDSKS

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SVYLQMNNLR VEDMGIYYCT GSYYGMDYWG QGTSVTVSSG

GCGGGEVAAL

EKEVAALEKE VAALEKEVAA LEK

Amino acid sequence of the second polypeptide chain of the “2-DART conjugate” (─ 4420VL).

5 Coupling ─ CD123VH ─ Coating - K-coil (Sequence No.: 59):

DVVMTQTPFS LPVSLGDQAS ISCRSSQSLV HSNGNTYLRW

YLQKPGQSPK

VLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEEAEDLGV

YFCSQSTHVP

WTFGGGTKLE IKGGGSGGGG EVQLVQSGAE LKKPGASVKV 10

SCKASGYTFT

DYYMKWVRQA PGQGLEWIGD IIPSNGATFY NQKFKGRVTI

TVDKSTSTAY

MELSSRSED TAVYYCARSH LLRASWFAYW GQGTLVTVSS GGCGGGKVAA 15

LKEKVAALKE KVAALKEKVA ALKE

Dual-function ELISA

A coated Nunc (MaxiSorp ELISA) dish was blocked overnight with human soluble IL3R-alpha or rhabdoid (0.5 μg/ml) in 0.5% bicarbonate buffer solution.

<p dir="rtl">20 BSA; 0.1% 20-Tween in BSA/PBST(PBS) for 30 min at room temperature. DART-A particles were applied, followed by sequential addition of human CD3εδ-biotin and Jackson ImmunoResearch (Streptavidin HRP). HRP activity was detected by titer shift</p>

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Methylbenzidine (BioFX) served as substrate for 5 min; the reaction was terminated with 40 μl/eye of 1% H2SO4 and absorbance was read at 450 nm.

Surface plasmon resonance analysis

The ability of DART-A to bind to human and monkey CD3 or CD123 proteins was analyzed

As described by BIAcore 3000 (GE, Healthcare) 5 Al-Rabahi with Biosensor Johnson, S. et al. (2010) (“Effector Cell Recruitment With Novel Fv-

Based Dual-Affinity Re-Targeting Protein Leads To Potent Tumor

Cytolysis And In Vivo B-Cell Depletion,” J. Mol. Biol. 399:436-449) and Moore, P. A. et al. (2011) (“Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell 10

(117:4542-4551 Lymphoma,” Blood. Briefly, the carboxyl groups on the CM5 sensor chip were activated by injection of 0.2 M N -ethyl-3(-N-diethylamino-propyl)carbodiimide

-Hydroxy-N and 0.05 M N-ethyl-N-(3dietylamino-propyl)carbodiimide

Succinimide N-hydroxy-succinimide. Soluble CD3 or CD123 (1 μg/ml) was injected

<p dir="rtl">15 ml) on the surface of CM5 activated in 10 mM sodium-acetate, pH 5.0, at a flow rate of 5 μl/min, followed by 1 M ethanolamide to deactivate. Binding experiments were performed in 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005% surfactant P20. Surfaces of fixed receptors were regenerated by pulsed injection of 10 mM glycine, pH 1.</p>

<p dir="rtl">20 1.5. KD values were determined by a general fit of the correlation curves to a 1:1 Langmuir correlation model</p>

(BIAevaluation software version 4.1).

Cell killing test

Cell strains used in cell killing assays were obtained from American Type

PBMCs were isolated from donor blood (Manassas, VA) (ATCC(Culture Collection).

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True using the GE Healthcare Ficoll-Paque Plus kit (Ficoll-Paque Plus kit); T cells were purified with a negative selection kit (Life Technologies).

Bangs (Quantum Simply Cellular) surface using CD123 beads

Cytotoxicity tests were performed as indicated (Laboratories, Inc., Fishers, IN).

Moore, P. A. et al. (2011) (“Application Of Dual Affinity Retargeting 5 By Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell

Lymphoma,” Blood (117:4542-4551). Briefly, target cell lines (105 cells/ml) were treated with serial dilutions of comparator DART-A or DART proteins in the presence of T cells at the effector cell:target cell ratios indicated and incubated at 37°C. All night long

In the supernatant (LDH, Promega) 10 Identify cell killing as release of lactate dehydrogenase.

For the farm. For flow-based killing, target cells were labeled with CMTMR

(Life Technologies) Cell killing was monitored using a FACSCalibur flow cytometer. Data were analyzed using GraphPad® PRISM software (5) and represented as percentage cytotoxicity.

<p dir="rtl">15 Pharmacological properties of baboons</p>

Nonhuman primate experiments were conducted at Charles River Laboratories (Reno, NV), in accordance with the guidelines of the local Institutional Animal Care and Use Committee (IACUC). Purpose-reared baboons (Macaca fascicularis) were given untreated rats of Chinese origin (S. Is between 2.5 and 9 years old, with a weight between 2.7 and 5 kg (conveyor material or

<p dir="rtl">20 DART-A is administered by intravenous infusion through the femoral and cervical ports using adjustable infusion pumps</p>

CADD-Legacy®, SIMS Deltec, Inc., St. Paul, MN) for battery operated programming

Peripheral blood or bone marrow samples were collected into tubes containing anticoagulant at the indicated time points. Cell surface phenotypic analyzes were performed using the LSR BD Biosciences(Fortessa) analysis instrument equipped with 488 nm, 640 nm and 405 nm lasers.

25 nM and the following antibodies: CD123-PE-Cy7, CD8-V450, CD4-V450.

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CD45-PerCP, CD8-FITC, CD25-PE-Cy7, CD69-

PerCP, CD3-Pacific Blue, CD95-APC, CD28-APC, TIM3-APC, PD-1-PE

CD16-FITC, BV421, CD38-PE, CD38-PE, CD123-PE-Cy7

CD45RA-APC-H7, CD90-BV421, CD34-APC, CD117-PerCP-Cy5.5 and

<p dir="rtl">5 BD Biosciences(CD33-APC). The absolute number of cells was determined using TruCOUNT</p>

BD Biosciences ((). Serum levels of IL-4, IL-4, IL-2 cytokines 6, TNF-α, and IFN-γ were measured with the BD Bioscience Cytokine Cytometric Kit. Bead Array Kit. The concentration of DART A in monkey serum samples was measured using an interference immunoassay through fluorescence detection.

MesoScale Diagnostics, MSD, ( electrochemiluminescence 10 electrochemical

Briefly, the MSD assay plate was coated with recombinant human 3-IL Ra and blocked with 5% BSA. Calibration standards or diluted test samples were applied, then Added biotinylated monoclonal antibody shown

<p dir="rtl">15 It is qualitatively related to the E-coil ranges shown above (sequence No. 34) and the K-coil is added (sequence No. 34).</p>

No.: 35) of the bright streptavidin conjugate^{^}The MSD™ SULFO-TAG™ was extracted from the molecule and complex formation was analyzed in the MSD SECTOR® imaging instrument. DART-A concentrations were determined from standard curves generated by matching light intensity data to a five-parameter logistic model.

<p dir="rtl">20 The physicochemical characterization of purified DART-A showed a uniform, heterogeneous dimer with a molecular mass of 58.9 kDa (Figure 23; Figures 24a–24b), and was stable at 2–8°C for up to 12 months in PBS. SPR analysis showed nearly identical binding affinity properties of DART-A with the corresponding soluble human CD3 and CD123 antigens (Figures 25a). – 25 d and Table 7). Moreover, DART-A is linked simultaneously</p>

<p dir="rtl">25 Both antigens in the ELISA format use human or simian CD123 to capture CD123 and</p>

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Human CD3 was detected (Figures 26A-26B), and showed similar binding to human and simian T lymphocytes (Figures 26C-26E). The data in Table 7 are averaged from 3 independent experiments each performed in pairs.

Table 7

5 Equilibrium dissociation (KD) constants for binding of DART-A to human CD3 and CD123 and from baboons

<tr><td><p>KD(±SD)</p><p dir="rtl">(nanomolar)</p></td><td><p>kd(±SD)</p><p dir="rtl">(W-1)</p></td><td><p>ka(±SD)</p><p dir="rtl">(molar-1s-1)</p></td><td><p dir="rtl">Antigens</p></td></tr><tr><td><p>2.3 ± 9.0</p></td><td><p>x (0.9±) 5.0</p><p>3-10</p></td><td><p>x (0.6±) 5.7</p><p>105</p></td><td><p dir="rtl">Human δ/CD3ε</p></td></tr><tr><td><p>2.3 ± 9.2</p></td><td><p>x (0.9±) 5.0</p><p>3-10</p></td><td><p>105x (0.5±) 5.5</p></td><td><p dir="rtl">CD3ε/δ Rabahi</p></td></tr><tr><td><p>± 0.13</p></td><td><p>x (0.4±) 1.9</p></td><td><p>x (0.4±) 1.6</p></td><td><p>CD123-His</p></td></tr><tr><td><p>0.01</p></td><td><p>4-10</p></td><td><p>106</p></td><td><p dir="rtl">Human</p></td></tr><tr><td><p>± 0.27</p></td><td><p>x (0.7±) 4.0</p></td><td><p>x (0.3±) 1.5</p></td><td><p>CD123-</p></td></tr><tr><td><p>0.02</p></td><td><p>4-10</p></td><td><p>106</p></td><td><p dir="rtl">His Al-Rabahi</p></td></tr>

DART-A causes redirected killing by human T lymphocytes or from baboons

DART-A induced killing of targets redirected by human or simian effector cells against leukemia-related CD123+ Kasumi-3 cell lineages (Figures 27a-27d), accompanied by

10 Search for signs of activation. No activity was observed against CD123 negative targets (U937 cells) or with the conjugate DART, demonstrating that T cell activation is strictly independent upon target cell binding and monovalent binding of CD3 by DART-A was insufficient to trigger

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T cell activation. Because CD123 is expressed by subsets of normal circulating leukocytes, including pDCs and monocytes (Figure 27e), the effect of DART-A was further examined in normal human and simian PBMCs.

A graded effect was observed among human PBMC, with rapid dose-dependent depletion of cells observed

5 pDC (CD14-CD123high and basophils) no more than 3 hours after the start of treatment, while monocytes (CD14+ cells) remained unaffected at this time point (Figures 27). Depletion of CD14-CD123high cells increased over time across all DART-A concentrations molecule, while monocytes decreased slightly over 6 h and were only depleted after 18 h and at concentrations higher than 1 ng/ml incubation of simian PBMCs with DART-A resulted in depletion

<p dir="rtl">10 A dose-dependent relative of CD14-CD123high cells (Figure 27h), further supporting the importance of this type for the pharmacological properties of CD14 (DART-A+ monkey cells do not express CD123 or express it only minimally and are not depleted).</p>

Pharmacokinetic properties of DART-A in baboons

The baboons were selected as a suitable drug model for DART-A analysis based on the equivalent distribution of all 15 target antigens in this species compared to humans based on immunohistochemistry.

Munoz, L. et al. (2001) “Interleukin-3 Receptor Alpha Chain (CD123) Is Widely Expressed” (2001) produced for others, consistent with published information

In Hematologic Malignancies,” Haematologica 86:1261-1269;

Korpelainen, E.I. et al. (1996) “IL-3 Receptor Expression, Regulation and Function In Cells Of The Vasculature,” Immunol. Cell Biol. 74:1-7 20

The study conducted in accordance with the present invention included 6 treatment groups consisting of 8 baboons each (4 males, 4 females) (Table 8). All groups received comparator for the first infusion; then vector or DART-A was administered in Intravenously for four doses weekly, animals in Group 1 received the comparator for all four subsequent infusions, while Group 2 animals received -

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5 Weekly escalating doses of DART-A 4 days per week for all subsequent infusions. Group 6 animals were treated with weekly uninterrupted escalating doses for 7 days of DART-A for all infusions. The 4-day on/3-day off and 7-day on time charts are designed to distinguish between ongoing and occasional effects associated with

<p dir="rtl">5 By giving DART-A. Two males and two females from each group were euthanized at the end of the treatment phase (day 36), while the remaining monkeys remained after a 4-week recovery (day 65). A subset of the monkeys developed antibodies (ADA) directed against the humanized Fv for both CD3 and CD123, and time points following the onset of ADA were excluded from the PK analysis. All monkeys were exposed to DART-A during the study period.</p>

Table 8

<tr><td colspan="5"><p>DART-A leak</p></td><td rowspan="2"><p dir="rtl">Subject</p><p dir="rtl">The tanker</p></td><td rowspan="3"><p dir="rtl">days</p><p dir="rtl">Study</p></td><td rowspan="3"><p dir="rtl">Infusion number</p></td></tr><tr><td><p dir="rtl">(7 operating days)</p><p dir="rtl">ng/kg/day</p><p dir="rtl">[ng/kg/7days]</p></td><td colspan="4"><p dir="rtl">(4 days on/3 days off)</p><p dir="rtl">ng/kg/day</p><p dir="rtl">[ng/kg/4 days]</p></td></tr><tr><td><p dir="rtl">group</p><p dir="rtl">6</p></td><td><p dir="rtl">the group</p><p dir="rtl">5</p></td><td><p dir="rtl">the group</p><p dir="rtl">4</p></td><td><p dir="rtl">group</p><p dir="rtl">3</p></td><td><p dir="rtl">the group</p><p dir="rtl">2</p></td><td><p dir="rtl">the group</p><p dir="rtl">1</p></td></tr><tr><td><p dir="rtl">Subject</p><p dir="rtl">The tanker</p></td><td><p dir="rtl">Subject</p><p dir="rtl">The tanker</p></td><td><p dir="rtl">Subject</p><p dir="rtl">The tanker</p></td><td><p dir="rtl">Subject</p><p dir="rtl">The tanker</p></td><td><p dir="rtl">Subject</p><p dir="rtl">The tanker</p></td><td><p dir="rtl">Subject</p><p dir="rtl">The tanker</p></td><td><p>1</p></td><td><p>1</p></td></tr>

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<tr><td><p>100</p><p>]700[</p></td><td><p>100</p><p>]400[</p></td><td><p>100</p><p>]400[</p></td><td><p>100</p><p>]400[</p></td><td><p>100</p><p>]400[</p></td><td><p dir="rtl">Subject</p><p dir="rtl">The tanker</p></td><td><p>8</p></td><td><p>2</p></td></tr><tr><td><p>300</p><p>]2100[</p></td><td><p>300</p><p>]1200[</p></td><td><p>300</p><p>]1200[</p></td><td><p>300</p><p>]1200[</p></td><td><p>100</p><p>]400[</p></td><td><p dir="rtl">Subject</p><p dir="rtl">The tanker</p></td><td><p>15</p></td><td><p>3</p></td></tr><tr><td><p>600</p><p>]4200[</p></td><td><p>600</p><p>]2400[</p></td><td><p>600</p><p>]2400[</p></td><td><p>300</p><p>]1200[</p></td><td><p>100</p><p>]400[</p></td><td><p dir="rtl">Subject</p><p dir="rtl">The tanker</p></td><td><p>22</p></td><td><p>4</p></td></tr><tr><td><p>1000</p><p>]7000[</p></td><td><p>1000</p><p>]4000[</p></td><td><p>600</p><p>]2400[</p></td><td><p>300</p><p>]1200[</p></td><td><p>100</p><p>]400[</p></td><td><p dir="rtl">Subject</p><p dir="rtl">The tanker</p></td><td><p>29</p></td><td><p>5</p></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td><p>65-36</p></td><td><p dir="rtl">Restoration</p></td></tr>

A two-compartment model was used to estimate PK variables (Table 9 and Figure 28). T1/2α was short (4–5 minutes), reflecting rapid binding to rotating targets; T1/2β was also fast, as expected for a molecule of this size. It is subject to renal clearance, according to analysis of serum samples

At the end of each infusion the 6 group monkeys collected a dose-dependent increase in DART-A

<p dir="rtl">5 Cmax. In Table 9, the carrier was PBS, pH 6.0, containing 0.1 mg/ml recombinant human albumin, 0.1 mg/ml 80-PS, and 0.24% benzyl alcohol was used for all carrier infusions during the days The first four days of each weekly infusion and then the same formula without benzyl alcohol for the remaining three days of each weekly infusion. DART-A was administered at the indicated times as a connected intravenous infusion of PBS solution, no</p>

<p dir="rtl">10 pH 6.0, containing 0.1 mg/ml recombinant human albumin, 0.1 mg/ml 80-PS, and 0.24% benzyl alcohol at desired concentration.</p>

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Table 9:

PK Parameters for Two-Compartment Analysis

DART-A

Cynomolgus Monkeys in Rabahi Monkeys

<tr><td><p dir="rtl">600 ng/kg/day (mean ± SD)</p></td><td><p dir="rtl">300 ng/kg/day (mean ± SD)</p></td><td><p dir="rtl">Theme</p></td></tr><tr><td><p>33.5 ± 113.8</p></td><td><p>9.4 ± 77.4</p></td><td><p dir="rtl">Cmax (pg/ml)</p></td></tr><tr><td><p>3282 ± 11188</p></td><td><p>913 ± 7465</p></td><td><p dir="rtl">AUC (hour* pkg/ml)</p></td></tr><tr><td><p>1.846 ± 2.098</p></td><td><p>0.511 ± 1.078</p></td><td><p dir="rtl">Vss(L/kg)</p></td></tr><tr><td><p>0.023 ± 0.067</p></td><td><p>0.018 ± 0.07</p></td><td><p dir="rtl">t1/2, alpha (hour)</p></td></tr><tr><td><p>18.779 ± 21.828</p></td><td><p>4.928 ± 13.79</p></td><td><p dir="rtl">2/1t, beta (hour)</p></td></tr><tr><td><p>8.891 ± 9.604</p></td><td><p>3.327 ± 6.73</p></td><td><p dir="rtl">MRT (hour)</p></td></tr>

5 Cytokine release in DART-A-treated baboons

In view of the T-cell activating properties of DART-A, an increase in circulating cytokines accompanying the infusion was expected and so a low starting dose was used as an allergic "desensitization" function, based on previous experience with similar compounds (see, for example, ). Topp, M. S. et al

(2011) “Targeted Therapy With The T-Cell-Engaging Antibody Blinatumomab Of Chemotherapy-Refractory Minimal Residual Disease In 10

B-Lineage Acute Lymphoblastic Leukemia Patients Results In High

Response Rate And Prolonged Leukemia-Free Survival,” J. Clin. Oncol.

29:2493-2498; Bargou, R. et al. (2008) “Tumor Regression In Cancer

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Patients By Very Low Doses Of AT Cell-Engaging Antibody,” Science

321:974-977). Of the cytokine tested, IL-6 showed the largest changes upon infusion, although this was transient in nature, with marginal magnitude and large variations between animals and between groups (Figures 29A-29C). Small transient increases were also observed In IL-6 after carrier leaks

<p dir="rtl">5 (All group 1 and day 1 infusions), demonstrating sensitivity of this cytokine to stress of administration. However, DART-A-dependent increases (&lt;80 pg/ml) in serum IL-6 were observed in some monkeys after DART-A infusion first (100ng/kg/day), and she returned to baseline by 72 hours. Interestingly, the amount of IL-6 release decreased with each subsequent DART-A infusion, even when the dose level was increased to 1000ng/kg/day.</p>

<p dir="rtl">10 Marginal and occasional DART-A-associated increases in serum TNF-α (&lt;10 pg/ml) were also observed; as with IL-6, the greatest magnitude of TNF-α release was observed after the first infusion. There were no changes associated with DART-A increased levels of IL-5, IL-4, IL-2, or IFN-γ in the study as a whole when compared with control subjects. In conclusion, cytokine release in response to treatment of monkeys with DART-A was marginal, incidental, and represents a possible first-dose effect.</p>

<p dir="rtl">15 Control it by increasing the doses of the individual undergoing treatment.</p>

DART-A-induced depletion of CD14+/CD123+ circulating leukocytes in vivo.

Circulating absolute levels of CD14+/CD123+ cells were measured over the course of the entire study as a pharmacokinetic endpoint. While the number of CD123+ cells in comparison group 1 remained constant over time

<p dir="rtl">20 time, DART-A treatment was associated with global depletion of circulating CD14+/CD123+ cells (94-100% of pre-study baseline) observed from the first time point measured (72 h) after the start of the first DART-A infusion (100 nM). /kg/day) in all animals across all active treatment groups (Figures 30a-30c). Depletion was persistent, remaining during the 3-day weekly dosing break in group 2-5, and returning to baseline levels</p>

<p dir="rtl">25 Basal only during the extended recovery period. To eliminate the possibility of DART-A masking or CD123 modification</p>

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(an unlikely scenario, given the low circulating DART-A levels), pDCs were counted by the vertical marker, CD303. Consistent with the CD123 data, CD303+ pDCs were similarly depleted in monkeys treated with DART-A (Figures 30D–30 And the(.

Analysis of circulating T lymphocyte levels, activation and subsets

<p dir="rtl">5 In contrast to continuous depletion of circulating CD123+ cells, DART-A given in a 4-day on/3 off schedule (groups 2-5) was associated with weekly oscillations in circulating T cells, while administration as continuous 7-day infusions resulted in a similar reduction T cell levels after the first administration returned slowly without fluctuation even during the dosing period (Figures 31a-31c). The difference between the two dosing strategies shows that the effect of DART-A on T lymphocytes is consistent with</p>

<p dir="rtl">10 With passage and/or migration, not exhaustion. After dosing was stopped, T cells returned to levels approximately 2 times higher than baseline for the recovery period. DART-A infusion was associated with a progressive, exposure-dependent increase in the number of T cells expressing the marker of delayed activation, PD-1, particularly in CD4+ cells, with the dose group 6 having the highest total levels (Figures 31d-31i and Figures 32a). – 32f and Figures 33a – 33f). 3-Tim was not detected, which is a</p>

<p dir="rtl">15 Associated with T cell exhaustion, on CD4+ T cells and only at a low frequency among CD8+ cells (9.7% - 5.5%), including 20.5-35.5% of CD8+/PD+1 double positive cells. There was no consistent change in the marker of early T cell activation. CD69, T cells, and there were only modest variations in the expression of CD25 between rotating cells.</p>

To abolish depletion after in vivo exposure, the ex vivo cytotoxic potential of 20 effector cells isolated from baboons receiving multiple infusions of DART was compared.

A in cells from untreated monkeys. As shown in Figure 34, PBMC isolated from DART-A-treated monkeys show cytotoxicity close to that of cells isolated from untreated monkeys, demonstrating that in vivo exposure to DART-A does not negatively affect the ability of T cells to Kill target cells.

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Exposure to DART-A resulted in an increase in the relative number of central memory CD4 cells and effector memory CD8+ cells at the expense of the corresponding untreated T cell population (Figures 35A-35F, Figures 32A-32F, and Figures 33A-33F). , indicating that exposure to DART-A led to improved expansion and/or motility of these cells.

<p dir="rtl">5 Effect on blood formation and bone marrow producing substances</p>

DART-A was well tolerated in monkeys at all doses tested; However, reversible reductions in red cell parameters were observed at higher doses (Figures 36a-36c). Frequent blood sampling may have been a possible contributing factor, because vector-treated animals showed a modest reduction in red cell mass. A retinal response was observed in all 10 animals; However, at the highest exposure (Group 6), the response appeared to be slightly less consistent relative to a similar decrease in red cell mass (Figures 36d-36f). Compositional analysis of bone marrow stains during the study was not noteworthy. However, it showed Flow cytometry analysis showed that the number of CD123+ cells in the immature lineage-negative bone marrow populations (Lin) decreased in animals treated with DART-A at the end of the dosing period.

<p dir="rtl">15 It returned to baseline levels by the end of the recovery period (Figure 37a-37b). Show HSC</p>

Pang, W. W. et al. ( Lin-/CD34+/CD38-/CD45RA-/CD90+ defined as cells) (2011) “Human Bone Marrow Hematopoietic Stem Cells Are Increased In

Frequency And Myeloid-Biased With Age,” Proc. Natl. Acad. Sci.

(108:20012-20017 (USA).) Varied widely between groups; Groups 4-6 of 20 monkeys treated with DART-A showed some apparent decrease compared to pre-dose levels, however, no decrease was observed in all groups. treatment compared to vector-treated animals. These data indicate that HSC are less susceptible to targeting by DART-A and are consistent with the observed reversibility of the negative effects of DART-A treatment on hematopoiesis.

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As indicated above, for 4-week infusions based on a weekly 4-day on/3-day off schedule or a 7-day on schedule starting at 100 ng/kg/day was gradually increased weekly to 300, 600, and 1 000 ng/kg/day, administration of DART-A to baboons was well tolerated. Depletion of CD123+ cells was observed

<p dir="rtl">5 circulation, including pDCs, after the start of the first administration and continued throughout the study at all doses and schedules. A reversible decrease in bone marrow CD123+-producing material was also observed. Cytokine release, an important safety concern with CD3-targeted therapies, appears to be controllable and consistent with the effect of a first dose. Modest reversible anemia was observed at the highest doses, but no other adverse effects (on-target or off-target) were observed.</p>

10

15

The baboons represent a suitable animal model for the pharmacological evaluation of DART-A, given the high homogeneity of isogenic genes and the ability of DART-A to bind with similar affinity to antigens and induce redirected T-cell killing in both species. Furthermore, both antigens are expressed compatibleally in monkeys and humans, including homologous expression by hematopoietic antigens and in the cytoplasm of the lining of multiple tissues. Marginal exceptions are expression in testicular Leydig cells in humans but not in monkeys and low to no CD123 in monocytes in monkeys compared with humans.

One of the primary concerns associated with therapeutic strategies involving T cell activation involves cytokine release and off-target cytotoxic effects. A recent study using a dual-specific scFv immune fusion construct CD3xCD123 with CD3 recognition has shown

<p dir="rtl">20 Bivalent has anti-leukemic activity in vitro, but causes non-specific activation of T cells</p>

Kuo, S.R. et al. (2012) “Engineering A CD123xCD3 IFN-γ Secretor and Bispecific scFv Immunofusion For The Treatment Of Leukemia And

Elimination Of Leukemia Stem Cells,” Protein Eng. Des. Sel. 25:561-

569). It confirms the monovalent nature of both the binding arms and the highly homogeneous monomeric form.

<p dir="rtl">25 From DART-A, T cell activation depends exclusively on target cell binding: no activation was observed</p>

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T cells in the absence of target cells or using a comparator DART molecule that includes only the CD3 targeting arm. Furthermore, high doses (up to 100 μg/kg/day) of the comparator DART molecule did not induce cytokine release in baboons.

The starting DART-A dose of 100ng/kg/day was well tolerated, with

<p dir="rtl">5 Marginal cytokine release. However, cytokine storm occurs at a high starting dose (5 mcg/kg/day); however, this dose can be safely reached through stepwise weekly dose escalations, indicating that DART-A-induced cytokine release appears to be mainly a dose effect. First, depletion of target CD123+ cells, and thus elimination of the source of CD3 binding, may explain the effect of the first dose: almost complete depletion of CD123+ cells was observed at doses of</p>

<p dir="rtl">10 Its decrease is 3-10 ng/kg/day, which indicates that the release of cytokines in the organism follows a more significant dose-response relationship compared to cytotoxicity. Dose-response values for cytotoxicity and cytokine release by human T cells were also consistent with this observation.</p>

T cell desensitization, in which DART-A-induced PD1 increase plays a role, also appears to contribute.

<p dir="rtl">15 In reducing cytokine release after the first infusion of DART-A. Recent studies show that increased expression of PD-1 after antigen-induced arrest of T cells at sites of inflammation contributes, through interactions with PD-L1, to termination of the arrest signal, thus triggering and desensitizing</p>

Honda, T. et al. (2014) “Tuning Of Antigen Sensitivity By T Cell (Receptor-Dependent Negative Feedback Controls T Cell Effector Function)

In Inflamed Tissues,” Immunity 40:235-247; Wei, F. et al. (2013) 20

“Strength Of PD-1 Signaling Differentially Affects T-Cell Effector

Functions,” Proc. Natl. Acad. Sci. (USA) 110:E2480-E2489

The 1-PD equation for the strength of TCR signal transmission is regular: while reproduction and cytokine production appear to be the most

Wei, F. et al. (2013) (Cytotoxicity is the least affected, PD-1 is sensitive to inhibition of “Strength Of PD-1 Signaling Differentially Affects T-Cell Effector 25

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Functions,” Proc. Natl. Acad. Sci. (USA) 110:E2480-E2489

However, the in vivo cytotoxic potential of T cells in monkeys exposed to multiple infusions of DART-A was close to that in T cells from untreated monkeys, despite increased PD-1 levels in the former. Furthermore, the increase of 1-PD was not accompanied by the expression of

<p dir="rtl">5 TIM3, a major marker of T cell exhaustion, as indicated for exposed T cells</p>

Gebel, HM (or chronic CD3 infections for prolonged stimulation with antibodies

et al. (1989) “T Cells From Patients Successfully Treated With OKT3 Do

“Not React With The T-Cell Receptor Antibody,” Hum. Immunol. 26:123

129; Wherry, E. J. (2011) “T Cell Exhaustion,” Nat. Immunol. 12:492-.)499 10

Depletion of circulating CD123+ cells in monkeys treated with DART-A was rapid and sustained over the course of weekly dosing in the 4-day on/3-day off schedule, consistent with target clearance. In contrast, the occasional oscillations in circulating T cells were likely the result of traffic to/from lymphoid tissues and organs as a function of DART-A. enhances

<p dir="rtl">15 Exposure to DART-A expands and/or mobilizes antigen-exposed T lymphocytes, which are cells</p>

Mirenda, (preferably contains tissues and more readily exerts a cytotoxic effect

<p>V. et al. (2007) “Physiologic And Aberrant Regulation Of Memory T-Cell Trafficking By The Costimulatory Molecule CD28,” Blood 109:2968-2977;</p>

Marelli-Berg, F.M. et al. (2010) “Memory T-Cell Trafficking: New Directions For Busy Commuters,” Immunology 130:158-165 20

Depletion of natural CD123+ cells can have potential drawbacks. pDCs and basophils express high levels of CD123, compared to lower levels in monocytes

Lopez, A. F. et al. (1989) “Reciprocal Inhibition Of Binding Between Interleukin 3 And Granulocyte-Macrophage Colony-Stimulating

Factor To Human Eosinophils,” Proc. Natl. Acad. Sci. (USA) 86:7022-25

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7026; Munoz, L. et al. (2001) “Interleukin-3 Receptor Alpha Chain (CD123) Is Widely Expressed In Hematologic Malignancies,”

Haematologica 86:1261-1269; Masten, B.J. et al. (2006) “Characterization Of Myeloid And Plasmacytoid Dendritic Cells In Human

Lung,” J. Immunol. 177:7784-7793; Korpelainen, E.I. et al. (1995) 5

“Interferon-Gamma Upregulates Interleukin-3 (IL-3) Receptor Expression.”

In Human Endothelial Cells And Synergizes With IL-3 In Stimulating Major Histocompatibility Complex Class II Expression And Cytokine

Production,” Blood (86:176-182). It has been shown that pDCs play a role in the control of certain viruses in mouse or monkey models of infection10, although they do not appear to be critical in

Colonna, M. et al. (1997) “Specificity” relates to the control of the immune response to influenza

And Function Of Immunoglobulin Superfamily NK Cell Inhibitory And

Stimulatory Receptors,” Immunol. Rev. 155:127-133; Smit, J. J. et al.

<p>(2006) “Plasmacytoid Dendritic Cells Inhibit Pulmonary Immunopathology</p>

And Promote Clearance Of Respiratory Syncytial Virus,” J. Exp. Med. 15

203:1153-1159). Occasional, modest, non-dose-dependent facial swelling has been observed in some monkeys treated with DART-A; however, increased histamine levels have not been observed in these monkeys or when human basophilia has been resolved by T-cell killing induced by DART-A. Depletion of monocytes may increase the risk of infection; therefore, the consequences of depletion of pDC, basophils or eosinophils should be monitored in humans.

Dedicated hematopoietic substances that express CD123 can be targeted, as can those that produce others

Jordan, CT et al. (2000) “The Interleukin-3 (CMP) Common Myeloid Receptor Alpha Chain Is A Unique Marker For Human Acute Myelogenous

“Leukemia Stem Cells,” Leukemia 14:1777-1784; Rieger, M.A. et al.

<p>(2012) “Hematopoiesis,” Cold Spring Harb. Perspect. Biol. 4:a008250 25</p>

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by DART-A, this is a possible explanation for the modest anemia observed after administration of DART-A at the highest dose. The reticuloerythropoietic response appeared to operate at all dose levels of DART-A; However, for equivalent reductions in red cell parameters, animals exposed to the highest DART-A exposure (Group 6, 7-day infusion) showed

<p dir="rtl">5 On) a decrease in retinal response, suggestive of possible cytotoxic activity on other substances producing (e.g., CMP). The effect was reversible after treatment with DART A was discontinued, consistent with repopulation of spared CD123low/negative HSC.</p>

Alternative methods for depleting CD123+ cells include a specific second-generation monoclonal antibody

Jin, L. et al. (2009) “Monoclonal Antibody-(Fc) Enhanced by CD123 for Mediated Targeting Of CD123, IL-3 Receptor Alpha Chain, Eliminates 10 Human Acute Myeloid Leukemic Stem Cells,” Cell Stem Cell 5:31-42;

Roberts, A. W. et al. (2010) “A Phase I Study Of Anti-CD123 Monoclonal Antibody (mAb) CSL360 Targeting Leukemia Stem Cells (LSC) In AML,” Frankel, A. (IL-3 Associated Diphtheria Toxin), J. Clin. Oncol. 28(Suppl):e13012

et al. (2008) “Phase I Clinical Study Of Diphtheria Toxin-Interleukin 3 15

Fusion Protein In Patients With Acute Myeloid Leukemia And

49:543-553 Myelodysplasia,” Leuk. Lymphoma, chimeric antigen receptor

CAR (chimeric antigen receptors) cytokine-induced killer cells

Tettamanti, S. et al. (CD123 that expresses CIK(cytokine-induced killer).

<p>(2013) “Targeting Of Acute Myeloid Leukaemia By Cytokine-Induced 20</p>

Killer Cells Redirected With A Novel CD123-Specific Chimeric Antigen

Gill, S. (CD123 CAR T and Receptor Cells,” Br. J. Haematol. 161:389-401

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- 25

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2354; Mardiros, A. et al. (2013) “T Cells Expressing CD123-Specific Chimeric Antigen Receptors Exhibit Specific Cytolytic Effector Functions

And Antitumor Effects Against Human Acute Myeloid Leukemia,” Blood

122:3138-3148). CAR T cells have shown a significant ability to kill related blast cells.

5 Leukemia in vitro and anti-leukogenic activity in an exogenous model of metastatic AML

Mardiros, A. et al. (2013) “T Cells Expressing CD123-Specific Chimeric Antigen Receptors Exhibit Specific Cytolytic Effector Functions And

Antitumor Effects Against Human Acute Myeloid Leukemia,” Blood

122:3138-3148). A recent study has recorded the abolition of normal hematopoiesis in NSG mice.

Gill, S. et al. (Human CD123 CAR T cells after transfer of engrafted CD34+ 10 cells

<p>(2014) “Efficacy Against Human Acute Myeloid Leukemia And</p>

Myeloablation Of Normal Hematopoiesis In A Mouse Model Using

Chimeric Antigen Receptor-Modified T Cells,” Blood 123(15): 2343-

2354), although other studies have not observed similar effects in vitro or in vivo.

Tettamanti, S. et al. (2013) “Targeting Of Acute Myeloid ( 15 Organism Leukaemia By Cytokine-Induced Killer Cells Redirected With A Novel

CD123-Specific Chimeric Antigen Receptor,” Br. J. Haematol. 161:389

401; Pizzitola, I. et al. (2014) “Chimeric Antigen Receptors Against CD33/CD123 Antigens Efficiently Target Primary Acute Myeloid Leukemia

<p dir="rtl">20 “Cells in vivo,” Leukemia doi:10.1038/leu, 2014.62. In the experiments described above,</p>

Depletion of bone marrow CD123+ populations was observed, but this was reversed during recovery; Moreover, depletion of this minority group did not lead to changes in bone marrow cellular composition or the ratio of erythroid-like cells to myeloid cells (M:E) at all DART-A dose levels tested. These differences highlight the potential advantage of DART -A comparison

<p dir="rtl">25 Cell therapies provide a titratable system based on autologous T cells versus “supercharged” induced ex vivo T cells that can be more difficult to control.</p>

8650

-123-

CD123 is overexpressed in several malignant leukemias, including AML, hairy cell leukemia, or plasmacytoid dendritic neoplasms (BPDCNs), a subset of B-cell-producing acute lymphoblastic leukemia (B-cell-producing ALL). (Chronic lymphocytic leukemia, Reed-Stemberg cells in Hodgkin's disease, as well as in syndrome

Kharfan-Dabaja, MA et al. 5 Myelodysplasia and Systemic Mastocytosis (2013) “Diagnostic And Therapeutic Advances In Blastic Plasmacytoid

Dendritic Cell Neoplasm: A Focus On Hematopoietic Cell Transplantation,”

Biol. Blood Marrow Transplant. 19:1006-1012; Florian, S. et al. (2006) “Detection Of Molecular Targets On The Surface Of CD34+/CD38-- Stem

Cells In Various Myeloid Malignancies,” Leuk. Lymphoma 47:207-222; 10

Munoz, L. et al. (2001) “Interleukin-3 Receptor Alpha Chain (CD123) Is Widely Expressed In Hematologic Malignancies,” Haematologica 86:1261-1269; Fromm, JR (2011) “Flow Cytometric Analysis of CD123

“Is Useful For Immunophenotyping Classical Hodgkin Lymphoma,”

It also supports the expected pharmacokinetic activity (Cytometry B Clin). Cytom. 80:91-99 15

The observed safety profile in non-human primates and the clinical benefit and efficacy of DART-A as an immunotherapy for these disorders.

Briefly, DART-A is an antibody-based moiety that binds the CD3ε subunit of the TCR to redirect T lymphocytes against cells expressing CD123, an antigen that is up-regulated20 in several hematological malignancies. DART-A binds to both human antigens

The baboons have similar alpha values and redirect T cells from both types to kill CD123+ cells. Monkeys treated with a 4- to 7-day per week infusion with weekly escalating doses of DART-A showed depletion of circulating CD123+ cells 72 hours after the start of treatment that persisted through the four weeks of treatment, regardless of dosing schedules. like that

<p dir="rtl">25 There was a decrease in circulating T cells, but they returned to baseline before the next infusion in the monkeys</p>

8650

-124-

subject to a 4-day dosing schedule, consistent with kinetics induced by DART-A. DART-A administration increased PD1+, but not 3-TIM+, circulating T cells; Furthermore, ex vivo analysis of T cells from treated monkeys showed no change in redirected degranulation of target cells, indicating no exhaustion. Toxicity was limited to release

<p dir="rtl">5 A marginal cytokine response after the first infusion of DART-A, but not after subsequent administrations even when the dose was increased, and a marginal, reversible decrease in red cell mass with a concomitant decrease in bone marrow CD123+ progenitor cells. Clinical testing of DART A in malignant aneurysms appears to be approved.</p>

All issues and patents of the present description are included in the present application by reference 10 to the same extent that they would be included if each individual issue or patent application was specifically and individually referenced by reference in its entirety. While the invention has been described in relation to specific embodiments, it should be recognized that further modifications may be made and the present application covers any variations, uses, or dispositions of the invention that generally comply with the principles of the invention, including variations of the present disclosure that It shall be within the framework of known or usual practice in the field to which the invention belongs and in the manner in which it belongs

<p dir="rtl">15 Applies to the main features outlined above of the current application.</p>

List of sequences

MacroGenics, Inc. &lt;110&gt;

Bonvini, Ezio

Johnson, Leslie

Huang, Ling 20

Moore, Paul

Chichili, Gurunadh

Alderson, Ralph

8650

-125-

<p dir="rtl">&lt;120&gt; Monovalent dimers that can bind CD123 and CD3 and their uses</p>

PCT1301.0109 &lt;130&lt;

US 61/907,749 &lt;150&gt;

22-11-2013&gt;151&lt;

US 61/869,510 &lt;150&lt;

23-08-2013&gt;151&lt;

59&gt;160&lt;

PatentIn version 3.5&gt;170&lt;

1&gt;210&lt;

272 &gt;211&lt;

PRT &lt;212&lt;

Artificial Sequence &lt;213&lt;

&gt;220&lt;

First Polypeptide Chain of DART-A &gt;223&lt;

1&gt;400&lt;

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

15 10 51

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

30 2520

10

15

8650

-126-

Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

45 4035

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe

60 5550

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 5

80 75 7065

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

95 9085

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly

110 105 10010

Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Gln Ser Gly Ala Glu

125 120115

Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly

140 135130

Tyr Thr Phe Thr Asp Tyr Tyr Met Lys Trp Val Arg Gln Ala Pro Gly 15

160 155 150145

Gln Gly Leu Glu Trp Ile Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr

175 170165

8650

-127-

Phe Tyr Asn Gln Lys Phe Lys Gly Arg Val Thr Ile Thr Val Asp Lys

190 185180

Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp

205 200195

Thr Ala Val Tyr Tyr Cys Ala Arg Ser His Leu Leu Arg Ala Ser Trp

220 215210

Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

240 235 230225

Cys Gly Gly Gly Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu

255 250245

Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys

270 265260

2 &gt;210&lt;

816 &gt;211&lt;

DNA &gt;212&lt;

Artificial Sequence &lt;213&lt;

&gt;220&lt;

Nucleic Acid Molecule Encoding First Polypeptide Chain of &lt;223&lt;

DART-A

10

15

8650

-128-

2 &gt;400&lt;

caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac tgtgaccctg

60

acatgcagat ccagcacagg cgcagtgacc acatctaact acgccaattg ggtgcagcag

120 5

aagccaggac aggcaccaag gggcctgatc gggggtacaa acaaaagggc tccctggacc

180

cctgcacggt tttctggaag tctgctgggc ggaaaggccg ctctgactat taccggggca

240

caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc 300 10

gggggtggca caaaactgac tgtgctggga gggggtggat ccggcggcgg aggcgaggtg

360

cagctggtgc agtccggggc tgagctgaag aaacccggag cttccgtgaa ggtgtcttgc

420

aaagccagtg gctacacctt cacagactac tatatgaagt gggtcaggca ggctccagga 1 5

480

cagggactgg aatggatcgg cgatatcatt ccttccaacg gggccacttt ctacaatcag

540

aagtttaaag gcagggtgac tattaccgtg gacaaatcaa caagcactgc ttatatggag

600 20

ctgagctccc tgcgctctga agatacagcc gtgtactatt gtgctcggtc acacctgctg 660

8650

-129-

agagccagct ggtttgctta ttggggacag ggcaccctgg tgacagtgtc ttccggagga

720

tgtggcggtg gagaagtggc cgcactggag aaagaggttg ctgctttgga gaaggaggtc

780

gctgcacttg aaaaggaggt cgcagccctg gagaaa 8165

3&gt;210&lt;

280 &gt;211&lt;

PRT &lt;212&lt;

Artificial Sequence &lt;213&lt;

&gt;220&lt;10

Second Polypeptide Chain of DART-A &gt;223&lt;

3&gt;400&lt;

Asp Phe Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

15 10 51

Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 15

30 2520

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

45 4035

8650

-130-

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

60 5550

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

80 75 7065

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 5

95 9085

Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

110 105100

Lys Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser

125 120 11510

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

140 135130

Ala Ser Gly Phe Thr Phe Ser Thr Tyr Ala Met Asn Trp Val Arg Gln

160 155 150145

Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile Arg Ser Lys Tyr 15

175 170165

Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr

190 185180

8650

-131-

Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser

205 200195

Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn

220 215210

Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr 5

240 235 230225

Leu Val Thr Val Ser Ser Gly Gly Cys Gly Gly Gly Lys Val Ala Ala

255 250245

Leu Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys

270 265 26010

Glu Lys Val Ala Ala Leu Lys Glu

280275

<tr><td><p>4</p></td><td><p>&gt;210&lt;</p></td><td></td></tr><tr><td><p>840</p></td><td><p>&gt;211&lt;</p></td><td></td></tr><tr><td><p>DNA</p></td><td><p>&gt;212&lt;</p></td><td><p>15</p></td></tr><tr><td><p>Artificial Sequence</p></td><td><p>&gt;213&lt;</p></td><td></td></tr><tr><td></td><td><p>&gt;220&lt;</p></td><td></td></tr>

Nucleic Acid Molecule Encoding Second Polypeptide Chain of &lt;223&lt;

DART-A

8650

-132-

4 &gt;400&lt;

gacttcgtga tgacacagtc tcctgatagt ctggccgtga gtctggggga gcgggtgact

60

atgtcttgca agagctccca gtcactgctg aacagcggaa atcagaaaaa ctatctgacc

120 5

tggtaccagc agaagccagg ccagccccct aaactgctga tctattggggc ttccaccagg

180

gaatctggcg tgcccgacag attcagcggc agcggcagcg gcacagattt taccctgaca

240

atttctagtc tgcaggccga ggacgtggct gtgtactatt gtcagaatga ttacagctat 300 10

ccctacactt tcggccaggg gaccaagctg gaaattaaag gaggcggatc cggcggcgga

360

ggcgaggtgc agctggtgga gtctggggga ggcttggtcc agcctggagg gtccctgaga

420

ctctcctgtg cagcctctgg attcaccttc agcacatacg ctatgaattg ggtccgccag 480 15

gctccaggga aggggctgga gtgggttgga aggatcaggt ccaagtacaa caattatgca

540

acctactatg ccgactctgt gaaggataga ttcaccatct caagagatga ttcaaagaac

600

tcactgtatc tgcaaatgaa cagcctgaaa accgaggaca cggccgtgta ttactgtgtg 2 0

660

8650

-133-

agacacggta acttcggcaa ttcttacgtg tcttggtttg cttattgggg acaggggaca 720

ctggtgactg tgtcttccgg aggatgtggc ggtggaaaag tggccgcact gaaggagaaa

780

gttgctgctt tgaaagagaa ggtcgccgca cttaaggaaa aggtcgcagc cctgaaagag 8405

5&gt;210&lt;

268&gt;211&lt;

PRT &lt;212&lt;

Artificial Sequence &lt;213&lt;

&gt;220&lt;10

First Polypeptide Chain of DART-B &lt;223&gt;

5&gt;400&lt;

Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

15 10 51

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 15

30 2520

Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

45 4035

8650

-134-

Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser

60 5550

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

80 75 7065

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 5

95 9085

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Ser Gly Gly

110 105100

Gly Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys Pro

125 120 11510

Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

140 135130

Asp Tyr Tyr Met Lys Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

160 155 150145

Trp Ile Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn Gln 15

175 170165

Lys Phe Lys Gly Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr

190 185180

8650

-135-

Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr

205 200195

Tyr Cys Ala Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp

220 215210

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Cys Gly Gly Gly 5

240 235 230225

Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val

255 250245

Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys

265 26010

6 &gt;210&lt;

804 &gt;211&lt;

DNA &gt;212&lt;

Artificial Sequence &lt;213&lt;

&gt;220&lt; 15

Nucleic Acid Molecule Encoding First Polypeptide Chain of &lt;223&lt;

DART-B

6 &gt;400&lt;

8650

-136-

gacattcagc tgacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc

60

atgacctgca gagccagttc aagtgtaagt tacatgaact ggtaccagca gaagtcaggc

120

acctccccca aaagatggat ttatgacaca tccaaagtgg cttctggagt cccttatcgc 5

180

ttcagtggca gtgggtctgg gacctcatac tctctcacaa tcagcagcat ggaggctgaa

240

gatgctgcca cttattactg ccaacagtgg agtagtaacc cgctcacgtt cggtgctggg

300 10

accaagctgg agctgaaagg aggcggatcc ggcggcggag gccaggtgca gctggtgcag

360

tccggggctg agctgaagaa acccggagct tccgtgaagg tgtcttgcaa agccagtggc

420

tacaccttca cagactacta tatgaagtgg gtcaggcagg ctccaggaca gggactggaa 1 5

480

tggatcggcg atatcattcc ttccaacggg gccactttct acaatcagaa gtttaaaggc 540

agggtgacta ttaccgtgga caaatcaaca agcactgctt atatggagct gagctccctg

600

cgctctgaag atacagccgt gtactattgt gctcggtcac acctgctgag agccagctgg 2 0

660

8650

-137-

tttgcttatt ggggacaggg caccctggtg acagtgtctt ccggaggatg tggcggtgga

720

gaagtggccg cactggagaa agaggttgct gctttggaga aggaggtcgc tgcacttgaa

780

aaggaggtcg cagccctgga gaaa 804 5

7 &gt;210&lt;

274 &gt;211&lt;

PRT &lt;212&lt;

Artificial Sequence &lt;213&lt;

&gt;220&lt;10

Second Polypeptide Chain of DART-B &lt;223&gt;

7 &gt;400&lt;

Asp Phe Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

15 10 51

Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 15

30 2520

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

45 4035

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

8650

-138-

60 5550

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

80 75 7065

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

95 90 855

Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

110 105100

Lys Gly Gly Gly Ser Gly Gly Gly Gly Asp Ile Lys Leu Gln Gln Ser

125 120115

Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys 10

140 135130

Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln

160 155 150145

Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg

175 170 16515

Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr

190 185180

Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr

205 200195

8650

-139-

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His

220 215210

Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

240 235 230225

Gly Gly Cys Gly Gly Gly Lys Val Ala Ala Leu Lys Glu Lys Val Ala 5

255 250245

Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu

270 265260

LysGlu 10

8&gt;210&lt;

822 &gt;211&lt;

DNA &gt;212&lt;

Artificial Sequence &gt;213&lt;15

&gt;220&lt;

Nucleic Acid Molecule Encoding Second Polypeptide Chain of &lt;223&lt;

DART-B

8&gt;400&lt;

8650

-140-

gacttcgtga tgacacagtc tcctgatagt ctggccgtga gtctggggga gcgggtgact

60

atgtcttgca agagctccca gtcactgctg aacagcggaa atcagaaaaa ctatctgacc

120

tggtaccagc agaagccagg ccagccccct aaactgctga tctattggggc ttccaccagg 5

180

gaatctggcg tgcccgacag attcagcggc agcggcagcg gcacagattt taccctgaca

240

atttctagtc tgcaggccga ggacgtggct gtgtactatt gtcagaatga ttacagctat 300

ccctacactt tcggccaggg gaccaagctg gaaattaaag gaggcggatc cggcggcgga 1 0

360

ggcgatatca aactgcagca gtcaggggct gaactggcaa gacctggggc ctcagtgaag

420

atgtcctgca agacttctgg ctacaccttt actaggtaca cgatgcactg ggtaaaacag 480 15

aggcctggac agggtctgga atggattgga tacattaatc ctagccgtgg ttatactaat 540

tacaatcaga agttcaagga caaggccaca ttgactacag acaaatcctc cagcacagcc

600

tacatgcaac tgagcagcct gacatctgag gactctgcag tctattactg tgcaagatat 660 20

tatgatgatc attactgcct tgactactgg ggccaaggca ccactctcac agtctcctcc 720

8650

-141-

ggaggatgtg gcggtggaaa agtggccgca ctgaaggaga aagttgctgc tttgaaagag 780

aaggtcgccg cacttaagga aaaggtcgca gccctgaaag ag822

9 &gt;210&lt;

322 &gt;211&lt;5

PRT &lt;212&lt;

Artificial Sequence &lt;213&lt;

&gt;220&lt;

CD123 x CD3 Diabody Polypeptide Chain Having Albumin &gt;223&lt;

Binding Site 10

9 &gt;400&lt;

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

15 10 51

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

30 25 2015

Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

45 4035

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe

60 5550

8650

-142-

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala

80 75 7065

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

95 9085

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly 5

110 105100

Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Gln Ser Gly Ala Glu

125 120115

Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly

140 135 13010

Tyr Thr Phe Thr Asp Tyr Tyr Met Lys Trp Val Arg Gln Ala Pro Gly

160 155 150145

Gln Gly Leu Glu Trp Ile Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr

175 170165

Phe Tyr Asn Gln Lys Phe Lys Gly Arg Val Thr Ile Thr Val Asp Lys 15

190 185180

Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp

205 200195

Thr Ala Val Tyr Tyr Cys Ala Arg Ser His Leu Leu Arg Ala Ser Trp

8650

-143-

220 215210

Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

240 235 230225

Cys Gly Gly Gly Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu

255 250 2455

Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys

270 265260

Gly Gly Gly Ser Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu Leu

285 280275

Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn Leu Ile Asp Asn Ala 10

300 295290

Lys Ser Ala Glu Gly Val Lys Ala Leu Ile Asp Glu Ile Leu Ala Ala

320 315 310305

Leu Pro

15

10 &gt;210&lt;

966 &gt;211&lt;

DNA &gt;212&lt;

Artificial Sequence &lt;213&lt;

8650

-144-

&gt;220&lt;

Polynucleotide Encoding CD123 x CD3 Diabody Polypeptide &lt;223&gt;

Chain

Having Albumin Binding Site

10 &gt;400&lt; 5

caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac tgtgaccctg

60

acatgcagat ccagcacagg cgcagtgacc acatctaact acgccaattg ggtgcagcag

120

aagccaggac aggcaccaag gggcctgatc gggggtacaa acaaaagggc tccctggacc 1 0

180

cctgcacggt tttctggaag tctgctgggc ggaaaggccg ctctgactat taccggggca

240

caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc 300

gggggtggca caaaactgac tgtgctggga gggggtggat ccggcggcgg aggcgaggtg 1 5

360

cagctggtgc agtccggggc tgagctgaag aaacccggag cttccgtgaa ggtgtcttgc

420

aaagccagtg gctacacctt cacagactac tatatgaagt gggtcaggca ggctccagga

480 20

8650

-145-

cagggactgg aatggatcgg cgatatcatt ccttccaacg gggccacttt ctacaatcag

540

aagtttaaag gcagggtgac tattaccgtg gacaaatcaa caagcactgc ttatatggag

600

ctgagctccc tgcgctctga agatacagcc gtgtactatt gtgctcggtc acacctgctg 660 5

agagccagct ggtttgctta ttggggacag ggcaccctgg tgacagtgtc ttccggagga

720

tgtggcggtg gagaagtggc cgcactggag aaagaggttg ctgctttgga gaaggaggtc

780

gctgcacttg aaaaggaggt cgcagccctg gagaaaggcg gcgggtctct ggccgaagca 1 0

840

aaagtgctgg ccaaccgcga actggataaa tatggcgtga gcgattatta taagaacctg

900

attgacaacg caaaatccgc ggaaggcgtg aaagcactga ttgatgaaat tctggccgcc

96015

ctgcct966

11&gt;210&lt;

217 &gt;211&lt;

PRT &lt;212&lt;

Artificial Sequence &lt;213&lt; 20

&gt;220&lt;

8650

-146-

CH2-CH3 Domains of a Modified Human Antibody Fc Region &lt;223&gt;

11&gt;400&lt;

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

15 10 51

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 5

30 2520

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

45 4035

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

60 55 5010

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

80 75 7065

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

95 9085

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 15

110 105100

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

125 120115

Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro

8650

-147-

140 135130

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

160 155 150145

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

175 170 1655

Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

190 185180

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Tyr Thr Gln

205 200195

Lys Ser Leu Ser Leu Ser Pro Gly Lys 10

215210

12 &gt;210&lt;

681&gt;211&lt;

DNA &gt;212&lt;

Artificial Sequence &gt;213&lt;15

&gt;220&lt;

Nucleic Acid Molecule Encoding Peptide 1 and the CH2 and CH3 &gt;223&lt;

Domains of an IgG Fc region

12 &gt;400&lt;

8650

-148-

gacaaaactc acacatgccc accgtgccca gcacctgaag ccgcgggggg accgtcagtc

60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca

120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 5

180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac

240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag

300 10

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa

360

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag

420

aaccaggtca gcctgagttg cgcagtcaaa ggcttctatc ccagcgacat cgccgtggag 1 5

480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc

540

gacggctcct tcttcctcgt cagcaagctc accgtggaca agagcaggtg gcagcagggg

600 20

aacgtcttct catgctccgt gatgcatgag gctctgcaca accgctacac gcagaagagc

660

8650

-149-

ctctccctgt ctccgggtaa a681

13&gt;210&lt;

510 &gt;211&lt;

PRT &lt;212&lt;

Artificial Sequence &gt;213&lt;5

&gt;220&lt;

First Polypeptide Chain of DART-A w/Fc Version 1 Construct &lt;223&lt;

13&gt;400&lt;

Asp Phe Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

15 10 5 110

Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

30 2520

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

45 4035

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 15

60 5550

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

80 75 7065

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

8650

-150-

95

90

85

Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

110 105100

Lys Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser

125 120 1155

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

140 135130

Ala Ser Gly Phe Thr Phe Ser Thr Tyr Ala Met Asn Trp Val Arg Gln

160 155 150145

Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile Arg Ser Lys Tyr 10

175 170165

Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr

190 185180

Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser

205 200 19515

Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn

220 215210

Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr

240 235 230225

8650

-151-

Leu Val Thr Val Ser Ser Gly Gly Cys Gly Gly Gly Glu Val Ala Ala

255 250245

Leu Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu

270 265260

Lys Glu Val Ala Ala Leu Glu Lys Gly Gly Gly Asp Lys Thr His Thr 5

285 280275

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe

300 295290

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

320 315 310 30510

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

335 330325

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

350 345340

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 15

365 360355

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

380 375370

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

8650

-152-

400 395 390385

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

415 410405

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val

430 425 4205

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

445 440435

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

460 455450

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 10

480 475 470465

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

495 490485

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

510 505 50015

14 &gt;210&lt;

1530 &gt;211&lt;

DNA &gt;212&lt;

Artificial Sequence &lt;213&lt;

8650

-153-

&gt;220&lt;

Nucleic Acid Molecule Encoding First Polypeptide Chain of &lt;223&lt;

DART-A

w/Fc Version 1 Construct

14 &gt;400&lt; 5

gacttcgtga tgacacagtc tcctgatagt ctggccgtga gtctggggga gcgggtgact

60

atgtcttgca agagctccca gtcactgctg aacagcggaa atcagaaaaa ctatctgacc

120

tggtaccagc agaagccagg ccagccccct aaactgctga tctattggggc ttccaccagg 1 0

180

gaatctggcg tgcccgacag attcagcggc agcggcagcg gcacagattt taccctgaca

240

atttctagtc tgcaggccga ggacgtggct gtgtactatt gtcagaatga ttacagctat 300

ccctacactt tcggccaggg gaccaagctg gaaattaaag gaggcggatc cggcggcgga 1 5

360

ggcgaggtgc agctggtgga gtctggggga ggcttggtcc agcctggagg gtccctgaga

420

ctctcctgtg cagcctctgg attcaccttc agcacatacg ctatgaattg ggtccgccag 480

gctccaggga aggggctgga gtgggttgga aggatcaggt ccaagtacaa caattatgca 2 0

540

8650

-154-

acctactatg ccgactctgt gaaggataga ttcaccatct caagagatga ttcaaagaac

600

tcactgtatc tgcaaatgaa cagcctgaaa accgaggaca cggccgtgta ttactgtgtg

660

agacacggta acttcggcaa ttcttacgtg tcttggtttg cttattgggg acaggggaca 720 5

ctggtgactg tgtcttccgg aggatgtggc ggtggagaag tggccgcact ggagaaagag

780

gttgctgctt tggagaagga ggtcgctgca cttgaaaagg aggtcgcagc cctggagaaa

840

ggcggcgggg acaaaactca cacatgccca ccgtgcccag cacctgaagc cgcgggggga 1 0

900

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct

960

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg

1020 15

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac

1080

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag

1140

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 20

1200

8650

-155-

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag

1260

atgaccaaga accaggtcag cctgtggtgc ctggtcaaag gcttctatcc cagcgacatc

1320

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 5

1380

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg

1440

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg

150010

cagaagagcc tctccctgtc tccgggtaaa1530

15&gt;210&lt;

272 &gt;211&lt;

PRT &lt;212&lt;

Artificial Sequence &gt;213&lt;15

&gt;220&lt;

Second Polypeptide Chain of DART-A w/Fc Version 1 Construct &lt;223&lt;

15&gt;400&lt;

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

15 10

5 1 20

8650

-156-

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

30 2520

Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

45 4035

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 5

60 5550

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala

80 75 7065

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

95 90 8510

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly

110 105100

Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Gln Ser Gly Ala Glu

125 120115

Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 15

140 135130

Tyr Thr Phe Thr Asp Tyr Tyr Met Lys Trp Val Arg Gln Ala Pro Gly

160 155 150145

Gln Gly Leu Glu Trp Ile Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr

8650

-157-

175

170

165

Phe Tyr Asn Gln Lys Phe Lys Gly Arg Val Thr Ile Thr Val Asp Lys

190 185180

Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp

205 200 1955

Thr Ala Val Tyr Tyr Cys Ala Arg Ser His Leu Leu Arg Ala Ser Trp

220 215210

Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

240 235 230225

Cys Gly Gly Gly Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu 10

255 250245

Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu

270 265260

16 &gt;210&lt;

816 &gt;211&lt;15

DNA &gt;212&lt;

Artificial Sequence &lt;213&lt;

&gt;220&lt;

8650

-158-

Nucleic Acid Molecule Encoding Second Polypeptide Chain of &lt;223&lt;

DART-A

w/Fc Version 1 Construct

16 &gt;400&lt;

caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac tgtgaccctg 5

60

acatgcagat ccagcacagg cgcagtgacc acatctaact acgccaattg ggtgcagcag

120

aagccaggac aggcaccaag gggcctgatc gggggtacaa acaaaagggc tccctggacc 180 10

cctgcacggt tttctggaag tctgctgggc ggaaaggccg ctctgactat taccggggca

240

caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc 300

gggggtggca caaaactgac tgtgctggga gggggtggat ccggcggcgg aggcgaggtg 360 15

cagctggtgc agtccggggc tgagctgaag aaacccggag cttccgtgaa ggtgtcttgc

420

aaagccagtg gctacacctt cacagactac tatatgaagt gggtcaggca ggctccagga

480

cagggactgg aatggatcgg cgatatcatt ccttccaacg gggccacttt ctacaatcag 2 0

540

8650

-159-

aagtttaaag gcagggtgac tattaccgtg gacaaatcaa caagcactgc ttatatggag

600

ctgagctccc tgcgctctga agatacagcc gtgtactatt gtgctcggtc acacctgctg 660

agagccagct ggtttgctta ttggggacag ggcaccctgg tgacagtgtc ttccggagga

7205

tgtggcggtg gaaaagtggc cgcactgaag gagaaagttg ctgctttgaa agagaaggtc

780

gccgcactta aggaaaaggt cgcagccctg aaagag816

17 &gt;210&lt;

515 &gt;211&lt;10

PRT &lt;212&lt;

Artificial Sequence &lt;213&lt;

&gt;220&lt;

First Polypeptide Chain of DART-A w/Fc Version 2 Construct &lt;223&lt;

17 &gt;400&lt;15

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

15 10 51

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

30 2520

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Asp Val Ser His 20

8650

-160-

45 4035

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

60 5550

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

80 75 70 655

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

95 9085

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

110 105100

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 10

125 120115

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

140 135130

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

160 155 150 14515

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

175 170165

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

190 185180

8650

-161-

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

205 200195

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

220 215210

Pro Gly Lys Ala Pro Ser Ser Ser Pro Met Glu Asp Phe Val Met Thr 5

240 235 230225

Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Val Thr Met

255 250245

Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn

270 265 26010

Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu

285 280275

Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser

300 295290

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 15

320 315 310305

Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro

335 330325

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Ser

8650

-162-

350

345

340

Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

365 360355

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

380 375 3705

Phe Ser Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly

400 395 390385

Leu Glu Trp Val Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr

415 410405

Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp 10

430 425420

Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp

445 440435

Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr

460 455 45015

Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

480 475 470465

Ser Gly Gly Cys Gly Gly Gly Lys Val Ala Ala Leu Lys Glu Lys Val

495 490485

8650

-163-

Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala

510 505500

Leu Lys Glu

515

18 &gt;210&lt;5

1545&gt;211&lt;

DNA &gt;212&lt;

Artificial Sequence &lt;213&lt;

&gt;220&lt;

Nucleic Acid Molecule Encoding First Polypeptide Chain of &gt;223&lt;10

DART-A

w/Fc Version 2 Construct

18&gt;400&lt;

gacaaaactc acacatgccc accgtgccca gcacctgaag ccgcgggggg accgtcagtc

6015

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca

120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac

180

8650

-164-

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac

240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag

300

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 5

360

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag

420

aaccaggtca gcctgtggtg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag

480 10

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc

540

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg

600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1 5

660

ctctccctgt ctccgggtaa agccccttcc agctccccta tggaagactt cgtgatgaca 720

cagtctcctg atagtctggc cgtgagtctg ggggagcggg tgactatgtc ttgcaagagc

780

tcccagtcac tgctgaacag cggaaatcag aaaaactatc tgacctggta ccagcagaag 2 0

840

8650

-165-

ccaggccagc cccctaaact gctgatctat tgggcttcca ccagggaatc tggcgtgccc

900

gacagattca gcggcagcgg cagcggcaca gattttaccc tgacaatttc tagtctgcag

960

gccgaggacg tggctgtgta ctattgtcag aatgattaca gctatcccta cactttcggc 1020 5

caggggacca agctggaaat taaaggaggc ggatccggcg gcggaggcga ggtgcagctg

1080

gtggagtctg ggggaggctt ggtccagcct ggagggtccc tgagactctc ctgtgcagcc

1140

tctggattca ccttcagcac atacgctatg aattgggtcc gccaggctcc agggaagggg 10

1200

ctggagtggg ttggaaggat caggtccaag tacaacaatt atgcaaccta ctatgccgac

1260

tctgtgaagg atagattcac catctcaaga gatgattcaa agaactcact gtatctgcaa

1320 15

atgaacagcc tgaaaaccga ggacacggcc gtgtattact gtgtgagaca cggtaacttc

1380

ggcaattctt acgtgtcttg gtttgcttat tggggacagg ggacactggt gactgtgtct 1440

tccggaggat gtggcggtgg aaaagtggcc gcactgaagg agaaagttgc tgctttgaaa

1500 20

gagaaggtcg ccgcacttaa ggaaaaaggtc gcagccctga aagag

1545

8650

-166-

19 &gt;210&lt;

279 &gt;211&lt;

PRT &lt;212&lt;

Artificial Sequence &lt;213&lt;

&gt;220&lt;5

First Polypeptide Chain of Control DART &lt;223&gt;

19 &gt;400&lt;

Asp Val Val Met Thr Gln Thr Pro Phe Ser Leu Pro Val Ser Leu Gly

15 10 51

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 10

30 2520

Asn Gly Asn Thr Tyr Leu Arg Trp Tyr Leu Gln Lys Pro Gly Gln Ser

45 4035

Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

60 55 5015

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

80 75 7065

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

95 9085

8650

-167-

Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

110 105100

Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly

125 120115

Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 5

140 135130

Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala

160 155 150145

Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn

175 170 16510

Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile

190 185180

Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu

205 200195

Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn Phe 15

220 215210

Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu

240 235 230225

Val Thr Val Ser Ser Gly Gly Cys Gly Gly Gly Glu Val Ala Ala Leu

8650

-168-

255

250

245

Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys

270 265 260

Glu Val Ala Ala Leu Glu Lys

275 5

20 &gt;210&lt;

270 &gt;211&lt;

PRT &lt;212&lt;

Artificial Sequence &lt;213&lt;

&gt;220&lt;10

Second Polypeptide Chain of Control DART &lt;223&lt;

20 &gt;400&lt;

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

15 10 51

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 15

30 2520

Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

45 4035

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe

8650

-169-

60 5550

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala

80 75 7065

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

95 90 855

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly

110 105100

Gly Ser Gly Gly Gly Gly Glu Val Lys Leu Asp Glu Thr Gly Gly Gly

125 120115

Leu Val Gln Pro Gly Arg Pro Met Lys Leu Ser Cys Val Ala Ser Gly 10

140 135130

Phe Thr Phe Ser Asp Tyr Trp Met Asn Trp Val Arg Gln Ser Pro Glu

160 155 150145

Lys Gly Leu Glu Trp Val Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr

175 170 16515

Glu Thr Tyr Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

190 185180

Asp Asp Ser Lys Ser Ser Val Tyr Leu Gln Met Asn Asn Leu Arg Val

205 200195

8650

-170-

Glu Asp Met Gly Ile Tyr Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp

220 215210

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Cys Gly

240 235 230225

Gly Gly Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu 5

255 250245

Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu

270 265260

21&gt;210&lt;

110 &gt;211&lt;10

PRT &lt;212&lt;

Artificial Sequence &lt;213&lt;

&gt;220&lt;

Light Chain CD3-Binding Domain of DART-A &lt;223&gt;

21 &gt;400&lt; 15

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

15 10 5 1

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

30 25 20

8650

-171-

Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

45 4035

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe

60 5550

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 5

80 75 7065

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

95 9085

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

110 105 10010

22 &gt;210&lt;

125&gt;211&lt;

PRT &lt;212&lt;

Artificial Sequence &lt;213&lt;

&gt;220&lt;15

Heavy Chain CD3-Binding Domain of DART-A &lt;223&gt;

22 &gt;400&lt;

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

15 10 51

8650

-172-

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

30 2520

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

45 4035

Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 5

60 5550

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

80 75 7065

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

95 90 8510

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

110 105100

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

125 120115

23 &gt;210&lt;15

106 &gt;211&lt;

PRT &lt;212&lt;

Artificial Sequence &lt;213&lt;

&gt;220&lt;

8650

-173-

Light Chain CD3-Binding Domain of DART-B &lt;223&gt;

23&gt;400&lt;

Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

15 10 51

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 5

30 2520

Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

45 4035

Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser

60 55 5010

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

80 75 7065

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

95 9085

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 15

105100

24 &gt;210&lt;

119 &gt;211&lt;

PRT &lt;212&lt;

8650

-174-

Artificial Sequence &lt;213&lt;

&gt;220&lt;

Heavy Chain CD3-Binding Domain of DART-B &lt;223&gt;

24 &gt;400&lt;

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 5

15 10 51

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

30 2520

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

45 40 3510

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

60 5550

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

80 75 7065

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 15

95 9085

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

110 105100

Thr Thr Leu Thr Val Ser Ser