Antibodies that bind to CLEC12A

Abstract

Anti-CLEC12A antibodies are disclosed, along with methods for making such antibodies, compositions, including pharmaceutical compositions, comprising such antibodies, and their use for treating disorders characterized by expression of CLEC12A.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 396,536, filed August 9, 2022, the disclosure of which is incorporated by reference herein in its entirety.

[0002] Technical Field The present invention relates to antibodies that bind to CLEC12A. The invention further relates to methods of making such antibodies, compositions, including pharmaceutical compositions, comprising such antibodies, and their use for treating disorders characterized by expression of CLEC12A. [Background technology]

[0003] CLEC12A CLEC12A, also known as C-type lectin domain family 12 member A, DCAL-2, CLL-1, MICL, and CD371, is a member of the C-type lectin / C-type lectin-like domain (CTL / CTLD) superfamily. Members of this family share a common protein fold and have diverse functions, including roles in cell adhesion, cell-cell signaling, glycoprotein turnover, and inflammation and immune responses. The protein encoded by this gene is a negative regulator of granulocyte and monocyte function.

[0004] RNA expression analysis from the TCGA database indicates that CLEC12A is highly expressed and specific to acute myeloid leukemia. Therefore, therapeutic development of antibodies targeting CLEC12A may be effective in treating AML patients, including a significant number of AML patients who are resistant to current standard care treatments. Summary of the Invention

[0005] Aspects of the present invention relate to CLEC12A-binding antibodies. Further aspects of the present invention relate to methods of making such antibodies, compositions comprising such antibodies, and their use in the treatment of disorders characterized by the expression of CLEC12A.

[0006] In some embodiments, an antibody that binds to CLEC12A comprises a first binding unit comprising a heavy chain variable region comprising: (a) a CDRH1 sequence of any one of SEQ ID NOs: 1-5; and / or (b) a CDRH2 sequence of any one of SEQ ID NOs: 6-14; and / or (c) a CDRH3 sequence of any one of SEQ ID NOs: 15-22; and a light chain variable region comprising: (d) a CDRL1 sequence of any one of SEQ ID NOs: 23-27; and / or (e) a CDRL2 sequence of any one of SEQ ID NOs: 28-33; and / or (f) a CDRL3 sequence of any one of SEQ ID NOs: 34-40. In some embodiments, the CDR1, CDR2, and CDR3 sequences of this first binding unit are present in a human VH framework. In some embodiments, the CDR1, CDR2, and CDR3 sequences of this first binding unit are present in a human VL framework.

[0007] In some embodiments, the first binding unit comprises a heavy chain variable region comprising: (a) a CDRH1 sequence of any one of SEQ ID NOs: 1-5; and (b) a CDRH2 sequence of any one of SEQ ID NOs: 6-14; and (c) a CDRH3 sequence of any one of SEQ ID NOs: 15-22.

[0008] In some embodiments, the first binding unit is: (a) a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 6, and a CDRH3 sequence of SEQ ID NO: 15; or (b) a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 7, and a CDRH3 sequence of SEQ ID NO: 16; or (c) a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 8, and a CDRH3 sequence of SEQ ID NO: 17; or (d) a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 9, and a CDRH3 sequence of SEQ ID NO: 18; or (e) a CDRH1 sequence of SEQ ID NO: 2, a CDRH2 sequence of SEQ ID NO: 1 or (f) a CDRH1 sequence of SEQ ID NO: 3, a CDRH2 sequence of SEQ ID NO: 11, and a CDRH3 sequence of SEQ ID NO: 20; or (g) a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 12, and a CDRH3 sequence of SEQ ID NO: 21; or (h) a CDRH1 sequence of SEQ ID NO: 4, a CDRH2 sequence of SEQ ID NO: 13, and a CDRH3 sequence of SEQ ID NO: 22; or (i) a CDRH1 sequence of SEQ ID NO: 5, a CDRH2 sequence of SEQ ID NO: 14, and a CDRH3 sequence of SEQ ID NO: 22.

[0009] In some embodiments, the first binding unit comprises a light chain variable region comprising: (a) a CDRL1 sequence of any one of SEQ ID NOs: 23 to 27; and (b) a CDRL2 sequence of any one of SEQ ID NOs: 28 to 33; and (c) a CDRL3 sequence of any one of SEQ ID NOs: 34 to 40. In some embodiments, the first binding unit comprises a light chain variable region comprising: (a) the CDRL1 sequence of SEQ ID NO:23, the CDRL2 sequence of SEQ ID NO:28, and the CDRL3 sequence of SEQ ID NO:34; or (b) the CDRL1 sequence of SEQ ID NO:24, the CDRL2 sequence of SEQ ID NO:29, and the CDRL3 sequence of SEQ ID NO:35; or (c) the CDRL1 sequence of SEQ ID NO:25, the CDRL2 sequence of SEQ ID NO:30, and the CDRL3 sequence of SEQ ID NO:36; or (d) the CDRL1 sequence of SEQ ID NO:26, the CDRL2 sequence of SEQ ID NO:31, and the CDRL3 sequence of SEQ ID NO:37; or (e) the CDRL1 sequence of SEQ ID NO:25, the CDRL2 sequence of SEQ ID NO:30, and the CDRL3 sequence of SEQ ID NO:38; or (f) the CDRL1 sequence of SEQ ID NO:27, the CDRL2 sequence of SEQ ID NO:32, and the CDRL3 sequence of SEQ ID NO:39; or (g) the CDRL1 sequence of SEQ ID NO:24, the CDRL2 sequence of SEQ ID NO:33, and the CDRL3 sequence of SEQ ID NO:40.

[0010] In some embodiments, the first binding unit comprises (a) a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 1, the CDRH2 sequence of SEQ ID NO: 6, and the CDRH3 sequence of SEQ ID NO: 15; and a light chain variable region comprising the CDRL1 sequence of SEQ ID NO: 23; the CDRL2 sequence of SEQ ID NO: 28, and the CDRL3 sequence of SEQ ID NO: 34; or (b) a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 1, the CDRH2 sequence of SEQ ID NO: 7, and the CDRH3 sequence of SEQ ID NO: 16; and a CDRH1 sequence of SEQ ID NO: 23, the CDRH2 sequence of SEQ ID NO: 28, and the CDRH3 sequence of SEQ ID NO: 34. or (c) a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 1, the CDRH2 sequence of SEQ ID NO: 8, and the CDRH3 sequence of SEQ ID NO: 17; and a light chain variable region comprising the CDRL1 sequence of SEQ ID NO: 24, the CDRH2 sequence of SEQ ID NO: 29, and the CDRL3 sequence of SEQ ID NO: 35; or (d) a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 1, the CDRH2 sequence of SEQ ID NO: 9, and the CDRH3 sequence of SEQ ID NO: 18; and a CDRL1 sequence of SEQ ID NO: 25, the CDRH2 sequence of SEQ ID NO: 30, and the CDRL3 sequence of SEQ ID NO: 36. or (e) a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 2, the CDRH2 sequence of SEQ ID NO: 10, and the CDRH3 sequence of SEQ ID NO: 19; and a light chain variable region comprising the CDRL1 sequence of SEQ ID NO: 26, the CDRH2 sequence of SEQ ID NO: 31, and the CDRL3 sequence of SEQ ID NO: 37; or (f) a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 1, the CDRH2 sequence of SEQ ID NO: 6, and the CDRH3 sequence of SEQ ID NO: 15; and a light chain variable region comprising the CDRL1 sequence of SEQ ID NO: 23, the CDRL2 sequence of SEQ ID NO: 28, and the CDRL3 sequence of SEQ ID NO: 34. or (g) a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 3, the CDRH2 sequence of SEQ ID NO: 11, and the CDRH3 sequence of SEQ ID NO: 20; and a light chain variable region comprising the CDRH1 sequence of SEQ ID NO: 25, the CDRH2 sequence of SEQ ID NO: 30, and the CDRL3 sequence of SEQ ID NO: 38; or (h) a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 1, the CDRH2 sequence of SEQ ID NO: 12, and the CDRH3 sequence of SEQ ID NO: 21; and a light chain variable region comprising the CDRL1 sequence of SEQ ID NO: 27, the CDRL2 sequence of SEQ ID NO: 32, and the CDRL3 sequence of SEQ ID NO: 39;or (i) a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 4, the CDRH2 sequence of SEQ ID NO: 13, and the CDRH3 sequence of SEQ ID NO: 22; and a light chain variable region comprising the CDRL1 sequence of SEQ ID NO: 24, the CDRL2 sequence of SEQ ID NO: 33, and the CDRL3 sequence of SEQ ID NO: 40; or (j) a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 5, the CDRH2 sequence of SEQ ID NO: 14, and the CDRH3 sequence of SEQ ID NO: 22, and a light chain variable region comprising the CDRL1 sequence of SEQ ID NO: 24, the CDRL2 sequence of SEQ ID NO: 33, and the CDRL3 sequence of SEQ ID NO: 40;

[0011] In some embodiments, the first binding unit comprises a heavy chain variable region sequence having at least 95% identity to any one of SEQ ID NOs: 41-52. In some embodiments, the first binding unit comprises a heavy chain variable region sequence comprising any one of SEQ ID NOs: 41-52. In some embodiments, the first binding unit comprises a light chain variable region sequence having at least 95% identity to any one of SEQ ID NOs: 53-64. In some embodiments, the first binding unit comprises a light chain variable region sequence comprising any one of SEQ ID NOs: 53-64.

[0012] In some embodiments, the first binding unit is selected from the group consisting of (a) the heavy chain variable region sequence of SEQ ID NO: 41 and the light chain variable region sequence of SEQ ID NO: 53; or (b) the heavy chain variable region sequence of SEQ ID NO: 42 and the light chain variable region sequence of SEQ ID NO: 54; or (c) the heavy chain variable region sequence of SEQ ID NO: 43 and the light chain variable region sequence of SEQ ID NO: 55; or (d) the heavy chain variable region sequence of SEQ ID NO: 44 and the light chain variable region sequence of SEQ ID NO: 56; or (e) the heavy chain variable region sequence of SEQ ID NO: 45 and the light chain variable region sequence of SEQ ID NO: 57; or (f) the heavy chain variable region sequence of SEQ ID NO: 46 and the light chain variable region sequence of SEQ ID NO: 58 or (g) the heavy chain variable region sequence of SEQ ID NO: 47 and the light chain variable region sequence of SEQ ID NO: 59; or (h) the heavy chain variable region sequence of SEQ ID NO: 48 and the light chain variable region sequence of SEQ ID NO: 60; or (i) the heavy chain variable region sequence of SEQ ID NO: 49 and the light chain variable region sequence of SEQ ID NO: 61; or (j) the heavy chain variable region sequence of SEQ ID NO: 50 and the light chain variable region sequence of SEQ ID NO: 62; or (k) the heavy chain variable region sequence of SEQ ID NO: 51 and the light chain variable region sequence of SEQ ID NO: 63; or (l) the heavy chain variable region sequence of SEQ ID NO: 52 and the light chain variable region sequence of SEQ ID NO: 64.

[0013] In some embodiments, the antibody further comprises a heavy chain constant region. In some embodiments, the heavy chain constant region comprises a hinge region, a CH1 region, a CH2 region, and / or a CH3 region. In some embodiments, the heavy chain constant region comprises one or more knobs-in-holes (KiH) mutations. In some embodiments, the heavy chain constant region comprises one or more silencing mutations. In some embodiments, the heavy chain constant region comprises one or more Protein A-binding mutations. In some embodiments, the one or more Protein A-binding mutations comprise an H435R mutation, a Y436F mutation, or both an H435R and a Y436F mutation.

[0014] In some embodiments, the antibody further comprises a light chain constant region. In some embodiments, the light chain constant region comprises a CL region. In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific. In some embodiments, the antibody is bispecific.

[0015] In some embodiments, the antibody further comprises a second binding unit that binds to CD3ε. In some embodiments, this second binding unit comprises: a heavy chain variable region comprising: (a) the CDRH1 sequence of SEQ ID NO: 65; and / or (b) the CDRH2 sequence of any one of SEQ ID NOs: 66-67; and / or (c) the CDR3 sequence of SEQ ID NO: 68; and a light chain variable region comprising: (d) the CDRL1 sequence of SEQ ID NO: 69; and / or (e) the CDRL2 sequence of SEQ ID NO: 70; and / or (f) the CDRL3 sequence of SEQ ID NO: 71. In some embodiments, the CDRH1, CDRH2, and CDRH3 sequences of this second binding unit are present in a human VH framework. In some embodiments, the CDR1, CDR2, and CDR3 sequences of this second binding unit are present in a human VL framework. In some embodiments, the second binding unit comprises a heavy chain variable region comprising: (a) the CDRH1 sequence of SEQ ID NO: 65; and (b) the CDRH2 sequence of any one of SEQ ID NOs: 66-67; and (c) the CDRH3 sequence of SEQ ID NO: 68. In some embodiments, the second binding unit comprises: (a) the CDRH1 sequence of SEQ ID NO: 65; the heavy chain CDRH2 sequence of SEQ ID NO: 66; and the heavy chain CDRH3 sequence of SEQ ID NO: 68; or (b) a heavy chain variable region comprising the CDRL1 sequence of SEQ ID NO: 65, the CDRH2 sequence of SEQ ID NO: 67, and the CDRH3 sequence of SEQ ID NO: 68.

[0016] In some embodiments, this second binding unit comprises a light chain variable region comprising: the CDRL1 sequence of SEQ ID NO: 69; the CDRL2 sequence of SEQ ID NO: 70; and the CDRL3 sequence of SEQ ID NO: 71. In some embodiments, this second binding unit comprises (a) a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 65, the CDRH2 sequence of SEQ ID NO: 66, and the CDRH3 sequence of SEQ ID NO: 68; and a light chain variable region comprising the CDRL1 sequence of SEQ ID NO: 69, the CDRL2 sequence of SEQ ID NO: 70, and the CDRL3 sequence of SEQ ID NO: 71; or (b) a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 65, the CDRH2 sequence of SEQ ID NO: 67, and the CDRH3 sequence of SEQ ID NO: 68; and a light chain variable region comprising the CDRL1 sequence of SEQ ID NO: 69, the CDRL2 sequence of SEQ ID NO: 70, and the CDRL3 sequence of SEQ ID NO: 71.

[0017] In some embodiments, the second binding unit comprises a heavy chain variable region having at least 95% identity to any one of SEQ ID NOs: 72-73. In some embodiments, the second binding unit comprises a heavy chain variable region sequence comprising any one of SEQ ID NOs: 72-73. In some embodiments, the second binding unit comprises a light chain variable region having at least 95% identity to any one of SEQ ID NOs: 74-75. In some embodiments, the second binding unit comprises a light chain variable region sequence comprising any one of SEQ ID NOs: 74-75.

[0018] In some embodiments, this second binding unit comprises (a) a heavy chain variable region sequence of SEQ ID NO: 72 and a light chain variable region sequence of SEQ ID NO: 74; or (b) a heavy chain variable region sequence of SEQ ID NO: 73 and a light chain variable region sequence of SEQ ID NO: 75.

[0019] An embodiment of the invention includes an antibody that binds to CLEC12A and CD3ε, comprising: a first light chain subunit comprising SEQ ID NO:79; a first heavy chain subunit comprising SEQ ID NO:76; a second light chain subunit comprising SEQ ID NO:79; and a second heavy chain subunit comprising SEQ ID NO:78.

[0020] An embodiment of the invention includes an antibody that binds to CLEC12A and CD3ε, comprising: a first light chain subunit comprising SEQ ID NO:79; a first heavy chain subunit comprising SEQ ID NO:77; a second light chain subunit comprising SEQ ID NO:79; and a second heavy chain subunit comprising SEQ ID NO:78.

[0021] An embodiment of the invention includes an antibody that binds to CLEC12A and CD3ε, comprising: a first light chain subunit comprising SEQ ID NO:83; a first heavy chain subunit comprising SEQ ID NO:80; a second light chain subunit comprising SEQ ID NO:83; and a second heavy chain subunit comprising SEQ ID NO:82.

[0022] An embodiment of the invention includes an antibody that binds to CLEC12A and CD3ε, comprising: a first light chain subunit comprising SEQ ID NO:83; a first heavy chain subunit comprising SEQ ID NO:81; a second light chain subunit comprising SEQ ID NO:83; and a second heavy chain subunit comprising SEQ ID NO:82.

[0023] Aspects of the invention include pharmaceutical compositions comprising the antibodies described herein.

[0024] Aspects of the invention include methods of treatment comprising administering to an individual in need thereof an effective dose of an antibody or pharmaceutical composition described herein.

[0025] Aspects of the invention include methods of treating a disorder characterized by expression of CLEC12A, comprising administering to a subject having the disorder an antibody or pharmaceutical composition described herein.

[0026] An embodiment of the invention includes the use of an antibody described herein in the preparation of a medicament for the treatment of a disorder characterized by expression of CLEC12A.

[0027] An embodiment of the invention includes an antibody described herein for use in treating a disorder characterized by expression of CLEC12A.

[0028] In some embodiments, the disorder is cancer. In some embodiments, the cancer is a hematological cancer. In some embodiments, the hematological cancer is acute myeloid leukemia (AML). In some embodiments, the AML is refractory AML.

[0029] Aspects of the invention include polynucleotides encoding the antibodies described herein, vectors comprising such polynucleotides, and cells comprising such vectors.

[0030] Aspects of the invention include methods for producing the antibodies described herein, comprising growing a cell described herein under conditions that allow for expression of the antibody and isolation of the antibody.

[0031] Aspects of the invention include kits comprising an antibody or pharmaceutical composition described herein and instructions for use. In some embodiments, the kit further comprises an additional therapeutic agent.

[0032] These and additional aspects are further described in the remainder of the disclosure, including the examples. [Brief explanation of the drawings]

[0033] [Figure 1] 1 is a table showing monovalent binding kinetics of humanized anti-CLEC12A antibodies according to embodiments of the invention. [Figure 2] Panels AC are a series of graphs showing binding data from humanized anti-CLEC12A antibodies binding to CLEC12A-expressing acute myeloid leukemia cells. [Figure 3] Panels AD are a series of graphs showing binding data from humanized anti-CLEC12A antibodies binding to human monocytes and neutrophils expressing CLEC12A. [Figure 4] 1 is a table showing EC50 values of humanized anti-CLEC12A antibodies binding to acute myeloid leukemia cell lines and primary human and cynomolgus monkey monocytes and neutrophils expressing CLEC12A. [Figure 5]FIG. 1 is a schematic diagram of a CLEC12AxCD3 multispecific antibody according to an embodiment of the invention. [Figure 6] Panels AC are a series of graphs showing binding of bispecific CLEC12AxCD3 bispecific antibodies to CLEC12A-expressing acute myeloid leukemia cells and T cells. [Figure 7] Figure 10 is a graph showing binding of CLEC12AxCD3 bispecific antibodies to CLEC12A and CD3 by sandwich ELISA. [Figure 8] Panels AB are graphs showing CD8+ T cell proliferation mediated by a CLEC12AxCD3 bispecific antibody according to an embodiment of the invention in the presence of autologous monocytes expressing CLEC12A. [Figure 9] Panels AD are a series of graphs showing cytokine release from T cells mediated by a CLEC12AxCD3 bispecific antibody according to an embodiment of the invention in the presence of U937 cells expressing CLEC12A. [Figure 10] Panels A-C are a series of graphs showing CD8+ T cell activation mediated by CLEC12AxCD3 bispecific antibodies according to embodiments of the invention in the presence of CLEC12A-expressing acute myeloid leukemia cells, and panels D-F are a series of graphs showing the corresponding EC50 values. [Figure 11] Panels A-C are a series of graphs showing cell killing of CLEC12A-expressing acute myeloid leukemia cells mediated by a CLEC12AxCD3 bispecific antibody according to an embodiment of the invention in the presence of CD8+ T cells, and panels D-F are a series of graphs showing the corresponding IC50 values. [Figure 12] Panels A-B are graphs showing cell killing of CLEC12A-expressing CD14+ monocytes mediated by a CLEC12AxCD3 bispecific antibody according to an embodiment of the invention in the presence of autologous CD8+ T cells. [Figure 13] 1 is a table summarizing IC50 values for cell killing of CLEC12A-positive cells. [Figure 14]1 is a graph showing the anti-tumor effect of a CLEC12AxCD3 bispecific antibody according to an embodiment of the invention in a U937 / human PBMC co-graft tumor model in NSG mice. [Figure 15] 1 is a graph showing the anti-tumor effect of a CLEC12AxCD3 bispecific antibody according to an embodiment of the invention in a U937 / human PBMC-reconstituted tumor model in NSG mice. [Figure 16] 1 is a table summarizing the monovalent binding kinetics of CLEC12AxCD3 bispecific antibodies to CLEC12A and CD3 epsilon according to embodiments of the invention. [Figure 17] 1 is a graph showing the anti-tumor effect of a CLEC12AxCD3 bispecific antibody according to an embodiment of the invention in an orthotopic HL-60-Luc2 AML model. DETAILED DESCRIPTION OF THE INVENTION

[0034] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described in “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (MJ Gait, ed., 1984); “Animal Cell Culture” (RIFreshney, ed., 1987); Biology” (FMAusubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction”, (Mullis et al., ed., 1994); “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual” (Barbas et al., 2001); Harlow, Lane and Harlow, Using Antibodies:A Laboratory Manual:Portable Protocol No.I,Cold Spring Harbor Laboratory(1998);and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

[0035] Where a range of values is expressed, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed herein, subject to any specific excluded limits in the stated range. Where a stated range includes one or both of those upper and lower limits, ranges excluding either or both of those included upper and lower limits are also encompassed within the invention.

[0036] Unless otherwise specified, antibody residues herein are numbered according to the Kabat numbering system (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0037] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one skilled in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures known to those skilled in the art are not described in order to avoid obscuring the present invention.

[0038] All references cited throughout this disclosure, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0039] I. Definition "Comprising" means that the recited element is necessary for the composition / method / kit, but other elements may be included to form the composition / method / kit, etc., within the scope of the claim.

[0040] "Consisting essentially of" means limiting the scope of the described composition or method to certain substances or steps that do not materially affect the basic and novel characteristic(s) of the invention.

[0041] "Consisting of" means that any element, step, or ingredient not specified in the claim is excluded from the composition, method, or kit.

[0042] Antibody residues herein are numbered according to the Kabat numbering system and the EU numbering system. The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1 to 113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise specified herein, references to residue numbers in the variable domain of an antibody refer to residue numbering according to the Kabat numbering system. Unless otherwise specified herein, references to residue numbers in the constant domain of an antibody refer to residue numbering according to the EU numbering system.

[0043] Antibodies, also called immunoglobulins, traditionally comprise at least one heavy chain and one light chain, with the amino-terminal domains of the heavy and light chains being variable in sequence and therefore commonly referred to as variable region domains, or variable heavy (VH) domains or variable light (VL) domains. The two domains traditionally associate to form a specific binding region, although, as discussed herein, specific binding can also be obtained with variable sequence in the heavy chain alone, and various non-native antibody structures are known and used in the art.

[0044] A "functional" or "biologically active" antibody or antigen-binding molecule (including a multispecific (e.g., bispecific) antibody) is one that can exert one or more of its native activities in structural, regulatory, biochemical, or biophysical events. For example, a functional antibody or other binding molecule may have the ability to specifically bind to an antigen, and the binding may then trigger or modify a cellular or molecular event, such as signal transduction or enzymatic activity. A functional antibody or other binding molecule may also block ligand activation of a receptor, or function as an agonist or antagonist. The ability of an antibody or other binding molecule to exert one or more of its native activities depends on several factors, including proper folding and assembly of the polypeptide chain.

[0045] The term "antibody" as used herein is used in the broadest sense and includes, among others, monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain-only antibodies, three-chain antibodies, TCAs, single-chain Fvs (scFvs), nanobodies, and the like, as well as antibody fragments so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species.

[0046] The term antibody may refer to a full-length heavy chain, a full-length light chain, an intact immunoglobulin molecule, or an immunologically active portion of any of these polypeptides, i.e., a polypeptide comprising an antigen-binding site that immunospecifically binds to an antigen of a desired target or portion thereof, such as, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune disease. The immunoglobulins disclosed herein can be immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass, including engineered subclasses with modified Fc portions that provide reduced or enhanced effector cell activity. The light chain of the subject antibody can be a kappa light chain (Vκ) or a lambda light chain (Vλ). The immunoglobulin can be derived from any species. In one aspect, the immunoglobulin is predominantly human in origin.

[0047] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies according to the present invention can be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or can also be produced, for example, via recombinant protein production methods (see, e.g., U.S. Pat. No. 4,816,567).

[0048] The term "variable" in reference to antibodies refers to the fact that the sequences of certain portions of antibody variable domains vary widely among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in both the light-chain and heavy-chain variable domains. The more highly conserved portions of variable domains are called framework regions (FRs). Native heavy-chain and light-chain variable domains each contain four FRs, which adopt a primarily β-sheet configuration and are connected by three hypervariable regions that form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions in each chain are held in close proximity to the hypervariable regions of the other chain by the FRs and contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participating in antibody-dependent cellular cytotoxicity (ADCC).

[0049] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody responsible for antigen binding. Hypervariable regions generally include amino acid residues from the "complementarity-determining regions" or "CDRs" (e.g., residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) of the heavy chain variable domain; Kabat et al., "Sequences of Proteins of Immunological Interest," 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or residues from the "hypervariable loops" of the heavy chain variable domain, residues 26-32 (H1), 53-55 (H2), and 96-101 (H3); Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some embodiments, "CDR" refers to the complementarity determining regions of an antibody as defined in Lefranc, MP et al., IMGT, the international ImMunoGeneTics database, Nucleic Acids Res., 27:209-212 (1999). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region / CDR residues as defined herein.

[0050] While exemplary CDR designations are provided herein, those skilled in the art will appreciate that several definitions of CDRs are commonly used, including the Kabat definition (see "Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions." Mol. Immunol. 2010;47:694-700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of structural loop regions (Chothia et al. "Conformations of immunoglobulin hypervariable regions." Nature. 1989;342:877-883).Alternative CDR definitions of interest include, but are not limited to, those described in Honegger, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol. 2001;309:657-670; Ofran et al., "Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B-cell epitopes," J Immunol. 2008;181:6230-6235; Almagro, "Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires," J Mol Recognit. 2004;17:132-143; and Padlan et al., "Identification of specificity-determining residues in antibodies," Faseb J. 1995;9:133-139, each of which is specifically incorporated herein by reference.

[0051] As used herein, an "intact antibody chain" includes a full-length variable region and a full-length constant region (Fc). An intact, "traditional" antibody includes an intact light chain and an intact heavy chain, as well as a light chain constant domain (CL) for secreted IgG and heavy chain constant domains, CH1, hinge, CH2, and CH3. Other isotypes, such as IgM or IgA, may have different CH domains. The constant domains may be native-sequence constant domains (e.g., human native-sequence constant domains) or amino acid sequence variants thereof. An intact antibody may have one or more "effector functions," which refer to biological activities attributable to the Fc constant region (a native-sequence Fc region or an amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and down-regulation of cell surface receptors. Constant region variants include variants that alter effector properties, binding to Fc receptors, and the like.

[0052] Depending on the amino acid sequence of the Fc (constant domain) of the heavy chain, antibodies and various antigen-binding proteins can be provided as different classes. There are five major classes of heavy chain Fc regions: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The Fc constant domains corresponding to the different classes of antibodies can be called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Ig forms include hinge-modified or hingeless forms (Roux et al (1998) J. Immunol. 161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; U.S. Patent Application Publication No. 2005 / 0048572; U.S. Patent Application Publication No. 2004 / 0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two types, called κ (kappa) and λ (lambda), based on the amino acid sequence of their constant domains. Antibodies according to embodiments of the invention can comprise a κ light chain sequence or a λ light chain sequence.

[0053] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Non-limiting examples of effector functions include C1q binding; CDC; Fc receptor binding; ADCC; ADCP; downregulation of cell surface receptors (e.g., B cell receptors); and the like. Such effector functions generally require the Fc region to interact with a receptor, e.g., FcγRI, FcγRIIA, FcγRIIB1, FcγRIIB2, FcγRIIIA, FcγRIIIB receptors, and the low-affinity FcRn receptor, and can be assessed using various assays known in the art. A "dead" or "silenced" Fc is an Fc that has been mutated to retain activity, e.g., with respect to extended serum half-life, but does not activate high-affinity Fc receptors or has reduced affinity for Fc receptors.

[0054] A "native sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native sequence human Fc regions include, for example, native sequence human IgG1 Fc regions (non-A and A allotypes), native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0055] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to the native-sequence Fc region or the Fc region of a parent polypeptide, e.g., about 1 to about 10 amino acid substitutions, and preferably about 1 to about 5 amino acid substitutions, in the native-sequence Fc region or in the Fc region of the parent polypeptide. A variant Fc region herein preferably has at least about 80% homology with the native-sequence Fc region and / or Fc region of the parent polypeptide, most preferably at least about 90% homology thereto, and more preferably at least about 95% homology thereto.

[0056] The variant Fc sequence may contain three amino acid substitutions in the CH2 region to reduce FcγRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332:563; Hezareh et al. (2001) J. Virology 75:12161; U.S. Patent No. 5,624,821, the disclosures of which are incorporated herein by reference in their entireties). In some embodiments, the variant Fc sequence may contain the following amino acid substitutions: L234A; L235A; and G237A. When these three amino acid substitutions are present in an IgG1 Fc sequence, they may be referred to as G1AAA.

[0057] Two amino acid substitutions in the complement C1q binding site at EU index positions 330 and 331 reduce complement binding (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitutions of human IgG1 or IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330, and 331 significantly reduce ADCC and CDC (see, e.g., Armour KL et al., 1999 Eur J Immunol. 29(8):2613-24; and Shields RL et al., 2001 J Biol Chem. 276(9):6591-604). The human IgG4 Fc amino acid sequence (UniProtKB No. P01861) is provided herein as SEQ ID NO: 76. Silenced IgG1 is described, for example, in Boesch, AW, et al., "Highly parallel characterization of IgG Fc binding interactions." MAbs, 2014.6(4):pp.915-27, the disclosure of which is incorporated herein by reference in its entirety.

[0058] Other Fc variants are possible, including, but not limited to, variants in which regions capable of forming disulfide bonds are deleted, or in which specific amino acid residues are removed or a methionine residue is added at the N-terminus of a native Fc. Thus, in some embodiments, one or more Fc portions of an antibody may contain one or more mutations in the hinge region to eliminate disulfide bonds. In yet another embodiment, the hinge region of the Fc may be completely removed. In yet another embodiment, an antibody may comprise an Fc variant.

[0059] Furthermore, Fc variants can be constructed to eliminate or substantially reduce effector function by substituting (mutating), deleting, or adding amino acid residues to confer complement binding or Fc receptor binding. For example, deletions can be made in complement binding sites, such as, but not limited to, the C1q binding site. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publication Nos. WO 97 / 34631 and WO 96 / 32478. Additionally, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0060] Additionally, Fc variants can be engineered to facilitate desired heterodimerization of heavy chain polypeptide subunits, e.g., using knob-into-hole or KiH mutations. In some embodiments, heavy chain polypeptides can include one or more "hole" mutations, e.g., Y349C, T366S, L368A, and / or Y407V, or any combination thereof. In some embodiments, heavy chain polypeptides can include one or more "knob" mutations, e.g., S354C and / or T366W, or any combination thereof. Antibodies according to embodiments of the invention can utilize any suitable combination of knob and hole residues to promote desired heterodimer formation.

[0061] Fc variants may also be constructed to promote extended half-life, for example through enhanced FcRn binding. In some embodiments, the heavy chain polypeptide may comprise a T250Q mutation for this purpose. In some embodiments, the heavy chain polypeptide may comprise a M428L mutation for this purpose. In some embodiments, the heavy chain polypeptide may comprise both a T250Q mutation and an M428L mutation for this purpose.

[0062] Fc variants may also be constructed to facilitate improved purification procedures, for example, through enhanced Protein A binding. In some embodiments, the heavy chain polypeptide may comprise an H435R mutation for this purpose. In some embodiments, the heavy chain polypeptide may comprise a Y436F mutation for this purpose. In some embodiments, the heavy chain polypeptide may comprise both H435R and Y436F mutations for this purpose.

[0063] The term "antibody comprising an Fc region" refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during antibody purification or by recombinant engineering of the nucleic acid encoding the antibody. Thus, antibodies with an Fc region according to the present invention may include antibodies with or without K447.

[0064] Aspects of the present invention include antibodies with multispecific configurations, including but not limited to bispecific, trispecific, etc. A wide variety of methods and protein configurations for bispecific monoclonal antibodies (BsMAB), trispecific antibodies, etc. are known and used.

[0065] Various methods for producing multivalent artificial antibodies have been developed by recombinantly fusing the variable domains of two or more antibodies. In some embodiments, the first and second antigen-binding domains on a polypeptide are connected by a polypeptide linker. One non-limiting example of such a polypeptide linker is a GS linker, which has an amino acid sequence of four glycine residues followed by one serine residue, repeated n times, where n is an integer ranging from 1 to about 10, such as 2, 3, 4, 5, 6, 7, 8, or 9. Non-limiting examples of such linkers include GGGGS (SEQ ID NO: 84) (n=1) and GGGGSGGGGS (SEQ ID NO: 85) (n=2). Further non-limiting examples of linkers include EPKSCDKTHT (SEQ ID NO: 86) and EPKSSDKTHT (SEQ ID NO: 87), which are derived from the natural hinge region of human IgG1. Other suitable linkers may also be used, for example, as described in Chen et al., Adv Drug Deliv Rev. 2013 October 15;65(10):1357-69, the disclosure of which is incorporated herein by reference in its entirety. Additional linker sequences may be described elsewhere herein and may be incorporated into the subject antibodies in any suitable configuration.

[0066] Antibodies (e.g., multispecific antibodies) as described herein may be in the form of dimers in which two heavy chains are disulfide-bonded or otherwise covalently or noncovalently bound to one another, and may optionally contain an asymmetric interface (commonly referred to as a "knobs-into-hole" interface) between two or more CH domains to facilitate proper pairing between the polypeptide chains. Knobs-into-hole antibody engineering techniques for heavy chain heterodimerization are discussed, for example, in Ridgway et al., Protein Eng. 1996 Jul;9(7):17-21, and U.S. Pat. No. 8,216,805, the disclosures of which are incorporated herein by reference in their entireties. Fc regions comprising an asymmetric interface may be referred to herein by the abbreviation "KiH," which stands for knobs-into-hole. For example, embodiments of the present invention include variant Fc region sequences, such as the G1AAA sequence, which contain an asymmetric interface and are referred to herein as "G1AAA KiH."

[0067] As used herein, the term "CLEC12A" refers to C-type lectin domain family 12 member A, which is a cell surface receptor that regulates signal transduction cascades and mediates tyrosine phosphorylation of target MAP kinases. The term "CLEC12A" includes CLEC12A proteins of any human and non-human animal species, and specifically includes human CLEC12A and CLEC12A of non-human mammals.

[0068] As used herein, the term "human CLEC12A" includes any variant, isoform, and species homologue of human CLEC12A (UniProt Q5QGZ9), regardless of its source or mode of preparation. Thus, "human CLEC12A" includes human CLEC12A naturally expressed by cells and CLEC12A expressed on cells transfected with the human CLEC12A gene.

[0069] The terms "anti-CLEC12A antibody," "CLEC12A antibody," and "CLEC12A-binding antibody" are used interchangeably herein and refer to an antibody as defined above that immunospecifically binds to CLEC12A, including human CLEC12A as defined above.

[0070] "Amino acid sequence identity percentage (%)" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage, without considering any conservative substitutions as part of sequence identity.Alignment for determining amino acid sequence identity percentage can be achieved by various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.However, for the purposes herein, the sequence comparison computer program ALIGN-2 is used to generate amino acid sequence identity value (%).

[0071] An "isolated" antibody is one that has been identified, separated, and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with diagnostic or therapeutic uses for the antibody, and these may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody is purified (1) to greater than 95% by weight, and most preferably greater than 99% by weight, of the antibody as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue, or preferably silver stain. Isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0072] The antibodies of the present invention include multispecific antibodies. Multispecific antibodies have multiple binding specificities. The term "multispecific" specifically includes "bispecific" and "trispecific" antibodies, as well as higher-order independent specific binding affinities such as higher-order polyepitopic specificities, as well as tetravalent antibodies and antibody fragments. The term "multispecific antibody" is used in the broadest sense herein and includes all antibodies with multiple binding specificities. Multispecific anti-CLEC12A antibodies of the present invention specifically include antibodies that immunospecifically bind to two or more non-overlapping epitopes on a CLEC12A protein, such as human CLEC12A (i.e., bivalent and biparatopic). Multispecific anti-CLEC12A antibodies of the present invention also specifically include antibodies that immunospecifically bind to an epitope on a CLEC12A protein, such as human CLEC12A, and an epitope on a different protein, such as a CD3 protein, for example, human CD3 (i.e., bivalent and biparatopic). Multispecific anti-CLEC12A antibodies of the invention also include antibodies that immunospecifically bind to two or more non-overlapping or partially overlapping epitopes on a CLEC12A protein, such as human CLEC12A protein, and epitopes on different proteins, such as CD3 protein, e.g., human CD3 protein (i.e., trivalent and biparatopic).

[0073] The antibodies of the present invention include monospecific antibodies having one binding specificity. Monospecific antibodies specifically include antibodies containing a single binding specificity and antibodies containing two or more binding units with the same binding specificity. The term "monospecific antibody" is used in the broadest sense herein and includes all antibodies with one binding specificity. Monospecific anti-CLEC12A antibodies of the present invention specifically include antibodies that immunospecifically bind to one epitope on a CLEC12A protein, such as human CLEC12A (monovalent and monospecific). Monospecific anti-CLEC12A antibodies of the present invention also specifically include antibodies with two or more binding units that immunospecifically bind to an epitope on a CLEC12A protein, such as human CLEC12A (e.g., multivalent antibodies). For example, a monospecific antibody according to embodiments of the present invention may contain two variable regions each containing an antigen-binding domain, and each antigen-binding domain binds to the same epitope on a CLEC12A protein (i.e., bivalent and monospecific).

[0074] An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule binds. Typically, an antigen has several or many different epitopes and will react with many different antibodies. The term specifically includes linear and conformational epitopes.

[0075] "Epitope mapping" is the process of identifying antibody binding sites, or epitopes, on a target antigen. Antibody epitopes can be linear or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are formed by amino acids that are discontinuous in the protein sequence but come together when the protein folds into its three-dimensional structure.

[0076] "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). As described above, the present invention specifically includes anti-CLEC12A antibodies with polyepitopic specificity, i.e., anti-CLEC12A antibodies that bind to one or more non-overlapping epitopes on a CLEC12A protein, such as human CLEC12A; and anti-CLEC12A antibodies that bind to one or more epitopes on a CLEC12A protein and to an epitope on another protein, such as the CD3 protein. The term "non-overlapping epitope(s)" or "non-competing epitope(s)" of an antigen is defined herein to mean epitope(s) that are recognized by one member of an antigen-specific antibody pair but not by the other member. Antibody pairs that recognize non-overlapping epitopes, or antigen-binding regions that target the same antigen on a multispecific antibody, do not compete for binding to the antigen and can simultaneously bind to the antigen.

[0077] If two antibodies recognize the same or sterically overlapping epitopes, the antibodies bind to "essentially the same epitope" as a reference antibody. The most widely used rapid method for determining whether two epitopes bind to the same or sterically overlapping epitopes is a competitive assay, which can be configured in a variety of formats using either labeled antigen or labeled antibody. Typically, the antigen is immobilized on a 96-well plate, and the ability of an unlabeled antibody to block the binding of the labeled antibody is measured using a radioactive or enzyme label.

[0078] As used herein, the term "valency" refers to a specific number of binding sites within an antibody molecule.

[0079] A "monovalent" antibody has one binding site. A monovalent antibody is therefore also monospecific.

[0080] A "multivalent" antibody has two or more binding sites. Thus, the terms "bivalent," "trivalent," and "tetravalent" refer to the presence of two binding sites, three binding sites, and four binding sites, respectively. Thus, bispecific antibodies according to the invention are at least bivalent, and may be trivalent, tetravalent, or multivalent. Bivalent antibodies according to embodiments of the invention may have two binding sites for the same epitope (i.e., bivalent, monoparatopic), or for two different epitopes (i.e., bivalent, biparatopic).

[0081] A wide variety of methods and protein structures are known and used to prepare bispecific monoclonal antibodies (BsMABs), trispecific antibodies, and the like.

[0082] The term "chimeric antigen receptor" or "CAR" is used herein in the broadest sense to refer to an engineered receptor that grafts a desired binding specificity (e.g., the antigen-binding region of a monoclonal antibody or other ligand) onto a transmembrane domain and an intracellular signaling domain. Typically, receptors are used to graft the specificity of a monoclonal antibody onto a T cell to create a chimeric antigen receptor (CAR). (J Natl Cancer Inst, 2015;108(7):dvj439, and Jackson et al., Nature Reviews Clinical Oncology, 2016;13:370-383). CAR-T cells are T cells that have been genetically engineered to produce an artificial T cell receptor for use in immunotherapy. In one embodiment, "CAR-T cells" refer to therapeutic T cells that express a transgene encoding one or more chimeric antigen receptors minimally consisting of an extracellular domain, a transmembrane domain, and at least one cytosolic domain.

[0083] The term "human antibody" is used herein to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences, e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo.

[0084] "Chimeric antibody" or "chimeric immunoglobulin" refers to an immunoglobulin molecule that contains amino acid sequences from at least two different Ig loci, e.g., a transgenic antibody that contains a portion encoded by a human Ig locus and a portion encoded by a rat Ig locus. Chimeric antibodies include transgenic antibodies with non-human or artificial Fc regions and human idiotypes. Such immunoglobulins can be isolated from animals of the invention that have been modified to produce such chimeric antibodies.

[0085] As used herein, the term "effector cell" refers to an immune cell that is involved in the effector stage of an immune response, as opposed to the recognition and activation stages of an immune response. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, effector cells, such as natural killer cells, can induce antibody-dependent cellular cytotoxicity (ADCC). For example, FcR-expressing monocytes and macrophages are involved in the specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens. In some embodiments, effector cells can phagocytose target antigens or target cells.

[0086] "Human effector cells" are leukocytes that express receptors such as T cell receptors and FcRs and perform effector function. Preferably, these cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with NK cells being preferred. Effector cells can be isolated from their native source, e.g., from blood or PBMCs, as described herein.

[0087] The term "immune cell" is used herein in the broadest sense and includes, but is not limited to, cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils.

[0088] An "effector function" of an antibody refers to a biological activity attributable to the Fc region of an antibody (a native sequence Fc region or an amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptor, BCR), and the like.

[0089] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibodies on target cells and subsequently cause lysis of the target cells. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo in an animal model, such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0090] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.

[0091] "Binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by common methods known in the art. Low-affinity antibodies usually bind antigens slowly and tend to dissociate easily, while high-affinity antibodies usually bind antigens more quickly and tend to remain bound to them.

[0092] As used herein, "Kd" or "Kd value" refers to the dissociation constant measured by biolayer interferometry using an Octet QK384 instrument (Fortebio Inc., Menlo Park, CA) in kinetic mode. For example, an anti-mouse Fc sensor is loaded with mouse Fc fusion antigen and immersed in a well containing antibody to measure the concentration-dependent association rate (k). In the final step, the sensor is immersed in a well containing only buffer solution to measure the dissociation rate (koff) of the antibody. Kd is the ratio of koff / koff. (For more details, see Concepcion, J, et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009.)

[0093] The terms "treatment," "treating," and the like are generally used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing the disease or condition, and / or therapeutic, in terms of partially or completely curing the disease and / or side effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, including (a) preventing the onset of the disease in a subject who is susceptible to, but has not yet been diagnosed with, the disease; (b) inhibiting the disease, i.e., preventing its development; or (c) relieving the disease, i.e., causing regression of the disease. Therapeutic agents can be administered before, during, or after the onset of a disease or injury. Treatment of an ongoing disease is particularly interesting if the treatment stabilizes or reduces undesirable clinical symptoms in the patient. Such treatment is desirably administered before complete loss of function in the affected tissue. A subject may be treated during, and in some cases after, the symptomatic phase of the disease.

[0094] "Therapeutically effective amount" refers to the amount of active agent required to provide a therapeutic benefit to a subject, e.g., an amount that induces, ameliorates, or causes an improvement in pathological symptoms, disease progression, or physiological condition associated with a disease, or improves resistance to a disorder.

[0095] The terms "cancer," "tumor," "cancerous," and "malignant" are used interchangeably herein to refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0096] The terms "hematologic cancer," "hematological malignancy," and "blood cancer" are used interchangeably herein to refer to cancers that begin in blood-forming tissues, including, but not limited to, bone marrow and / or cells of the immune system. In one embodiment, the leukemia is acute myeloid leukemia (AML).

[0097] The term "characterized by expression of CLEC12A" broadly refers to any disease or disorder in which expression of CLEC12A is associated with or contributes to one or more pathological processes that are characteristic of that disease or disorder, including, but not limited to, acute myeloid leukemia (AML).

[0098] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or being treated. In one embodiment, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infections, and the like. Subjects can be humans, but can also include other mammals, particularly those mammals useful as laboratory models of human disease, e.g., mice, rats, and the like.

[0099] The term "pharmaceutical formulation" refers to a preparation in which the biological activity of the active ingredient is effective and which does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such formulations are sterile. A "pharmaceutically acceptable" excipient (vehicle, additive) is one that can be reasonably administered to a mammalian subject to provide an effective dose of the active ingredient employed.

[0100] A "sterile" formulation is aseptic or free or essentially free of all viable microorganisms and their spores. A "frozen" formulation is a formulation at a temperature below 0°C.

[0101] A "stable" formulation is one in which the protein therein essentially retains its physical stability and / or chemical stability and / or biological activity upon storage. Preferably, the formulation essentially retains its physical and chemical stability and its biological activity upon storage. The storage period is generally selected based on the intended shelf life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pub. (1991) and Jones. A. Adv. Drug Delivery Rev. 10:29-90) (1993). Stability can be measured at a selected temperature over a selected period of time. Stability can be qualitatively and / or quantitatively assessed in a variety of different ways, including assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and / or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF), or capillary zone electrophoresis; amino- or carboxy-terminal sequence analysis; mass spectrometry; SDS-PAGE analysis comparing reduced and intact antibodies; peptide mapping (e.g., trypsin or LYS-C) analysis; assessment of antibody biological activity or antigen-binding function, etc. Instability can include any one or more of aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, differential glycosylation, etc.

[0102] II. Detailed Description Anti-CLEC12A antibody The present invention provides a family of closely related antibodies that bind to human CLEC12A, the antibodies of this family comprising a set of CDRH sequences as defined herein and shown in Table 1, and a set of CDRL sequences as defined herein and shown in Table 2.

[0103] Anti-CLEC12A antibodies according to embodiments of the invention are exemplified by the provided heavy chain variable region (VH) sequences shown in Table 3 and the provided light chain variable region (VL) sequences shown in Table 4. This family of antibodies offers many advantages that contribute to their usefulness as clinical therapeutic agent(s). The antibodies include members with a range of binding affinities, allowing for the selection of specific sequences with desired binding affinities. [Table 1] [Table 2] [Table 3] [Table 4]

[0104] Suitable antibodies can be selected from the antibodies provided herein for development and therapeutic or other uses, including, but not limited to, use as bispecific antibodies, for example, as shown in Figure 5 or as part of a CAR-T structure. Figure 5 is a schematic diagram of an anti-CLEC12A x anti-CD3ε multispecific antibody, in which the anti-CLEC12A binding region is located at the N-terminus of the polypeptide subunit and the CD3ε binding unit is in an scFv format and located on one of the heavy chain polypeptide subunits between the variable region and the hinge region. In some embodiments, the two heavy chain polypeptide subunits of the heterodimeric multispecific antibody are paired, for example, using knob-into-hole (KiH) technology.

[0105] Determining affinity for a candidate protein can be performed using methods known in the art, such as Biacore measurements. Members of an antibody family are approximately 10 -6 ~about 10 -11 For example, but not limited to, about 10 -6 ~about 10 -10 ;about 10 -6 ~about 10 -9 ;about 10 -6 ~about 10 -8 ;about 10 -8 ~about 10 -11 ;about 10 -8 ~about 10 -10 ;about 10 -8 ~about 10 -9 ;about 10 -9 ~about 10 -11 ;about 10 -9 ~about 10 -10 or may have an affinity for CLEC12A with a Kd of any value within these ranges. Affinity selection may be confirmed by biological evaluation to modulate, e.g., block, CLEC12A biological activity, including in vitro assays, preclinical models, and clinical trials, as well as evaluation of potential toxicity.

[0106] Members of the antibody family herein are not cross-reactive with cynomolgus monkey CLEC12A protein, but may be engineered to provide cross-reactivity with CLEC12A protein of any other animal species, if desired.

[0107] The family of anti-CLEC12A antibodies herein includes one or more binding units comprising a VH domain comprising CDRH1, CDRH2, and CDRH3 sequences in a human VH framework, and CDRL1, CDRL2, and CDRL3 sequences in a human Vkappa or Vlambda framework. The CDR sequences may be located, for example, in regions around amino acid residues 26-33; 51-58; and 97-116 for CDR1, CDR2, and CDR3, respectively, of the exemplary variable region sequences provided in Tables 3 and 4. One skilled in the art will appreciate that these CDR sequences may be in different positions if different framework sequences are selected, but generally the order of the sequences remains the same.

[0108] In some embodiments, the anti-CLEC12A antibody comprises a CDRH1 sequence of any one of SEQ ID NOs: 1-5. In some embodiments, the anti-CLEC12A antibody comprises a CDRH2 sequence of any one of SEQ ID NOs: 6-14. In some embodiments, the anti-CLEC12A antibody comprises a CDRH3 sequence of any one of SEQ ID NOs: 15-22.

[0109] In one preferred embodiment, the anti-CLEC12A antibody comprises the CDRH1 sequence of SEQ ID NO: 1, the CDRH2 sequence of SEQ ID NO: 6, and the CDRH3 sequence of SEQ ID NO: 15. In one preferred embodiment, the anti-CLEC12A antibody comprises the CDRH1 sequence of SEQ ID NO: 1, the CDRH2 sequence of SEQ ID NO: 7, and the CDRH3 sequence of SEQ ID NO: 16. In one preferred embodiment, the anti-CLEC12A antibody comprises the CDRH1 sequence of SEQ ID NO: 1, the CDRH2 sequence of SEQ ID NO: 8, and the CDRH3 sequence of SEQ ID NO: 17. In one preferred embodiment, the anti-CLEC12A antibody comprises the CDRH1 sequence of SEQ ID NO: 1, the CDRH2 sequence of SEQ ID NO: 9, and the CDRH3 sequence of SEQ ID NO: 18. In one preferred embodiment, the anti-CLEC12A antibody comprises the CDRH1 sequence of SEQ ID NO: 2, the CDRH2 sequence of SEQ ID NO: 10, and the CDRH3 sequence of SEQ ID NO: 19. In one preferred embodiment, the anti-CLEC12A antibody comprises a CDRH1 sequence of SEQ ID NO: 3, a CDRH2 sequence of SEQ ID NO: 11, and a CDRH3 sequence of SEQ ID NO: 20. In one preferred embodiment, the anti-CLEC12A antibody comprises a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 12, and a CDRH3 sequence of SEQ ID NO: 21. In one preferred embodiment, the anti-CLEC12A antibody comprises a CDRH1 sequence of SEQ ID NO: 4, a CDRH2 sequence of SEQ ID NO: 13, and a CDRH3 sequence of SEQ ID NO: 22. In one preferred embodiment, the anti-CLEC12A antibody comprises a CDRH1 sequence of SEQ ID NO: 5, a CDRH2 sequence of SEQ ID NO: 14, and a CDRH3 sequence of SEQ ID NO: 22.

[0110] In some embodiments, the anti-CLEC12A antibody comprises a CDRL1 sequence of any one of SEQ ID NOs: 23-27. In some embodiments, the anti-CLEC12A antibody comprises a CDRL2 sequence of any one of SEQ ID NOs: 28-33. In some embodiments, the anti-CLEC12A antibody comprises a CDRL3 sequence of any one of SEQ ID NOs: 34-40.

[0111] In one preferred embodiment, the anti-CLEC12A antibody comprises the CDRL1 sequence of SEQ ID NO:23, the CDRL2 sequence of SEQ ID NO:28, and the CDRL3 sequence of SEQ ID NO:34. In one preferred embodiment, the anti-CLEC12A antibody comprises the CDRL1 sequence of SEQ ID NO:24, the CDRL2 sequence of SEQ ID NO:29, and the CDRL3 sequence of SEQ ID NO:35. In one preferred embodiment, the anti-CLEC12A antibody comprises the CDRL1 sequence of SEQ ID NO:25, the CDRL2 sequence of SEQ ID NO:30, and the CDRL3 sequence of SEQ ID NO:36. In one preferred embodiment, the anti-CLEC12A antibody comprises the CDRL1 sequence of SEQ ID NO:26, the CDRL2 sequence of SEQ ID NO:31, and the CDRL3 sequence of SEQ ID NO:37. In one preferred embodiment, the anti-CLEC12A antibody comprises the CDRL1 sequence of SEQ ID NO:25, the CDRL2 sequence of SEQ ID NO:30, and the CDRL3 sequence of SEQ ID NO:38. In one preferred embodiment, the anti-CLEC12A antibody comprises the CDRL1 sequence of SEQ ID NO: 27, the CDRL2 sequence of SEQ ID NO: 32, and the CDRL3 sequence of SEQ ID NO: 39. In one preferred embodiment, the anti-CLEC12A antibody comprises the CDRL1 sequence of SEQ ID NO: 24, the CDRL2 sequence of SEQ ID NO: 33, and the CDRL3 sequence of SEQ ID NO: 40.

[0112] In one preferred embodiment, the anti-CLEC12A antibody comprises a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 1, the CDRH2 sequence of SEQ ID NO: 6, and the CDRH3 sequence of SEQ ID NO: 15; and a light chain variable region comprising the CDRL1 sequence of SEQ ID NO: 23, the CDRL2 sequence of SEQ ID NO: 28, and the CDRL3 sequence of SEQ ID NO: 34.

[0113] In one preferred embodiment, the anti-CLEC12A antibody comprises a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 1, the CDRH2 sequence of SEQ ID NO: 7, and the CDRH3 sequence of SEQ ID NO: 16; and a light chain variable region comprising the CDRL1 sequence of SEQ ID NO: 23, the CDRL2 sequence of SEQ ID NO: 28, and the CDRL3 sequence of SEQ ID NO: 34.

[0114] In one preferred embodiment, the anti-CLEC12A antibody comprises a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 1, the CDRH2 sequence of SEQ ID NO: 8, and the CDRH3 sequence of SEQ ID NO: 17; and a light chain variable region comprising the CDRL1 sequence of SEQ ID NO: 24, the CDRL2 sequence of SEQ ID NO: 29, and the CDRL3 sequence of SEQ ID NO: 35.

[0115] In one preferred embodiment, the anti-CLEC12A antibody comprises a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 1, the CDRH2 sequence of SEQ ID NO: 9, and the CDRH3 sequence of SEQ ID NO: 18; and a light chain variable region comprising the CDRL1 sequence of SEQ ID NO: 25, the CDRL2 sequence of SEQ ID NO: 30, and the CDRL3 sequence of SEQ ID NO: 36.

[0116] In one preferred embodiment, the anti-CLEC12A antibody comprises a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 2, the CDRH2 sequence of SEQ ID NO: 10, and the CDRH3 sequence of SEQ ID NO: 19; and a light chain variable region comprising the CDRL1 sequence of SEQ ID NO: 26, the CDRL2 sequence of SEQ ID NO: 31, and the CDRL3 sequence of SEQ ID NO: 37.

[0117] In one preferred embodiment, the anti-CLEC12A antibody comprises a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 1, the CDRH2 sequence of SEQ ID NO: 6, and the CDRH3 sequence of SEQ ID NO: 15; and a light chain variable region comprising the CDRL1 sequence of SEQ ID NO: 23, the CDRL2 sequence of SEQ ID NO: 28, and the CDRL3 sequence of SEQ ID NO: 34.

[0118] In one preferred embodiment, the anti-CLEC12A antibody comprises a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 3, the CDRH2 sequence of SEQ ID NO: 11, and the CDRH3 sequence of SEQ ID NO: 20; and a light chain variable region comprising the CDRL1 sequence of SEQ ID NO: 25, the CDRL2 sequence of SEQ ID NO: 30, and the CDRL3 sequence of SEQ ID NO: 38.

[0119] In one preferred embodiment, the anti-CLEC12A antibody comprises a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 1, the CDRH2 sequence of SEQ ID NO: 12, and the CDRH3 sequence of SEQ ID NO: 21; and a light chain variable region comprising the CDRL1 sequence of SEQ ID NO: 27, the CDRL2 sequence of SEQ ID NO: 32, and the CDRL3 sequence of SEQ ID NO: 39.

[0120] In one preferred embodiment, the anti-CLEC12A antibody comprises a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 4, the CDRH2 sequence of SEQ ID NO: 13, and the CDRH3 sequence of SEQ ID NO: 22; and a light chain variable region comprising the CDRL1 sequence of SEQ ID NO: 24, the CDRL2 sequence of SEQ ID NO: 33, and the CDRL3 sequence of SEQ ID NO: 40.

[0121] In one preferred embodiment, the anti-CLEC12A antibody comprises a heavy chain variable region comprising the CDRH1 sequence of SEQ ID NO: 5, the CDRH2 sequence of SEQ ID NO: 14, and the CDRH3 sequence of SEQ ID NO: 22; and a light chain variable region comprising the CDRL1 sequence of SEQ ID NO: 24, the CDRL2 sequence of SEQ ID NO: 33, and the CDRL3 sequence of SEQ ID NO: 40.

[0122] In some embodiments, the anti-CLEC12A antibody comprises any of the heavy chain variable region amino acid sequences of SEQ ID NOs: 41-52 (Table 3). In some embodiments, the anti-CLEC12A antibody comprises a heavy chain variable region amino acid sequence having at least about 80% identity, e.g., about 85%, 90%, 95%, 99%, or 100% identity, to any one of SEQ ID NOs: 41-52 (Table 3).

[0123] In some embodiments, the anti-CLEC12A antibody comprises any of the heavy chain variable region amino acid sequences of SEQ ID NOs: 53-64 (Table 4). In some embodiments, the anti-CLEC12A antibody comprises a light chain variable region amino acid sequence having at least about 80% identity, e.g., about 85%, 90%, 95%, 99%, or 100% identity, to any one of SEQ ID NOs: 53-64 (Table 4).

[0124] In some embodiments, the anti-CLEC12A antibody comprises the heavy chain variable region sequence of SEQ ID NO: 41 and the light chain variable region sequence of SEQ ID NO: 53. In some embodiments, the anti-CLEC12A antibody comprises the heavy chain variable region sequence of SEQ ID NO: 42 and the light chain variable region sequence of SEQ ID NO: 54. In some embodiments, the anti-CLEC12A antibody comprises the heavy chain variable region sequence of SEQ ID NO: 43 and the light chain variable region sequence of SEQ ID NO: 55. In some embodiments, the anti-CLEC12A antibody comprises the heavy chain variable region sequence of SEQ ID NO: 44 and the light chain variable region sequence of SEQ ID NO: 56. In some embodiments, the anti-CLEC12A antibody comprises the heavy chain variable region sequence of SEQ ID NO: 45 and the light chain variable region sequence of SEQ ID NO: 57. In some embodiments, the anti-CLEC12A antibody comprises the heavy chain variable region sequence of SEQ ID NO: 46 and the light chain variable region sequence of SEQ ID NO: 58. In some embodiments, the anti-CLEC12A antibody comprises the heavy chain variable region sequence of SEQ ID NO: 47 and the light chain variable region sequence of SEQ ID NO: 59. In some embodiments, the anti-CLEC12A antibody comprises the heavy chain variable region sequence of SEQ ID NO: 48 and the light chain variable region sequence of SEQ ID NO: 60. In some embodiments, the anti-CLEC12A antibody comprises the heavy chain variable region sequence of SEQ ID NO: 49 and the light chain variable region sequence of SEQ ID NO: 61. In some embodiments, the anti-CLEC12A antibody comprises the heavy chain variable region sequence of SEQ ID NO: 50 and the light chain variable region sequence of SEQ ID NO: 62. In some embodiments, the anti-CLEC12A antibody comprises the heavy chain variable region sequence of SEQ ID NO: 51 and the light chain variable region sequence of SEQ ID NO: 63. In some embodiments, the anti-CLEC12A antibody comprises the heavy chain variable region sequence of SEQ ID NO: 52 and the light chain variable region sequence of SEQ ID NO: 64.

[0125] In some embodiments, the CDR sequences in the anti-CLEC12A antibodies of the invention comprise one or two amino acid substitutions relative to the CDR1, CDR2 and / or CDR3 sequence or the set of CDR1, CDR2 and CDR3 sequences (Table 1 or 2).

[0126] In some embodiments, the anti-CLEC12A antibody preferably comprises a heavy chain variable domain (VH) whose CDRH3 sequence has 80% or more, e.g., at least 85%, at least 90%, at least 95%, or at least 99%, sequence identity at the amino acid level to the CDRH3 sequence of any one of the antibodies (whose CDRH3 sequence is shown in Table 1) that binds to CLEC12A.

[0127] In some embodiments, the anti-CLEC12A antibody preferably comprises a heavy chain variable domain (VH) in which the full set (combination) of CDRH1, 2, and 3 has eighty-five percent (85%) or greater sequence identity at the amino acid level to the CDR1, 2, and 3 (combination) of an antibody (whose CDRH sequences are shown in Table 1) that binds to CLEC12A.

[0128] In some embodiments, bispecific or multispecific antibodies are provided that may have any of the configurations discussed herein, including, but not limited to, multispecific antibodies comprising one or more binding units located on the heavy chain polypeptide subunits and positioned between the N-terminal binding unit and the hinge region, for example, as shown in Figure 5. In some embodiments, such binding units may comprise an scFv configuration in which the VH and VL regions are connected by a linker sequence and together form a binding unit that binds to a target protein, such as CD3.

[0129] In some embodiments, a multispecific antibody may comprise a first binding unit (comprising a heavy chain variable region paired with a light chain variable region) that binds to CLEC12A and a second binding unit (comprising a heavy chain variable region paired with a light chain variable region) that binds to a protein other than CLEC12A. In some embodiments, a multispecific antibody may comprise first and second binding units (each of which comprises a heavy chain variable region paired with a light chain variable region, both of which bind to CLEC12A) and at least one third binding unit (e.g., that binds to a protein other than CLEC12A, such as CD3). In some embodiments, the third binding unit is located at the C-terminus of one of the light chain polypeptide subunits that comprise the antibody. In some embodiments, the third binding unit is located within one of the heavy chain polypeptide subunits, for example, between the variable region and hinge region of the polypeptide subunit, as shown in FIG. 5. In some embodiments, the third binding unit comprises an scFv in which the heavy chain variable region is connected to the light chain variable region by a linker sequence.

[0130] In some embodiments, the anti-CLEC12A antibodies described herein comprise heavy chain constant region sequences, such as CH1, hinge, CH2, CH3, and / or CH4 domains. In some embodiments, the anti-CLEC12A antibodies described herein comprise light chain constant region sequences, e.g., CL domains. In some embodiments, the anti-CLEC12A antibodies described herein comprise heavy chain constant region sequences that form an Fc region.

[0131] As noted above, in some embodiments, the multispecific anti-CLEC12A antibody comprises a binding unit that binds to a protein other than CLEC12A, such as CD3 (e.g., CD3ε). In some embodiments, the CD3 binding unit comprises a set of CDRH sequences as defined herein and shown in Table 5, and a set of CDRL sequences as defined herein and shown in Table 6. [Table 5] [Table 6]

[0132] In some embodiments, the CD3 binding units comprise a heavy chain variable region (VH) sequence set forth in Table 7 and a light chain variable region (VL) sequence set forth in Table 8. These CD3 binding units offer many advantages that contribute to their usefulness as clinical therapeutic agent(s). [Table 7] [Table 8]

[0133] Multispecific anti-CLEC12A antibodies according to embodiments of the invention may comprise one or more CD3 binding units as described herein. Any of the binding domains of the anti-CLEC12A antibodies described herein may be combined with a CD3 binding domain described herein to generate a multispecific antibody that binds to CLEC12A and CD3. Full-length sequences that may be used to construct anti-CLEC12AxCD3 antibodies are shown in Table 9. [Table 9-1] [Table 9-2] [Table 9-3]

[0134] In one preferred embodiment, the multispecific antibody binds to CLEC12A and CD3ε and comprises a first light chain subunit comprising SEQ ID NO: 79; a first heavy chain subunit comprising SEQ ID NO: 76; a second light chain subunit comprising SEQ ID NO: 79; and a second heavy chain subunit comprising SEQ ID NO: 78.

[0135] In one preferred embodiment, the multispecific antibody binds to CLEC12A and CD3ε and comprises a first light chain subunit comprising SEQ ID NO: 79; a first heavy chain subunit comprising SEQ ID NO: 77; a second light chain subunit comprising SEQ ID NO: 79; and a second heavy chain subunit comprising SEQ ID NO: 78.

[0136] In one preferred embodiment, the multispecific antibody binds to CLEC12A and CD3ε and comprises a first light chain subunit comprising SEQ ID NO: 83, a first heavy chain subunit comprising SEQ ID NO: 80; a second light chain subunit comprising SEQ ID NO: 83; and a second heavy chain subunit comprising SEQ ID NO: 82.

[0137] In one preferred embodiment, the multispecific antibody binds to CLEC12A and CD3ε and comprises a first light chain subunit comprising SEQ ID NO: 83; a first heavy chain subunit comprising SEQ ID NO: 81; a second light chain subunit comprising SEQ ID NO: 83; and a second heavy chain subunit comprising SEQ ID NO: 82.

[0138] Various forms of multispecific antibodies are within the scope of the present invention, including, but not limited to, single-chain polypeptides, two-chain polypeptides, three-chain polypeptides, four-chain polypeptides, and multiples thereof. Specifically, the multispecific antibodies herein include T cell multispecific (e.g., bispecific) antibodies that bind to CLEC12A and CD3ε (anti-CLEC12A x anti-CD3ε antibodies). Such antibodies induce potent T cell-mediated cell death of cells expressing CLEC12A.

[0139] Preparation of anti-CLEC12A antibody Antibodies of the present invention can be prepared by methods known in the art, for example, by recombinant DNA technology, for example, by expression of an encoding nucleic acid in a suitable eukaryotic or prokaryotic host, including mammalian cells (e.g., CHO cells), E. coli, or yeast.

[0140] Antibodies that bind to non-overlapping epitopes on CLEC12A protein can be identified by competitive binding assays, such as enzyme-linked immunosorbent assays (ELISA assays) or flow cytometry competitive binding assays.For example, those skilled in the art can use the competition between a known antibody that binds to a target antigen and the antibody of interest.Using this approach, a set of antibodies can be classified into those that compete with a reference antibody and those that do not compete.Non-competitive antibodies are identified as those that bind to a distinct epitope that does not overlap with the epitope that the reference antibody binds.In many cases, one antibody is immobilized and allowed to bind to the antigen, and a second labeled (e.g., biotinylated) antibody is tested for its ability to bind to the captured antigen in an ELISA assay. This can also be performed using surface plasmon resonance (SPR) platforms including ProteOn XPR36 (BioRad, Inc), Biacore 2000 and Biacore T200 (GE Healthcare Life Sciences), and MX96 SPR Imager (Ibis Technologies BV), as well as biolayer interferometry platforms such as Octet Red384 and Octet HTX (ForteBio, Pall Inc).

[0141] Generally, an antibody "competes" with a reference antibody if it causes about a 15-100% reduction in binding of the reference antibody to a target antigen, as measured by standard techniques such as the competitive binding assays described above. In various embodiments, relative inhibition is at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more.

[0142] Pharmaceutical Compositions, Uses and Methods of Treatment It is another aspect of the present invention to provide pharmaceutical compositions comprising one or more antibodies of the present invention in admixture with a suitable pharmaceutically acceptable carrier. As used herein, pharmaceutically acceptable carriers are exemplified, but not limited to, adjuvants, solid carriers, water, buffers, or other carriers used in the art to carry therapeutic ingredients, or combinations thereof.

[0143] In one embodiment, the pharmaceutical composition comprises an antibody that binds to CLEC12A. In another embodiment, the pharmaceutical composition comprises a multispecific (including bispecific) antibody that has binding specificities for two or more non-overlapping epitopes on the CLEC12A protein. In a preferred embodiment, the pharmaceutical composition comprises a multispecific (including bispecific) antibody that binds to CLEC12A and a binding target on an effector cell (e.g., a binding target on a T cell, such as the CD3ε protein on a T cell).

[0144] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., a single dose). Formulations depend on the selected route of administration.

[0145] How to use The anti-CLEC12A antibodies and pharmaceutical compositions described herein can be used to treat diseases and conditions characterized by the expression of CLEC12A, including but not limited to the conditions and diseases further described herein.

[0146] CLEC12A, also known as C-type lectin domain family 12 member A, DCAL-2, CLL-1, MICL, and CD371, is a member of the C-type lectin / C-type lectin-like domain (CTL / CTLD) superfamily. Members of this family share a common protein fold and have diverse functions, including roles in cell adhesion, cell-cell signaling, glycoprotein turnover, and inflammation and immune responses. The protein encoded by this gene is a negative regulator of granulocyte and monocyte function.

[0147] RNA expression analysis from the TCGA database indicates that CLEC12A is highly expressed and specific to acute myeloid leukemia. Therefore, therapeutic development of antibodies targeting CLEC12A may be effective in treating AML patients, including a significant number of AML patients who are resistant to current standard care treatments.

[0148] In one aspect, the anti-CLEC12A antibodies and pharmaceutical compositions herein can be used to treat disorders characterized by expression of CLEC12A, including, but not limited to, hematological malignancies that begin in hematopoietic tissues, including bone marrow and / or cells of the immune system. In one preferred embodiment, the anti-CLEC12A antibodies and pharmaceutical compositions herein are used to treat AML. In one preferred embodiment, the AML is refractory AML, and the patient being treated has previously received another therapy (e.g., a chemotherapy regimen) and has not achieved remission of the AML.

[0149] The effective amount of the compositions of the present invention for treating a disease will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, such as companion animals such as dogs, cats, and horses, and laboratory animals such as rabbits, mice, and rats, may also be treated. Treatment dosages may be titrated to optimize safety and efficacy.

[0150] Dosage levels can be readily determined by one skilled in the art and can be modified as needed, for example, to modify a subject's response to treatment. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.

[0151] The therapeutic entities of the present invention are typically administered on multiple occasions. The intervals may also be irregular, as determined by measuring the patient's blood levels of the therapeutic entity. Alternatively, the therapeutic entities of the present invention may be administered as sustained-release formulations, in which case less frequent administration is required. The dosage and frequency will vary depending on the half-life of the polypeptide in the patient.

[0152] The toxicity of the antibodies and antibody constructs described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used in formulating a non-toxic dosage range for use in humans. The dosage of the antibodies described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. Dosages can vary within this range depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition.

[0153] The composition for administration will commonly comprise the antibody or other ablative agent dissolved in a pharmaceutically acceptable carrier.

[0154] Kits comprising the active agents of the present invention and their formulations, as well as instructions for use, are also within the scope of the present invention. The kits may further comprise at least one additional reagent. The kits typically comprise a label indicating the intended use of the contents of the kit. As used herein, the term "label" includes any writing or recorded material supplied on or with the kit, or otherwise accompanying the kit.

[0155] Now that the present invention is fully described, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the invention. [Example]

[0156] Example 1: Monovalent binding kinetics of humanized anti-CLEC12A antibodies The purified antibodies were characterized for their affinity to the antigen by loading them onto an anti-human heavy chain (AHC) capture sensor and measuring the association and dissociation rates of histidine-tagged recombinant human CLEC12A protein using Octet Red 96. The results are shown in Figure 1.

[0157] Example 2: Binding analysis of humanized anti-CLEC12A antibodies to CLEC12A-expressing cell lines and primary cells Humanized anti-CLEC12A antibodies were tested for their binding to cells expressing CLEC12A. Three acute myeloid leukemia cell lines expressing different levels of CLEC12A were tested alongside human and cynomolgus monkey monocytes and neutrophils isolated from PBMCs. Cells were washed and resuspended in FACS buffer. Cells were plated at 1E5 cells / well in a 96-well plate. Cells were stained with titrated anti-CLEC12A antibodies for 30 minutes on ice, followed by washing and secondary staining with goat anti-human IgG-AF647 diluted 1:500. After a final wash and addition of 7-AAD, cells were analyzed by flow cytometry. Geometric mean fluorescence intensity was plotted against antibody concentration to generate dose-response curves. Panels A–C of Figure 2 show binding curves for the indicated AML cell lines (U937, THP1, and MV411). Panels A to D of Figure 3 show binding curves to human and cynomolgus monkey neutrophils and monocytes. Figure 4 shows the EC 50 Summarizes values.

[0158] Example 3: Binding of bispecific CLEC12AxCD3 bispecific antibodies to CLEC12A-expressing acute myeloid leukemia cells and to T cells The CLEC12AxCD3 bispecific antibody was evaluated for binding to CLEC12A-expressing AML cell lines and to CD3-expressing T cells. Cells were plated at 1x10 in 96-well V-bottom plates. 5Cells were seeded at a density of 1000 μg / ml. Serially diluted antibodies were added to the cells and incubated on ice for 30 minutes. After two washes with FACS buffer, an anti-human Fc secondary antibody labeled with Alexa Fluor 647 was added and incubated on ice for 20 minutes. After two further washes with FACS buffer, the cells were resuspended in FACS buffer containing the 7AAD cell viability dye and analyzed on a flow cytometer. The geometric mean fluorescence intensity was plotted against the antibody concentration to generate a dose-response curve. The results are shown in Figure 6, panels A–C.

[0159] Example 4: Binding of CLEC12AxCD3 bispecific antibody to CLEC12 and CD3 in a sandwich ELISA Simultaneous binding to CLEC12A and CD3 was assessed using a sandwich ELISA. Recombinant His-tagged CD3 protein was coated onto a 96-well plate overnight with shaking at room temperature. After washing with 0.05% Tween-20 in PBS, the plate was blocked with 2% BSA for 60 minutes. Antibodies were added and incubated for 60 minutes at room temperature with shaking. After washing, biotinylated recombinant CLEC12A protein was added to the plate and incubated for 60 minutes at room temperature. The plate was washed, and HRP (horseradish peroxidase)-conjugated streptavidin was added and incubated for 30 minutes at room temperature. After washing, TMB (3,3',5,5'-tetramethylbenzidine) substrate was added and incubated for 5–10 minutes to develop color. The reaction was then stopped by adding 0.16 M sulfuric acid. The absorbance was read using a plate reader and plotted against antibody concentration to generate a dose-response curve. The results are shown in Figure 7.

[0160] Example 5: CLEC12AxCD3 bispecific antibody-mediated CD8+ T cell proliferation in the presence of autologous monocytes expressing CLEC12A The CLEC12AxCD3 bispecific antibodies were tested for their biological activity in stimulating T cells. First, T cell proliferation in the presence of autologous monocytes was tested. T cells and CD14+ monocytes were isolated from PBMCs of healthy donors. Monocytes were plated at 1 x 10 per well in a U-bottom 96-well plate in RPMI 1640 medium supplemented with 10% heat-inactivated human serum. 4 T cells were labeled with CellTrace Violet dye and plated at 5 × 10 4 Cells were added to wells containing monocytes. The CLEC12AxCD3 bispecific antibody was then added, and the cells were incubated for 5 days. On the day of analysis, cells were harvested and stained for CD4, CD8, and cell viability. Samples were analyzed on a flow cytometer. Dilutions of CellTrace Violet dye were gated, reported as % proliferation, and then plotted against antibody concentration. The results are shown in Figure 8, panels A-B.

[0161] Example 6: Cytokine release from T cells mediated by CLEC12AxCD3 bispecific antibody in the presence of CLEC12A-expressing U937 The CLEC12AxCD3 bispecific antibody was tested for stimulation of cytokine release from T cells. Human PBMCs were isolated and incubated with the CLEC12AxCD3 bispecific antibody and U937 cells expressing CLEC12A. After 48 hours, supernatants were collected for IL-2 and detection of IFNγ release using a commercially available ELISA kit. The results are shown in Figure 9, panels A-D.

[0162] Example 7: Activation and cytotoxic activity of CD8+ T cells The CLEC12AxCD3 bispecific antibody was tested for its efficacy in activating T cells and mediating killing of AML target cells and autologous monocytes. CD8+ T cells were isolated from PBMCs and incubated overnight in RPMI 1640 medium supplemented with 20 U / mL IL-2. U937, THP1, and MV411 AML cell lines and autologous CD14+ monocytes expressing CLEC12A were cocultured with isolated CD8+ T cells for 24 hours in the presence of titrated CLEC12AxCD3 bispecific antibody labeled with CellTrace Violet. Cells were harvested the following day and stained for CD8, CD25, and CD69, as well as with 7-AAD cell viability dye. Samples were analyzed by flow cytometry after the addition of counting beads. For T cell activation, the percentage of CD8+ labeled cells expressing CD25 and CD69 was plotted against antibody concentration. For cytotoxicity, the number of live AML cells was counted and reported as a percentage of AML cells in control wells that received neither T cells nor antibody. Panels A-F of Figure 10 show activation of CD8+ T cells. Panels A-F of Figure 11 show killing of AML cell lines. Panels A-B of Figure 12 show cytotoxicity against autologous monocytes. Figure 13 shows IC of survival of AML cell lines and autologous monocytes. 50 This summarizes the following.

[0163] Example 8: Antitumor effect of CLEC12A x CD3 bispecific antibody in a U937 / human PBMC co-transplant model in NSG mice The CLEC12AxCD3 bispecific antibody was tested for its ability to inhibit the growth of CLEC12A-positive U937 human cancer cells in vivo. In a co-transplantation model, U937 cells were co-transplanted with human PBMCs into the flanks of NSG mice and allowed to grow to approximately 100 mm3. The CLEC12AxCD3 bispecific antibody was then administered intravenously weekly for a total of two doses. The antibody construct 2A5-1B4 was more potent than 2A51-1B10 at both low and high doses. The results are shown in Figure 14.

[0164] Example 9: Antitumor effect of CLEC12A x CD3 bispecific antibody in a U937 / human PBMC reconstitution model in NSG mice In the second model, U937 cells were first implanted into the flank of NSG mice and allowed to grow to approximately 100 mm3. Human PBMCs were then intraperitoneally implanted. 48 hours later, intravenous injections of the CLEC12A x CD3 bispecific antibody were initiated twice weekly for a total of four doses. Consistent with the results from the co-implantation model, the antibody construct 2A5-1B4 was more effective in suppressing U937 cell proliferation. The results are shown in Figure 15.

[0165] Example 10: Monovalent binding kinetics of CLEC12AxCD3 bispecific antibodies to CLEC12A and CD3 epsilon The binding kinetics of the CLEC12A×CD3 bispecific antibody 2A5-1B4 was characterized using SPR. Specifically, a capture coupling method was used. Briefly, anti-human IgG (Fc) antibodies were first immobilized to the reference and experimental channels of a CM5 chip using amino coupling, followed by capture of the CLEC12A×CD3 bispecific antibody. Serially diluted recombinant human CLEC12A or CD3 epsilon proteins were then flowed over the surfaces of the two channels for association and dissociation. The results were analyzed and evaluated using Biacore 8K evaluation software. The average value of three technical replicates was reported as the assay result. The results are shown in Figure 16. The results indicated that the binding affinity to CLEC12A was higher than that to CD3 epsilon. Furthermore, the binding affinity was similar between the human and cynomolgus proteins.

[0166] Example 11: Antitumor Efficacy of CLEC12AxCD3 Bispecific Antibody in the Orthotopic HL-60-Luc2 AML Model In an orthotopic AML model using HL-60-Luc2 expressing luciferase for in vivo imaging, the CLEC12AxCD3 bispecific antibody 2A5-1B4 was administered by intravenous (iv) or subcutaneous (sc) injection every other week for a total of eight doses. The results are shown in Figure 17. Antibody 2A5-1B4 inhibited tumor growth at the lowest dose tested, 0.003 mpk, using either route of administration.

[0167] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will recognize numerous variations, changes, and substitutions that do not depart from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims define the scope of the invention, and it is intended to cover methods and structures within the scope of these claims and their equivalents.

Claims

1. 1. An antibody that binds to CLEC12A, comprising a first binding unit, said binding unit comprising: a heavy chain variable region, said heavy chain variable region comprising: (a) a CDRH1 sequence of any one of SEQ ID NOs: 1-5; and / or (b) a CDRH2 sequence of any one of SEQ ID NOs: 6-14; and / or (c) a CDRH3 sequence of any one of SEQ ID NOs: 15-22; and a light chain variable region, said light chain variable region comprising: (d) a CDRL1 sequence of any one of SEQ ID NOs: 23-27; and / or (e) a CDRL2 sequence of any one of SEQ ID NOs: 28-33; and / or (f) a CDRL3 sequence of any one of SEQ ID NOs: 34-40; The antibody comprising:

2. The antibody of claim 1 , wherein the CDRH1, CDRH2, and CDRH3 sequences of the first binding unit are present in a human VH framework.

3. 3. The antibody of claim 1, wherein the CDRL1, CDRL2, and CDRL3 sequences of the first binding unit are present in a human VL framework.

4. 4. The antibody of claim 1, wherein the first binding unit is: a heavy chain variable region, said heavy chain variable region comprising: (a) a CDRH1 sequence of any one of SEQ ID NOs: 1-5; and (b) a CDRH2 sequence of any one of SEQ ID NOs: 6-14; and (c) a CDRH3 sequence of any one of SEQ ID NOs: 15-22; The antibody comprising:

5. 5. The antibody of claim 4, wherein the first binding unit is: a heavy chain variable region, said heavy chain variable region comprising: (a) a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 6, and a CDRH3 sequence of SEQ ID NO: 15; or (b) a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 7, and a CDRH3 sequence of SEQ ID NO: 16; or (c) a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 8, and a CDRH3 sequence of SEQ ID NO: 17; or (d) a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 9, and a CDRH3 sequence of SEQ ID NO: 18; or (e) a CDRH1 sequence of SEQ ID NO: 2, a CDRH2 sequence of SEQ ID NO: 10, and a CDRH3 sequence of SEQ ID NO: 19; or (f) a CDRH1 sequence of SEQ ID NO: 3, a CDRH2 sequence of SEQ ID NO: 11, and a CDRH3 sequence of SEQ ID NO: 20; or (g) a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 12, and a CDRH3 sequence of SEQ ID NO: 21; or (h) a CDRH1 sequence of SEQ ID NO: 4, a CDRH2 sequence of SEQ ID NO: 13, and a CDRH3 sequence of SEQ ID NO: 22; or (i) a CDRH1 sequence of SEQ ID NO: 5, a CDRH2 sequence of SEQ ID NO: 14, and a CDRH3 sequence of SEQ ID NO: 22; The antibody comprising:

6. 6. The antibody of any one of claims 1 to 5, wherein the first binding unit is: a light chain variable region, said light chain variable region comprising: (a) a CDRL1 sequence of any one of SEQ ID NOs: 23-27; and (b) a CDRL2 sequence of any one of SEQ ID NOs: 28-33; and (c) a CDRL3 sequence of any one of SEQ ID NOs: 34 to 40; The antibody comprising:

7. 7. The antibody of claim 6, wherein the first binding unit is: a light chain variable region, said light chain variable region comprising: (a) a CDRL1 sequence of SEQ ID NO: 23, a CDRL2 sequence of SEQ ID NO: 28, and a CDRL3 sequence of SEQ ID NO: 34; or (b) a CDRL1 sequence of SEQ ID NO:24, a CDRL2 sequence of SEQ ID NO:29, and a CDRL3 sequence of SEQ ID NO:35; or (c) a CDRL1 sequence of SEQ ID NO: 25, a CDRL2 sequence of SEQ ID NO: 30, and a CDRL3 sequence of SEQ ID NO: 36; or (d) a CDRL1 sequence of SEQ ID NO:26, a CDRL2 sequence of SEQ ID NO:31, and a CDRL3 sequence of SEQ ID NO:37; or (e) a CDRL1 sequence of SEQ ID NO: 25, a CDRL2 sequence of SEQ ID NO: 30, and a CDRL3 sequence of SEQ ID NO: 38; or (f) a CDRL1 sequence of SEQ ID NO:27, a CDRL2 sequence of SEQ ID NO:32, and a CDRL3 sequence of SEQ ID NO:39; or (g) a CDRL1 sequence of SEQ ID NO: 24, a CDRL2 sequence of SEQ ID NO: 33, and a CDRL3 sequence of SEQ ID NO: 40; The antibody comprising:

8. 8. The antibody of claim 1, wherein the first binding unit is: (a) a heavy chain variable region comprising: the heavy chain variable region comprising a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 6, and a CDRH3 sequence of SEQ ID NO: 15; and A light chain variable region: the light chain variable region comprising a CDRL1 sequence of SEQ ID NO:23, a CDRL2 sequence of SEQ ID NO:28, and a CDRL3 sequence of SEQ ID NO:34; or (b) a heavy chain variable region comprising: the heavy chain variable region comprising a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 7, and a CDRH3 sequence of SEQ ID NO: 16; and A light chain variable region: the light chain variable region comprising a CDRL1 sequence of SEQ ID NO:23, a CDRL2 sequence of SEQ ID NO:28, and a CDRL3 sequence of SEQ ID NO:34; or (c) a heavy chain variable region comprising: the heavy chain variable region comprising a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 8, and a CDRH3 sequence of SEQ ID NO: 17; and A light chain variable region: the light chain variable region comprising a CDRL1 sequence of SEQ ID NO:24, a CDRL2 sequence of SEQ ID NO:29, and a CDRL3 sequence of SEQ ID NO:35; or (d) a heavy chain variable region comprising: the heavy chain variable region comprising a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 9, and a CDRH3 sequence of SEQ ID NO: 18; and A light chain variable region: the light chain variable region comprising a CDRL1 sequence of SEQ ID NO:25, a CDRL2 sequence of SEQ ID NO:30, and a CDRL3 sequence of SEQ ID NO:36; or (e) a heavy chain variable region comprising: the heavy chain variable region comprising a CDRH1 sequence of SEQ ID NO: 2, a CDRH2 sequence of SEQ ID NO: 10, and a CDRH3 sequence of SEQ ID NO: 19; and A light chain variable region: the light chain variable region comprising a CDRL1 sequence of SEQ ID NO:26, a CDRL2 sequence of SEQ ID NO:31, and a CDRL3 sequence of SEQ ID NO:37; or (f) a heavy chain variable region comprising: the heavy chain variable region comprising a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 6, and a CDRH3 sequence of SEQ ID NO: 15; and A light chain variable region: the light chain variable region comprising a CDRL1 sequence of SEQ ID NO:23, a CDRL2 sequence of SEQ ID NO:28, and a CDRL3 sequence of SEQ ID NO:34; or (g) a heavy chain variable region comprising: the heavy chain variable region comprising a CDRH1 sequence of SEQ ID NO: 3, a CDRH2 sequence of SEQ ID NO: 11, and a CDRH3 sequence of SEQ ID NO: 20; and A light chain variable region: the light chain variable region comprising a CDRL1 sequence of SEQ ID NO:25, a CDRL2 sequence of SEQ ID NO:30, and a CDRL3 sequence of SEQ ID NO:38; or (h) a heavy chain variable region comprising: the heavy chain variable region comprising a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 12, and a CDRH3 sequence of SEQ ID NO: 21; and A light chain variable region: the light chain variable region comprising a CDRL1 sequence of SEQ ID NO:27, a CDRL2 sequence of SEQ ID NO:32, and a CDRL3 sequence of SEQ ID NO:39; or (i) a heavy chain variable region comprising: the heavy chain variable region comprising a CDRH1 sequence of SEQ ID NO: 4, a CDRH2 sequence of SEQ ID NO: 13, and a CDRH3 sequence of SEQ ID NO: 22; and A light chain variable region comprising: the light chain variable region comprising a CDRL1 sequence of SEQ ID NO:24, a CDRL2 sequence of SEQ ID NO:33, and a CDRL3 sequence of SEQ ID NO:40; or (j) a heavy chain variable region comprising: the heavy chain variable region comprising a CDRH1 sequence of SEQ ID NO:5, a CDRH2 sequence of SEQ ID NO:14, and a CDRH3 sequence of SEQ ID NO:22; and A light chain variable region comprising: the light chain variable region comprising a CDRL1 sequence of SEQ ID NO: 24, a CDRL2 sequence of SEQ ID NO: 33, and a CDRL3 sequence of SEQ ID NO: 40; The antibody comprising:

9. The antibody of claim 8, wherein the first binding unit comprises a heavy chain variable region sequence having at least 95% identity to any one of SEQ ID NOs: 41 to 52.

10. The antibody of claim 8, wherein the first binding unit comprises a heavy chain variable region sequence comprising any one of SEQ ID NOs: 41 to 52.

11. The antibody of any one of claims 8 to 10, wherein the first binding unit comprises a light chain variable region sequence having at least 95% identity to any one of SEQ ID NOs: 53 to 64.

12. The antibody of any one of claims 8 to 10, wherein the first binding unit comprises a light chain variable region sequence comprising any one of SEQ ID NOs: 53 to 64.

13. 13. The antibody of any one of claims 8 to 12, wherein the first binding unit is: (a) a heavy chain variable region sequence of SEQ ID NO: 41 and a light chain variable region sequence of SEQ ID NO: 53; or (b) a heavy chain variable region sequence of SEQ ID NO: 42 and a light chain variable region sequence of SEQ ID NO: 54; or (c) a heavy chain variable region sequence of SEQ ID NO: 43 and a light chain variable region sequence of SEQ ID NO: 55; or (d) a heavy chain variable region sequence of SEQ ID NO: 44 and a light chain variable region sequence of SEQ ID NO: 56; or (e) a heavy chain variable region sequence of SEQ ID NO: 45 and a light chain variable region sequence of SEQ ID NO: 57; or (f) a heavy chain variable region sequence of SEQ ID NO: 46 and a light chain variable region sequence of SEQ ID NO: 58; or (g) a heavy chain variable region sequence of SEQ ID NO: 47 and a light chain variable region sequence of SEQ ID NO: 59; or (h) a heavy chain variable region sequence of SEQ ID NO: 48 and a light chain variable region sequence of SEQ ID NO: 60; or (i) a heavy chain variable region sequence of SEQ ID NO: 49 and a light chain variable region sequence of SEQ ID NO: 61; or (j) a heavy chain variable region sequence of SEQ ID NO: 50 and a light chain variable region sequence of SEQ ID NO: 62; or (k) a heavy chain variable region sequence of SEQ ID NO: 51 and a light chain variable region sequence of SEQ ID NO: 63; or (l) a heavy chain variable region sequence of SEQ ID NO: 52, and a light chain variable region sequence of SEQ ID NO: 64; The antibody comprising:

14. The antibody of any one of claims 1 to 13, further comprising a heavy chain constant region sequence.

15. The antibody of claim 14, wherein the heavy chain constant region comprises a hinge region, a CH1 region, a CH2 region, and / or a CH3 region.

16. The antibody of any one of claims 14 to 15, wherein the heavy chain constant region comprises one or more knob-in-hole (KiH) mutations.

17. The antibody of any one of claims 14 to 16, wherein the heavy chain constant region comprises one or more silencing mutations.

18. The antibody of any one of claims 14 to 17, wherein the heavy chain constant region comprises one or more protein A binding mutations.

19. 19. The antibody of claim 18, wherein the one or more Protein A binding mutations comprise a H435R mutation, a Y436F mutation, or both H435R and Y436F mutations.

20. The antibody of any one of claims 1 to 19, further comprising a light chain constant region sequence.

21. The antibody of claim 20, wherein the light chain constant region comprises a CL region.

22. The antibody of any one of claims 1 to 21, which is monospecific.

23. The antibody of any one of claims 1 to 21, which is multispecific.

24. 24. The antibody of claim 23, which is bispecific.

25. 25. The antibody of claim 23 or 24, further comprising a second binding unit that binds to CD3ε.

26. 26. The antibody of claim 25, wherein the second binding unit is: A heavy chain variable region comprising: (a) the CDRH1 sequence of SEQ ID NO: 65; and / or (b) a CDRH2 sequence of any one of SEQ ID NOs: 66-67; and / or (c) the heavy chain variable region comprising a CDRH3 sequence of SEQ ID NO: 68; and A light chain variable region comprising: (d) the CDRL1 sequence of SEQ ID NO: 69; and / or (e) the CDRL2 sequence of SEQ ID NO: 70; and / or (f) the light chain variable region comprising a CDRL3 sequence of SEQ ID NO: 71; The antibody comprising:

27. 27. The antibody of claim 26, wherein the CDRH1, CDRH2, and CDRH3 sequences of the second binding unit are present in a human VH framework.

28. 28. The antibody of claim 26 or 27, wherein the CDRL1, CDRL2, and CDRL3 sequences of the second binding unit are present in a human VL framework.

29. 29. The antibody of any one of claims 26 to 28, wherein the second binding unit is: A heavy chain variable region comprising: (a) a CDRH1 sequence of SEQ ID NO: 65; and (b) a CDRH2 sequence of any one of SEQ ID NOs: 66-67; and (c) the heavy chain variable region comprising a CDRH3 sequence of SEQ ID NO: 68; The antibody comprising:

30. 30. The antibody of claim 29, wherein the second binding unit is: A heavy chain variable region comprising: (a) a CDRH1 sequence of SEQ ID NO: 65, a heavy chain CDRH2 sequence of SEQ ID NO: 66, and a heavy chain CDRH3 sequence of SEQ ID NO: 68; or (b) the heavy chain variable region comprising a CDRH1 sequence of SEQ ID NO: 65, a CDRH2 sequence of SEQ ID NO: 67, and a CDRH3 sequence of SEQ ID NO: 68; The antibody comprising:

31. 31. The antibody of any one of claims 26 to 30, wherein the second binding unit is: a light chain variable region comprising the CDRL1 sequence of SEQ ID NO:69; the CDRL2 sequence of SEQ ID NO:70; and the CDRL3 sequence of SEQ ID NO:71; The antibody comprising:

32. 32. The antibody of any one of claims 26 to 31, wherein the second binding unit is: (a) a heavy chain variable region comprising: the heavy chain variable region comprising a CDRH1 sequence of SEQ ID NO: 65, a CDRH2 sequence of SEQ ID NO: 66, and a CDRH3 sequence of SEQ ID NO: 68; and A light chain variable region comprising: the light chain variable region comprising a CDRL1 sequence of SEQ ID NO:69, a CDRL2 sequence of SEQ ID NO:70, and a CDRL3 sequence of SEQ ID NO:71; or (b) a heavy chain variable region comprising: the heavy chain variable region comprising a CDRH1 sequence of SEQ ID NO: 65, a CDRH2 sequence of SEQ ID NO: 67, and a CDRH3 sequence of SEQ ID NO: 68; and A light chain variable region comprising: the light chain variable region comprising a CDRL1 sequence of SEQ ID NO: 69, a CDRL2 sequence of SEQ ID NO: 70, and a CDRL3 sequence of SEQ ID NO: 71; The antibody comprising:

33. The antibody of claim 32, wherein the second binding unit comprises a heavy chain variable region sequence having at least 95% identity to any one of SEQ ID NOs: 72-73.

34. The antibody of claim 32, wherein the second binding unit comprises a heavy chain variable region sequence comprising any one of SEQ ID NOs: 72-73.

35. The antibody of any one of claims 32 to 34, wherein the second binding unit comprises a light chain variable region sequence having at least 95% identity to any one of SEQ ID NOs: 74 to 75.

36. The antibody of any one of claims 32 to 34, wherein the second binding unit comprises a light chain variable region sequence comprising any one of SEQ ID NOs: 74 to 75.

37. 37. The antibody of any one of claims 32 to 36, wherein the second binding unit is: (a) a heavy chain variable region sequence of SEQ ID NO: 72 and a light chain variable region sequence of SEQ ID NO: 74; or (b) a heavy chain variable region sequence of SEQ ID NO: 73, and a light chain variable region sequence of SEQ ID NO: 75; The antibody comprising:

38. 1. An antibody that binds to CLEC12A and CD3ε: a first light chain subunit comprising SEQ ID NO: 79; a first heavy chain subunit comprising SEQ ID NO: 76; a second light chain subunit comprising SEQ ID NO:79; and a second heavy chain subunit comprising SEQ ID NO: 78; The antibody comprising:

39. 1. An antibody that binds to CLEC12A and CD3ε: a first light chain subunit comprising SEQ ID NO: 79; a first heavy chain subunit comprising SEQ ID NO: 77; a second light chain subunit comprising SEQ ID NO:79; and a second heavy chain subunit comprising SEQ ID NO: 78; The antibody comprising:

40. 1. An antibody that binds to CLEC12A and CD3ε: a first light chain subunit comprising SEQ ID NO: 83; a first heavy chain subunit comprising SEQ ID NO: 80; a second light chain subunit comprising SEQ ID NO: 83; and a second heavy chain subunit comprising SEQ ID NO: 82; The antibody comprising:

41. 1. An antibody that binds to CLEC12A and CD3ε: a first light chain subunit comprising SEQ ID NO: 83; a first heavy chain subunit comprising SEQ ID NO:81; a second light chain subunit comprising SEQ ID NO: 83; and a second heavy chain subunit comprising SEQ ID NO: 82; The antibody comprising:

42. A pharmaceutical composition comprising the antibody of any one of claims 1 to 41.

43. A method of treatment comprising administering an effective dose of the antibody of any one of claims 1 to 41, or the pharmaceutical composition of claim 42, to an individual in need thereof.

44. A method for treating a disorder characterized by expression of CLEC12A, comprising administering to a subject having the disorder an antibody described in any one of claims 1 to 41, or a pharmaceutical composition described in claim 42.

45. Use of an antibody according to any one of claims 1 to 41 in the preparation of a medicament for treating a disorder characterized by expression of CLEC12A.

46. The antibody of any one of claims 1 to 41 for use in the treatment of a disorder characterized by expression of CLEC12A.

47. 47. The method, use or antibody of any one of claims 44 to 46, wherein the disorder is cancer.

48. 48. The method, use or antibody of claim 47, wherein the cancer is a blood cancer.

49. 49. The method, use or antibody of claim 48, wherein the hematological cancer is acute myeloid leukemia (AML).

50. 50. The method, use, or antibody of claim 49, wherein the AML is refractory AML.

51. A polynucleotide encoding the antibody of any one of claims 1 to 41.

52. A vector comprising the polynucleotide of claim 51.

53. 53. A cell comprising the vector of claim 52.

54. 54. A method for producing an antibody according to any one of claims 1 to 41, comprising growing a cell according to claim 53 under conditions permissive for expression of said antibody, and isolating said antibody.

55. A kit comprising the antibody of any one of claims 1 to 41 or the pharmaceutical composition of claim 42, and instructions for use.

56. 56. The kit of claim 55, further comprising an additional therapeutic agent.

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