B7-H4 Antibody Dosing Regimen

Abstract

The present disclosure provides methods for administering antibodies and antigen-binding fragments thereof that specifically bind to human B7-H4 to a subject in need thereof, such as a cancer patient.

Description

1. Technical Field

[0001] The present disclosure generally relates to methods of administering antibodies that specifically bind to human B7-H4 to treat diseases such as cancer. Advantageous dosage regimens are provided. 2. Background Technology

[0002] B7-H4 (also known as B7x, B7-S1, and VTCN1) is an immunomodulatory molecule that shares homology with other B7 family members, including PD-L1. It is a type I transmembrane protein composed of IgV and IgC extracellular domains. Although B7-H4 expression in healthy tissues is relatively limited at the protein level, B7-H4 is expressed in several solid tumors, such as gynecological cancers of the breast, ovary, and endometrium. B7-H4 expression in tumors is often associated with a poor prognosis. The receptor for B7-H4 is unknown, but it is believed to be expressed on T cells. B7-H4 is believed to directly inhibit T cell activity.

[0003] Given the expression and function of B7-H4, antibodies that specifically bind to B7-H4 are being developed for use in therapies involving modulation of B7-H4 activity, such as for the treatment of cancer. Therefore, dosing regimens for the effective administration of such antibodies are needed. 3. Summary of the Invention

[0004] Provided herein are methods of administering B7-H4 antibodies and antigen-binding fragments thereof using therapeutically effective dosing regimens.

[0005] In certain aspects, a method of treating a solid tumor in a human subject comprises administering to the subject about 0.005 to about 20 mg / kg of an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and comprises the heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3, and light chain variable region (VL) CDR1, VL CDR2, and VL CDR3 sequences of the 20502 antibody.

[0006] In certain aspects, a method of treating a solid tumor in a human subject comprises administering to the subject a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to human B7-H4 and comprises the heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3, and light chain variable region (VL) CDR1, VL CDR2, and VL CDR3 sequences of the 20502 antibody; and (ii) a pharmaceutically acceptable excipient, wherein at least 95% of the antibody or antigen-binding fragment thereof in the composition is afucosylated, and wherein about 0.005 to about 20 mg / kg of the antibody or antigen-binding fragment thereof is administered.

[0007] In some aspects, the CDRs are Kabat-defined CDRs, Chothia-defined CDRs, or AbM-defined CDRs. In some aspects, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and CDR3 sequences comprise the amino acid sequences set forth in SEQ ID NOs: 5-10, respectively.

[0008] In some aspects, the subject is administered about 20 mg / kg or 20 mg / kg of the antibody or its antigen-binding fragment. In some aspects, the subject is administered about 10 mg / kg or 10 mg / kg of the antibody or its antigen-binding fragment. In some aspects, the subject is administered about 3 mg / kg or 3 mg / kg of the antibody or its antigen-binding fragment. In some aspects, the subject is administered about 1 mg / kg or 1 mg / kg of the antibody or its antigen-binding fragment. In some aspects, the subject is administered about 0.3 mg / kg or 0.3 mg / kg of the antibody or its antigen-binding fragment. In some aspects, the subject is administered about 0.1 mg / kg or 0.1 mg / kg of the antibody or its antigen-binding fragment. In some aspects, wherein the subject is administered about 0.03 mg / kg or 0.03 mg / kg of the antibody or its antigen-binding fragment. In some aspects, the subject is administered about 0.01 mg / kg or 0.01 mg / kg of the antibody or its antigen-binding fragment. In certain aspects, about 0.005 mg / kg or 0.005 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject.

[0009] In certain aspects, the antibody or antigen-binding fragment thereof is administered about once every three weeks.

[0010] In certain aspects, the antibody or antigen-binding fragment thereof is administered intravenously.

[0011] In certain aspects, B7-H4 has been detected in the solid tumor using immunohistochemistry (IHC) prior to administration.

[0012] In certain aspects, the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 11; and / or a VL comprising the amino acid sequence set forth in SEQ ID NO: 12. In certain aspects, the antibody or antigen-binding fragment comprises a heavy chain constant region and / or a light chain constant region. In certain aspects, the heavy chain constant region is a human immunoglobulin IgG1 heavy chain constant region, and / or the light chain constant region is a human immunoglobulin IgGκ light chain constant region. In certain aspects, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 25; and / or a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 23. In certain aspects, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 21; and / or a light chain comprising the amino acid sequence set forth in SEQ ID NO: 22.

[0013] In certain aspects, the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof.

[0014] In certain aspects, the antibody or antigen-binding fragment thereof is afucosylated.

[0015] In some aspects, the antibody or antigen-binding fragment thereof is a full-length antibody. In some aspects, the antibody or antigen-binding fragment thereof is an antigen-binding fragment. In some aspects, the antigen-binding fragment comprises or is Fab, Fab', F(ab')2, single-chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetravalent antibody, trivalent antibody, bivalent antibody, single domain antibody, DVD-Ig, Fcab, mAb 2 , (scFv)2 or scFv-Fc.

[0016] In certain aspects, fucosylation is not detectable in the composition.

[0017] In certain aspects, the solid tumor expresses B7-H4.

[0018] In certain aspects, the solid tumor is unresectable, locally advanced, or metastatic.

[0019] In some aspects, the solid tumor is selected from the group consisting of: breast cancer, ductal carcinoma, endometrial cancer, ovarian cancer, urothelial carcinoma, non-small cell lung cancer, pancreatic cancer, thyroid cancer, kidney cancer and bladder cancer. In some aspects, the solid tumor is breast cancer, ovarian cancer, endometrial cancer or urothelial cancer. In some aspects, the breast cancer is advanced breast cancer. In some aspects, the breast cancer is HER2-negative breast cancer. In some aspects, the breast cancer is triple-negative breast cancer. In some aspects, the breast cancer is hormone receptor (HR)-positive breast cancer. In some aspects, the non-small cell lung cancer is squamous cell carcinoma. In certain embodiments, the subject has not received prior therapy with a PD-1 / PD-L1 antagonist.

[0020] In some aspects, the method further comprises monitoring the number of immune cells in the tumor. In some aspects, the method further comprises monitoring the number of natural killer (NK) cells, CD4+ cells and / or CD8+ cells in the tumor. In some aspects, the method further comprises monitoring the cytokine levels of the experimenter. In some aspects, the method further comprises monitoring the IL-2, IL-6, IL-10, TNF and / or interferon gamma (IFNγ) levels of the experimenter.

[0021] In certain aspects, a method of treating a solid tumor in a human subject comprises intravenously administering to the subject about 20 mg / kg of an antibody that specifically binds to human B7-H4 and comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 11; and a VL comprising the amino acid sequence set forth in SEQ ID NO: 12.

[0022] In certain aspects, a method of treating a solid tumor in a human subject comprises administering to the subject a pharmaceutical composition comprising (i) an antibody that specifically binds to human B7-H4 and comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 11; and a VL comprising the amino acid sequence set forth in SEQ ID NO: 12; and (ii) a pharmaceutically acceptable excipient, wherein at least 95% of the antibody or antigen-binding fragment thereof in the composition is afucosylated, and wherein about 20 mg / kg of the antibody or antigen-binding fragment thereof is administered intravenously about once every three weeks.

[0023] In certain aspects, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 21 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 22. In certain aspects, the solid tumor is breast cancer, ovarian cancer, endometrial cancer, or urothelial cancer. 4. Brief Description of the Figures

[0024] Figure 1 ADCC activity of fucosylated and afucosylated B7-H4 antibodies against cells expressing various levels of B7-H4 is shown. (See Example 3.)

[0025] Figure 2 The effect of B7-H4 antibodies on tumor growth inhibition in mice, where the tumors were derived from CT26 cancer cells engineered to express B7-H4. (See Example 4.)

[0026] Figure 3 Phase 1a and 1b study protocols are shown. CNS = central nervous system; DLT = dose-limiting toxicity; IV = intravenous; LTFU = long-term follow-up; MTD = maximum tolerated dose; PD = progressive disease; Q3W = every 3 weeks; TNBC = triple-negative breast cancer; RD = recommended dose. (See Examples 7 and 8.) 5. Specific implementation methods

[0027] Provided herein are methods for administering antibodies (e.g., monoclonal antibodies) and antigen-binding fragments thereof that specifically bind to B7-H4 (e.g., human B7-H4). Anti-B7-H4 antibodies and antigen-binding fragments thereof can be administered, for example, to treat a solid tumor in a subject. In a specific embodiment, the subject is administered about 20 mg / kg, about 10 mg / kg, about 3 mg / kg, about 1 mg / kg, about 0.3 mg / kg, about 0.1 mg / kg, about 0.03 mg / kg, about 0.01 mg / kg, or about 0.005 mg / kg of the antibody or antigen-binding fragment thereof, for example, wherein the administration occurs about once every three weeks.

[0028] 5.1 Terminology

[0029] As used herein, the term "B7-H4" refers to mammalian B7-H4 polypeptides, including but not limited to native B7-H4 polypeptides and isoforms of B7-H4 polypeptides. "B7-H4" encompasses full-length, unprocessed B7-H4 polypeptides as well as forms of B7-H4 polypeptides produced by processing within cells. As used herein, the term "human B7-H4" refers to a polypeptide comprising the amino acid sequence of SEQ ID NO: 1. "B7-H4 polynucleotide," "B7-H4 nucleotide," or "B7-H4 nucleic acid" refers to a polynucleotide encoding B7-H4.

[0030] The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target (such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid or a combination thereof) by at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses complete polyclonal antibodies, complete monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising antibodies, and any other modified immunoglobulin molecules, as long as the antibody exhibits the desired biological activity. Antibodies can have any of the following five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM or its subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), which are referred to as α, δ, ε, γ and μ based on the properties of their heavy chain constant structures. Different types of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules (such as toxins, radioisotopes, etc.).

[0031] The term "antibody fragment" refers to a portion of an intact antibody. An "antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to a portion of an intact antibody that binds to an antigen. An antigen-binding fragment may contain the antigen recognition site of an intact antibody (e.g., a complementary determining region (CDR) sufficient to specifically bind to an antigen). Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. Antigen-binding fragments of antibodies may be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or may be artificially produced.

[0032] The terms "anti-B7-H4 antibody," "B7-H4 antibody," and "antibody that binds to B7-H4" refer to antibodies that are capable of specifically binding to PD-1 with sufficient affinity to render the antibodies useful as diagnostic and / or therapeutic agents in targeting PD-1. As used herein, the terms "specifically bind," "immunospecifically binds," "immunospecifically recognizes," and "specifically recognizes" are analogous terms in the context of antibodies or antigen-binding fragments thereof. These terms indicate that the antibody or antigen-binding fragment thereof binds to the epitope via its antigen-binding domain, and that binding requires a certain degree of complementarity between the antigen-binding domain and the epitope. Thus, an antibody that "specifically binds" to human B7-H4 (SEQ ID NO: 1) may also bind to B7-H4 from other species (e.g., cynomolgus monkey, mouse, and / or rat B7-H4) and / or B7-H4 proteins produced by other human alleles, but binds to unrelated, non-B7-H4 proteins (e.g., other B7 protein family members, such as PD-L1) to an extent that is less than about 10% of the antibody's binding to B7-H4, as measured, for example, by radioimmunoassay (RIA). In a specific embodiment, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to human, cynomolgus monkey, mouse, and rat B7-H4.

[0033] A "monoclonal" antibody or antigen-binding fragment thereof refers to a homogeneous population of antibodies or antigen-binding fragments that participate in highly specific binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants. The term "monoclonal antibody" or antigen-binding fragment thereof encompasses complete and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. In addition, a "monoclonal" antibody or antigen-binding fragment thereof refers to such antibodies and antigen-binding fragments thereof prepared in any number of ways, including but not limited to hybridomas, phage selection, recombinant expression, and transgenic animals.

[0034] As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. A variable region generally refers to a part of an antibody, generally a part of a light chain or a heavy chain, typically about 110 to 120 amino acids or 110 to 125 amino acids in a mature heavy chain and about 90 to 115 amino acids in a mature light chain, which differ in sequence between antibodies and are used for the binding and specificity of a particular antibody to its specific antigen. The variability of the sequence is concentrated in those regions referred to as complementary determining regions (CDRs), while the regions with higher conservatism in the variable domains are referred to as framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In specific embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable regions comprise rodent or murine CDRs and primate (eg, non-human primate) framework regions (FRs).

[0035] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0036] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0037] The term "Kabat numbering" and similar terms are generally recognized in the art and refer to a system in which the amino acid residues in the heavy chain and light chain variable region of an antibody or its antigen-binding fragment are numbered. In some aspects, CDR can be determined according to the Kabat numbering system (see, for example, Kabat EA and Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, the 5th edition, U.S. Department of Health and Human Services, NIH publication number 91-3242). Using the Kabat numbering system, the CDR in the antibody heavy chain molecule is generally present in amino acid position 31 to 35 (which optionally can include one or two other amino acids (referred to as 35A and 35B in the Kabat numbering scheme) after 35) (CDR1), amino acid position 50 to 65 (CDR2) and amino acid position 95 to 102 (CDR3). Using the Kabat numbering system, the CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In a specific embodiment, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

[0038] In contrast, Chothia refers to the position of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). When numbered using the Kabat numbering convention, the ends of the Chothia CDR-H1 loop vary between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if 35A or 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.

[0039]

[0040]

[0041] As used herein, the terms "constant region" and "constant domain" are interchangeable and have their common meaning in the art. The constant region is the portion of an antibody that is not directly involved in binding of the antibody to an antigen but can exhibit a variety of effector functions, such as interactions with Fc receptors, such as the carboxyl terminal portion of a light chain and / or a heavy chain. Relative to the immunoglobulin variable domain, the constant region of an immunoglobulin molecule typically has a more conserved amino acid sequence. In certain aspects, the antibody or antigen-binding fragment comprises a constant region or portion thereof sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

[0042] As used herein, the term "heavy chain" when used with respect to antibodies can refer to any of the different types, e.g., α (α), δ (δ), ε (ε), γ (γ), and μ (μ), based on the amino acid sequence of the constant domain, which give rise to antibodies of the IgA, IgD, IgE, IgG, and IgM classes, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4. Heavy chain amino acid sequences are well known in the art. In a specific embodiment, the heavy chain is a human heavy chain.

[0043] As used herein, the term "light chain" when used with respect to an antibody can refer to any of the different types, such as kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain.

[0044] The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof in which the amino acid sequences are derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to the variable regions of an antibody or antigen-binding fragment thereof derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in an antibody or antigen-binding fragment thereof derived from another species (usually human) to avoid eliciting an immune response in that species.

[0045] The term "humanized antibody" or its antigen-binding fragment refers to a non-human (e.g., murine) antibody or its antigen-binding fragment form that is a specific immunoglobulin chain, chimeric immunoglobulin or its fragment containing minimal non-human (e.g., murine) sequence. Typically, a humanized antibody or its antigen-binding fragment is a human immunoglobulin ("CDR graft") in which residues from the complementary determining region (CDR) are replaced with residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) with the desired specificity, affinity and ability. In some cases, certain Fv framework region (FR) residues of a human immunoglobulin are replaced with corresponding residues from an antibody or fragment of a non-human species with the desired specificity, affinity and ability. Humanized antibodies or their antigen-binding fragments can be further modified by replacing additional residues in the Fv framework region and / or in the non-human CDR residues to improve and optimize the specificity, affinity and / or ability of the antibody or its antigen-binding fragment. In general, a humanized antibody or its antigen-binding fragment will comprise a variable domain containing all or substantially all CDR regions corresponding to non-human immunoglobulins, while all or substantially all FR regions are those of a human immunoglobulin consensus sequence. A humanized antibody or its antigen-binding fragment may also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically at least a portion of a human immunoglobulin. Examples of methods for producing humanized antibodies are described in U.S. Patent No. 5,225,539; Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994); and Roguska et al., Protein Eng. 9(10):895-904 (1996). In some embodiments, a "humanized antibody" is a resurfaced antibody.

[0046] The term "human" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof having an amino acid sequence derived from a human immunoglobulin locus, wherein such antibody or antigen-binding fragment thereof is prepared using any technique known in the art. This definition of human antibody or antigen-binding fragment thereof includes intact or full-length antibodies and fragments thereof.

[0047] An "afucosylated" antibody or antigen-binding fragment thereof or an antibody or antigen-binding fragment thereof that "lacks fucose" refers to an IgG1 or IgG3 isotype antibody or antigen-binding fragment thereof that lacks fucose in its constant region glycosylation. Glycosylation of human IgG1 occurs at Asn297, as the core fucosylated biantennary complex oligosaccharide glycosylation terminates at most two Gal residues. In some embodiments, an afucosylated antibody lacks fucose at Asn297. Depending on the amount of terminal Gal residues, these structures are designated as G0, G1 (α 1,6 or α 1,3), or G2 glycan residues. See, e.g., Raju, TS, BioProcess Int. 1:44-53 (2003). CHO-type glycosylation of antibody Fc is described, e.g., in Routier, F. FL, Glycoconjugate J. 14:201-207 (1997).

[0048] The method for measuring fucose includes any method known in the art. For the purposes of this article, fucose is detected by the method described in Example 1 of WO2015 / 017600, which is incorporated herein by reference in its entirety. In short, glycan analysis is performed by releasing glycans from antibodies (e.g., by enzymatic release), labeling the glycans with o-aminobenzoic acid (2-AA), and then purifying the labeled glycans. Normal phase HPLC with fluorescence detection is used to separate glycans, and the relative content of each glycan in the antibody is measured. Glycans can be clearly identified as lacking or including fucose by mass spectrometry. In some embodiments, fucose is not detectable in a composition comprising a plurality of afucosylated antibodies or their antigen-binding fragments. In some embodiments, afucosylated antibodies or their antigen-binding fragments have enhanced affinity for FcγRIIIA. In some embodiments, afucosylated antibodies or their antigen-binding fragments have enhanced affinity for FcγRIIIA (V158). In some embodiments, the afucosylated antibody or antigen-binding fragment thereof has enhanced affinity for FcγRIIIA (F158).

[0049] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antigen-binding fragment thereof) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody or antigen-binding fragment thereof and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D Affinity can be measured and / or expressed in a variety of ways known in the art, including but not limited to the equilibrium dissociation constant (K D ) and the equilibrium association constant (KA ). K D It is based on k off / k on The quotient of K is calculated, and K A It is based on k on / k off The quotient of k on refers to the association rate constant of, for example, an antibody or antigen-binding fragment thereof with an antigen, and k off Refers to, for example, the dissociation of an antibody or its antigen-binding fragment from an antigen. on and k off It can be done by techniques known to those skilled in the art, such as or KinExA to determine.

[0050] As used herein, "epitope" is a term in this area, and refers to the local region of the antigen to which an antibody or its antigen-binding fragment can specifically bind. An epitope can be, for example, the continuous amino acids of a polypeptide (linear or continuous epitope), or an epitope can be, for example, from two or more discontinuous regions (conformational, nonlinear, discontinuous or discontinuous epitopes) of one or more polypeptides. In certain embodiments, the epitope to which an antibody or its antigen-binding fragment specifically binds can be determined by, for example, NMR spectroscopy, X-ray crystallography, ELISA assay, hydrogen / deuterium exchange (for example, liquid chromatography electrospray mass spectrometry) in combination with mass spectrometry, array-based oligopeptide scanning assays and / or mutagenesis mapping (for example, site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any method known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303). Antibody / antigen-binding fragment thereof: Antigen crystals can be studied using well-known X-ray diffraction techniques and can be improved using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; See, e.g., Meth Enzymol (1985) Vols. 114 and 115, edited by Wyckoff HW et al.; US 2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, edited by Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10): 1316-1323). Mutagenesis mapping studies can be performed using any method known to those skilled in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis, see, eg, Champe M et al. (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-108.

[0051] The terms "programmed cell death protein 1" and "PD-1" refer to an immunoinhibitory receptor belonging to the CD28 family. PD-1 is primarily expressed in vivo on previously activated T cells and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes human PD-1 (hPD-1), naturally occurring variants and isoforms of hPD-1, and species homologs of hPD-1. The hPD-1 sequence is

[0052] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTE RRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL(SEQ IDNO:30).

[0053] The terms "programmed cell death 1 ligand 1" and "PD-L1" refer to one of the two cell surface glycoprotein ligands of PD-1 (the other is PD-L2) that downregulate T cell activation and cytokine secretion after binding to PD-1. The term "PD-L1" as used herein includes human PD-L1 (hPD-L1), naturally occurring variants and isoforms of hPD-1, and species homologs of hPD-L1. The sequence of hPD-L1 is

[0054] MRIFAVFFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPV TSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET(SEQ IDNO:31).

[0055] The term "PD-1 / PD-L1 antagonist" refers to a moiety that disrupts the PD-1 / PD-L1 signaling pathway. In some embodiments, the antagonist inhibits the PD-1 / PD-L1 signaling pathway by binding to PD-1 and / or PD-L1. In some embodiments, the PD-1 / PD-L1 antagonist also binds to PD-L2. In some embodiments, the PD-1 / PD-L1 antagonist blocks the binding of PD-1 to PD-L1 and, optionally, to PD-L2. Non-limiting exemplary PD-1 / PD-L1 antagonists include PD-1 antagonists, such as antibodies that bind to PD-1, e.g., nivolumab (OPDIVO) and pembrolizumab (KEYTRUDA); PD-L1 antagonists, such as antibodies that bind to PD-L1 (e.g., atezolizumab (TECENTRIQ), durvalumab, and avelumab); fusion proteins, such as AMP-224; and peptides, such as AUR-012.

[0056] An "isolated" polypeptide, antibody, polynucleotide, vector, cell or composition is one that is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells or compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some embodiments, the isolated antibodies, polynucleotides, vectors, cells or compositions are substantially pure. As used herein, "substantially pure" refers to material that is at least 50% pure (i.e., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure or at least 99% pure.

[0057] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of amino acids (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It will be understood that because the polypeptides of the present invention are based on antibodies, in certain embodiments, the polypeptides may exist as single chains or associated chains.

[0058] As used herein, the term "host cell" can be any type of cell, such as a primary cell, a cell in culture, or a cell from a cell line. In specific embodiments, the term "host cell" refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, for example due to mutations or environmental influences that may occur during passage or integration of the nucleic acid molecule into the host cell genome.

[0059] The term "pharmaceutical formulation" refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective and that contains no other components that are unacceptably toxic to a subject to which the formulation would be administered. The formulation may be sterile.

[0060] As used herein, the terms "administer," "administering," "administration," and the like refer to methods (e.g., intravenous administration) that can be used to achieve delivery of an agent, such as an anti-B7-H4 antibody or antigen-binding fragment thereof, to a desired site of biological action. Administration techniques that can be used with the agents and methods described herein are found, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0061] As used herein, the terms "subject" and "patient" are used interchangeably. The subject can be an animal. In some embodiments, the subject is a mammal, such as a non-human animal (e.g., a cow, pig, horse, cat, dog, rat, mouse, monkey or other primate, etc.). In some embodiments, the subject is a cynomolgus monkey. In some embodiments, the subject is a human.

[0062] The term "therapeutically effective amount" refers to an amount of an agent, such as an anti-B7-H4 antibody or antigen-binding fragment thereof, that is effective in treating a disease or condition in a subject. In the case of cancer, a therapeutically effective amount of an agent can reduce the number of cancer cells; reduce tumor size or burden; inhibit cancer cell infiltration into surrounding organs to some extent; inhibit tumor metastasis to some extent; inhibit tumor growth to some extent; alleviate one or more symptoms associated with cancer to some extent; and / or produce a favorable response, such as an increase in progression-free survival (PFS), disease-free survival (DFS), overall survival (OS), complete response (CR), partial response (PR), or in some cases, stable disease (SD), decreased progressive disease (PD), decreased time to progression (TTP), or any combination thereof. To the extent that the agent can prevent the growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic.

[0063] Terms such as "treat," "treatment," "treatment," "alleviate," and "alleviate" refer to therapeutic measures that can cure, slow, alleviate the symptoms of, and / or prevent the progression of, a pathological condition or disorder. Thus, those in need of treatment include those already diagnosed with or suspected of having the disorder. In certain embodiments, a subject's cancer is successfully "treated" according to the methods of the present invention if the patient exhibits one or more of the following: a decrease in the number of cancer cells or the complete absence of cancer cells; a decrease in tumor size; inhibition or absence of cancer cell infiltration into surrounding organs, including, for example, spread of cancer into soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; alleviation of one or more symptoms associated with a particular cancer; reduced morbidity and mortality; improved quality of life; a decrease in tumorigenicity, tumorigenic frequency, or the tumorigenic capacity of a tumor; a decrease in the number or frequency of cancer stem cells in a tumor; differentiation of tumorigenic cells into a non-tumorigenic state; an increase in progression-free survival (PFS), disease-free survival (DFS), overall survival (OS), complete response (CR), partial response (PR), stable disease (SD), a decrease in progressive disease (PD), a decrease in time to progression (TTP), or any combination thereof.

[0064] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells is characterized by unregulated cell growth. Examples of cancer include, but are not limited to, gynecological cancers (e.g., breast cancer (including triple-negative breast cancer, ductal carcinoma, ovarian cancer, and endometrial cancer), non-small cell lung cancer, pancreatic cancer, thyroid cancer, kidney cancer (e.g., renal cell carcinoma), and bladder cancer (e.g., urothelial cell carcinoma). A cancer can be a "cancer that expresses B7-H4" or a "B7-H4 expressing cancer." Such terms refer to a cancer that comprises cells that express B7-H4. A cancer can be a solid tumor that expresses B7-H4. A cancer can be a primary tumor, or it can be an advanced or metastatic cancer.

[0065] A "refractory" cancer is one that progresses even after anti-tumor therapy, such as chemotherapy, is administered to the cancer patient.

[0066] A "recurrent" cancer is one that has grown back in the original site or at a distant site after responding to initial therapy.

[0067] As used in this disclosure and in the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0068] It should be understood that when the language "comprising" is used herein to describe an embodiment, other similar embodiments described with respect to "consisting of" and / or "consisting essentially of" are also provided. In this disclosure, "comprises," "comprising," "containing," and "having," etc. may have the meanings ascribed to them under U.S. patent law, and may mean "includes," "including," etc.; "consisting essentially of" or "consists essentially of" likewise have the meanings ascribed to them under U.S. patent law, and the terms are open-ended, allowing for inclusion beyond what is recited, as long as the basic or novel features recited are not altered by the inclusion beyond what is recited, but excluding prior art embodiments.

[0069] Unless expressly stated otherwise or obvious from the context, as used herein, the term "or" is understood to be inclusive. The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both "A and B," "A or B," "A," and "B." Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0070] As used herein, the terms "about" and "approximately" when used to modify a numerical value or numerical range mean that a deviation of 5% to 10% above and 5% to 10% below the stated value or range is still within the intended meaning of the stated value or range.

[0071] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0072] 5.2 Methods for treating cancer

[0073] In one aspect, provided herein are methods for treating cancer in a human subject, comprising administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof described herein, or a pharmaceutical composition thereof as described herein.

[0074] In one aspect, a method of treating cancer in a human subject comprises administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof described herein, or a pharmaceutical composition thereof as described herein, wherein about 0.005 to about 20 mg / kg of the anti-B7-H4 antibody or antigen-binding fragment thereof is administered, for example, about once every three weeks.

[0075] In one aspect, a method of treating cancer in a human subject comprises administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition thereof as described herein, wherein, for example, about 0.005 mg / kg of the anti-B7-H4 antibody or antigen-binding fragment thereof is administered about once every three weeks. In one aspect, a method of treating cancer in a human subject comprises administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition thereof as described herein, wherein, for example, about 0.01 mg / kg of the anti-B7-H4 antibody or antigen-binding fragment thereof is administered about once every three weeks. In one aspect, a method of treating cancer in a human subject comprises administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition thereof as described herein, wherein, for example, about 0.03 mg / kg of the anti-B7-H4 antibody or antigen-binding fragment thereof is administered about once every three weeks. In one aspect, a method of treating cancer in a human subject comprises administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition thereof as described herein, wherein, for example, about 0.1 mg / kg of the anti-B7-H4 antibody or antigen-binding fragment thereof is administered about once every three weeks. In one aspect, a method of treating cancer in a human subject comprises administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition thereof as described herein, wherein, for example, about 0.3 mg / kg of the anti-B7-H4 antibody or antigen-binding fragment thereof is administered about once every three weeks. In one aspect, a method of treating cancer in a human subject comprises administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition thereof as described herein, wherein, for example, about 1 mg / kg of the anti-B7-H4 antibody or antigen-binding fragment thereof is administered about once every three weeks. In one aspect, a method of treating cancer in a human subject comprises administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition thereof as described herein, wherein, for example, about 3 mg / kg of the anti-B7-H4 antibody or antigen-binding fragment thereof is administered about once every three weeks. In one aspect, a method of treating cancer in a human subject comprises administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition thereof as described herein, wherein, for example, about 10 mg / kg of the anti-B7-H4 antibody or antigen-binding fragment thereof is administered about once every three weeks. In one aspect, a method of treating cancer in a human subject comprises administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition thereof as described herein, wherein, for example, about 20 mg / kg of the anti-B7-H4 antibody or antigen-binding fragment thereof is administered about once every three weeks.

[0076] In one aspect, a method of treating cancer in a human subject comprises administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition thereof as described herein, wherein, for example, 0.005 mg / kg of the anti-B7-H4 antibody or antigen-binding fragment thereof is administered once every three weeks. In one aspect, a method of treating cancer in a human subject comprises administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition thereof as described herein, wherein, for example, 0.01 mg / kg of the anti-B7-H4 antibody or antigen-binding fragment thereof is administered once every three weeks. In one aspect, a method of treating cancer in a human subject comprises administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition thereof as described herein, wherein, for example, 0.03 mg / kg of the anti-B7-H4 antibody or antigen-binding fragment thereof is administered once every three weeks. In one aspect, a method of treating cancer in a human subject comprises administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition thereof as described herein, wherein, for example, 0.1 mg / kg of the anti-B7-H4 antibody or antigen-binding fragment thereof is administered once every three weeks. In one aspect, a method of treating cancer in a human subject comprises administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition thereof as described herein, wherein, for example, 0.3 mg / kg of the anti-B7-H4 antibody or antigen-binding fragment thereof is administered once every three weeks. In one aspect, a method of treating cancer in a human subject comprises administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition thereof as described herein, wherein, for example, 1 mg / kg of the anti-B7-H4 antibody or antigen-binding fragment thereof is administered once every three weeks. In one aspect, a method of treating cancer in a human subject comprises administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition thereof as described herein, wherein, for example, 3 mg / kg of the anti-B7-H4 antibody or antigen-binding fragment thereof is administered once every three weeks. In one aspect, a method of treating cancer in a human subject comprises administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition thereof as described herein, wherein, for example, 10 mg / kg of the anti-B7-H4 antibody or antigen-binding fragment thereof is administered once every three weeks. In one aspect, a method of treating cancer in a human subject comprises administering to a subject in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition thereof as described herein, wherein, for example, 20 mg / kg of the anti-B7-H4 antibody or antigen-binding fragment thereof is administered once every three weeks.

[0077] According to the methods provided herein, the anti-B7-H4 antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the anti-B7-H4 antibody or antigen-binding fragment thereof, can be administered intravenously.

[0078] In a certain embodiment, provided herein is a method for treating a cancer selected from the group consisting of: breast cancer (e.g., advanced breast cancer, triple-negative breast cancer, or ductal carcinoma), endometrial cancer, ovarian cancer, urothelial carcinoma, non-small cell lung cancer (e.g., squamous cell carcinoma), pancreatic cancer, thyroid cancer, kidney cancer (e.g., renal cell carcinoma), and bladder cancer (e.g., urothelial cell carcinoma). In a certain embodiment, provided herein is a method for treating advanced breast cancer (including triple-negative breast cancer), ovarian cancer, endometrial cancer, or urothelial cancer. In a certain embodiment, provided herein is a method for treating breast cancer. In a certain embodiment, provided herein is a method for treating hormone receptor (HR) positive breast cancer. In a certain embodiment, provided herein is a method for treating ovarian cancer. In a certain embodiment, provided herein is a method for treating endometrial cancer. In a certain embodiment, provided herein is a method for treating urothelial carcinoma. In a certain embodiment, the subject has not received prior therapy with a PD-1 / PD-L1 antagonist. In certain embodiments, such methods comprise administering to a patient (e.g., a human patient) in need thereof an anti-B7-H4 antibody or antigen-binding fragment thereof provided herein, or a pharmaceutical composition provided herein comprising an anti-B7-H4 antibody or antigen-binding fragment thereof.

[0079] In some embodiments, the cancer is a B7-H4 expressing cancer. In certain embodiments, the cancer is a solid tumor that expresses B7-H4. In certain embodiments, B7-H4 has been detected in a biological sample obtained from a subject (e.g., using immunohistochemistry (IHC)).

[0080] The biological sample can be any biological sample obtained from a subject, cell line, tissue, or other cell source that potentially expresses B7-H4. Methods for obtaining tissue biopsies and body fluids from humans are well known in the art. Biological samples include peripheral blood mononuclear cells. The biological sample can also be a blood sample, in which circulating tumor cells (or "CTCs") can express B7-H4 and be detected.

[0081] Determination of the expression level of a B7-H4 protein is intended to include qualitatively or quantitatively measuring or estimating the level of a B7-H4 protein in a first biological sample, either directly (e.g., by determining or estimating absolute protein levels) or relatively (e.g., by comparing to protein levels in a second biological sample). The level of B7-H4 polypeptide expression in the first biological sample can be measured or estimated and compared to a standard B7-H4 protein level, determined from a second biological sample that is not diseased or by averaging levels from a population of non-diseased samples. As will be understood in the art, once the "standard" B7-H4 polypeptide level is known, it can be repeatedly used as a standard for comparison.

[0082] In another embodiment, an anti-B7-H4 antibody or antigen-binding fragment thereof or pharmaceutical composition is administered to a patient (e.g., a human patient) diagnosed with cancer to increase T cells, CD4 T cells, and T cells in the patient. + T cells or CD8 + Proliferation of T cells. In another embodiment, an anti-B7-H4 antibody, or an antigen-binding fragment thereof, or a pharmaceutical composition is administered to a patient (e.g., a human patient) diagnosed with cancer to increase interferon-γ (IFNγ) production in the patient. In another embodiment, an anti-B7-H4 antibody, or an antigen-binding fragment thereof, or a pharmaceutical composition is administered to a patient (e.g., a human patient) diagnosed with cancer to block the inhibitory activity of B7-H4 against T cells in the patient. In another embodiment, an anti-B7-H4 antibody, or an antigen-binding fragment thereof, or a pharmaceutical composition is administered to a patient (e.g., a human patient) diagnosed with cancer to deplete B7-H4-expressing cancer cells in the patient.

[0083] In some embodiments, the present invention relates to an anti-B7-H4 antibody or antigen-binding fragment thereof or a pharmaceutical composition provided herein for use as a medicament, wherein the medicament is for administration at about 0.005 mg / kg to about 20 mg / kg (e.g., about 0.005 mg / kg, about 0.01 mg / kg, about 0.03 mg / kg, about 0.1 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 3 mg / kg, about 10 mg / kg, or about 20 mg / kg) of the antibody or antigen-binding fragment thereof. In some aspects, the present invention relates to an antibody or antigen-binding fragment thereof, or a pharmaceutical composition provided herein, for use in a method of treating cancer, wherein about 0.005 mg / kg to about 20 mg / kg (e.g., about 0.005 mg / kg, about 0.01 mg / kg, about 0.03 mg / kg, about 0.1 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 3 mg / kg, about 10 mg / kg, or about 20 mg / kg) of the antibody or antigen-binding fragment thereof is administered. In some aspects, the present invention relates to an antibody or antigen-binding fragment thereof, or a pharmaceutical composition provided herein, for use in a method of treating cancer in a subject, comprising administering to the subject about 0.005 mg / kg to about 20 mg / kg (e.g., about 0.005 mg / kg, about 0.01 mg / kg, about 0.03 mg / kg, about 0.1 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 3 mg / kg, about 10 mg / kg, or about 20 mg / kg) of an antibody or antigen-binding fragment thereof, or a pharmaceutical composition provided herein.

[0084] In some embodiments, the present invention relates to an anti-B7-H4 antibody or antigen-binding fragment thereof or pharmaceutical composition provided herein for use as a medicament, wherein the medicament is for administration at 0.005 mg / kg to 20 mg / kg (e.g., 0.005 mg / kg, 0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 10 mg / kg, or 20 mg / kg) of the antibody or antigen-binding fragment thereof. In some aspects, the present invention relates to an antibody or antigen-binding fragment thereof or pharmaceutical composition provided herein for use in a method of treating cancer, wherein 0.005 mg / kg to 20 mg / kg (e.g., 0.005 mg / kg, 0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 10 mg / kg, or 20 mg / kg) of the antibody or antigen-binding fragment thereof is administered. In some aspects, the present invention relates to an antibody or antigen-binding fragment thereof, or a pharmaceutical composition provided herein, for use in a method of treating cancer in a subject, the method comprising administering to the subject 0.005 mg / kg to 20 mg / kg (e.g., 0.005 mg / kg, 0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 10 mg / kg or 20 mg / kg) of an antibody or antigen-binding fragment thereof, or a pharmaceutical composition provided herein.

[0085] 5.3B7-H4 Antibody and Antigen-Binding Fragments

[0086] Provided herein are methods for treating cancer in a human subject, comprising administering to the subject an antibody (e.g., a monoclonal antibody, such as a chimeric, humanized, or human antibody) and antigen-binding fragments thereof that specifically bind to B7-H4 (e.g., human B7-H4). Exemplary B7-H4 antibodies and antigen-binding fragments thereof that can be used in the methods provided herein are known in the art. The amino acid sequences of human, cynomolgus monkey, murine, and rat B7-H4 are known in the art and are also provided herein, represented by SEQ ID NOs: 1-4, respectively.

[0087] Human B7-H4:

[0088] MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKASLCVSSFFAISWALLPLSPYLMLK(SEQ ID NO:1)

[0089] Cynomolgus monkey B7 - H4:

[0090] MASLGQILFWSIISIIFILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVIGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKASLCVSSFLAISWALLPLAPYLMLK(SEQ ID NO:2)

[0091] Murine B7 - H4

[0092] MASLGQIIFWSIINIIIILAGAIALIIGFGISGKHFITVTTFTSAGNIGEDGTLSCTFEPDIKLNGIVIQWLKEGIKGLVHEFKEGKDDLSQQHEMFRGRTAVFADQVVGNASLRLKNVQLTDAGTYTCYIRTSKGKGNANL EYKTGAFSMPEINVDYNASSESLRCEAPRWFPQPTVASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTDSEVKRRSQLQLLNSGPSPCVFSSAFVAGWALLSLSCCLMLR(SEQ ID NO:3)

[0093] Rat B7-H4

[0094] MASLGQIIFWSIINVIIILAGAIVLIIGFGISGKHFITVTTFTSAGNIGEDGTLSCTFEPDIKLNGIVIQWLKEGIKGLVHEFKEGKDDLSQQHEMFRGRTAVFADQVVVGNASLRLKNVQLTDAGTYTCYIHTSKGKGNANL EYKTGAFSMPEINVDYNASSESLRCEAPRWFPQPTVASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTDSEVKRRSQLELLNSGPSPCVSSVSAAGWALLSLSCLMLR(SEQ ID NO:4)

[0095] In certain embodiments, the antibodies or antigen-binding fragments thereof used in the methods described herein specifically bind to human B7-H4. In certain embodiments, the antibodies or antigen-binding fragments thereof used in the methods described herein specifically bind to human and cynomolgus monkey B7-H4. In certain embodiments, the antibodies or antigen-binding fragments thereof used in the methods described herein specifically bind to human, murine, and rat B7-H4. In certain embodiments, the antibodies or antigen-binding fragments thereof used in the methods described herein specifically bind to human, cynomolgus monkey, murine, and rat B7-H4.

[0096] B7-H4 contains an IgC extracellular domain (amino acids 153-241 of SEQ ID NO: 1) and an IgV extracellular domain (amino acids 35-146 of SEQ ID NO: 1). In certain embodiments, the antibodies or antigen-binding fragments thereof used in the methods described herein specifically bind to the IgV domain of human B7-H4. Thus, provided herein are methods of administering an antibody or antigen-binding fragment thereof comprising a polypeptide that specifically binds to amino acids 35-146 of SEQ ID NO: 1.

[0097] In certain embodiments, the antibodies or antigen-binding fragments thereof used in the methods described herein specifically bind to human B7-H4 and comprise the six CDRs of the 20502 antibody as listed in Tables 1 and 2. "20502" refers to the 20502 antibody described herein.

[0098] Table 1. VH CDR amino acid sequences 1

[0099]

[0100] 1 The VH CDRs in Table 1 are defined according to Kabat.

[0101] Table 2. VL CDR amino acid sequences 2

[0102]

[0103] 2 The VL CDRs in Table 2 were determined according to Kabat.

[0104] In certain embodiments, the antibody or antigen-binding fragment thereof used in the methods described herein specifically binds to human B7-H4 and comprises the VH of the 20502 antibody listed in Table 3.

[0105] Table 3: Variable heavy chain (VH) amino acid sequence

[0106]

[0107] In certain embodiments, the antibody or antigen-binding fragment thereof used in the methods described herein specifically binds to human B7-H4 and comprises the VL of 20502 listed in Table 4.

[0108] Table 4: Variable light chain (VL) amino acid sequence

[0109]

[0110] In certain embodiments, the antibody or antigen-binding fragment thereof used in the methods described herein specifically binds to human B7-H4 and comprises the VH and VL of the 20502 antibody listed in Tables 3 and 4.

[0111] In certain embodiments, the antibody or antigen-binding fragment thereof used in the methods described herein specifically binds to human B7-H4 and comprises the VH framework region of the 20502 antibody listed in Table 5.

[0112] Table 5. VH FR amino acid sequences 3

[0113]

[0114] 3 The VH framework regions described in Table 5 are defined based on the boundaries of the Kabat numbering system for the CDRs. Thus, the VH CDRs are defined by Kabat, and the framework regions are the amino acid residues in the variable region surrounding the CDRs in the format FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0115] In certain embodiments, the antibody or antigen-binding fragment thereof used in the methods described herein specifically binds to human B7-H4 and comprises the VL framework region of the 20502 antibody listed in Table 6.

[0116] Table 6. VL FR amino acid sequence 4

[0117]

[0118] 4 The VL framework regions described in Table 6 are defined based on the boundaries of the Kabat numbering system for the CDRs. Thus, the VL CDRs are defined by Kabat, and the framework regions are the amino acid residues in the variable region surrounding the CDRs in the format FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0119] In certain embodiments, the antibodies or antigen-binding fragments thereof used in the methods described herein specifically bind to human B7-H4 and comprise the four VH framework regions and four VL framework regions of the 20502 antibody listed in Tables 5 and 6.

[0120] In certain embodiments, the antibody or antigen-binding fragment thereof used in the methods described herein specifically binds to human B7-H4 and comprises the heavy chain sequence of the 20502 antibody listed in Table 7.

[0121] Table 7: Full-length heavy chain amino acid sequence

[0122]

[0123] In certain embodiments, the antibody or antigen-binding fragment thereof used in the methods described herein specifically binds to human B7-H4 and comprises the light chain sequence of the 20502 antibody listed in Table 8.

[0124] Table 8: Full-length light chain amino acid sequences

[0125]

[0126] In certain embodiments, the antibody or antigen-binding fragment used in the methods described herein specifically binds to human B7-H4 and comprises the heavy chain sequence and light chain sequence of the 20502 antibody listed in Tables 7 and 8.

[0127] In certain aspects, the antibodies or antigen-binding fragments thereof used in the methods described herein are described by their individual VL domains, or their individual VH domains, or their individual three VL CDRs, or their individual three VH CDRs. See, for example, Rader C et al., (1998) PNAS 95:8910-8915, which is incorporated herein by reference in its entirety, which describes humanization of mouse anti-αvβ3 antibodies by identifying complementary light or heavy chains from a library of human light or heavy chains, respectively, to generate humanized antibody variants with an affinity as high as or greater than that of the original antibody. See also Clackson T et al., (1991) Nature 352:624-628, which is incorporated herein by reference in its entirety, which describes methods for generating antibodies that specifically bind to a specific antigen by using a specific VL domain (or VH domain) and screening the library for a complementary VH domain or (VL domain). By ELISA, the screening yielded 14 new partners for specific VH domains and 13 new partners for specific VL domains that were strong binders as determined by ELISA. See also Kim SJ and Hong HJ, (2007) J Microbiol 45:572-577, which is incorporated herein by reference in its entirety, describing a method for generating antibodies that specifically bind to a specific antigen by using a specific VH domain and screening a library (e.g., a human VL library) for complementary VL domains; the selected VL domains can then be used to guide the selection of other complementary (e.g., human) VH domains.

[0128] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be identified according to the Chothia numbering scheme, which refers to the positions of immunoglobulin structural loops (see, e.g., Chothia C and Lesk AM, (1987), J Mol Biol 196:901-917; Al-Lazikani B et al., (1997) J Mol Biol 273:927-948; Chothia C et al., (1992) J Mol Biol 227:799-817; Tramontano A et al., (1990) J Mol Biol 215(1):175-82; and U.S. Pat. No. 7,709,226). Generally, when using the Kabat numbering convention, the Chothia CDR-H1 loop is present at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop is present at heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop is present at heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop is present at light chain amino acids 24 to 34, the Chothia CDR-L2 loop is present at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop is present at light chain amino acids 89 to 97. When numbered using the Kabat numbering convention, the ends of the Chothia CDR-H1 loop vary between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if either 35A or 35B is absent, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34).

[0129] In certain aspects, provided herein are methods of administering antibodies and antigen-binding fragments thereof that specifically bind to B7-H4 (e.g., human B7-H4) and comprise the Chothia VH and VL CDRs of the 20502 antibody listed in Tables 3 and 4. In certain embodiments, provided herein are methods of administering antibodies or antigen-binding fragments thereof that specifically bind to B7-H4 (e.g., human B7-H4) and comprise one or more CDRs in which the Chothia and Kabat CDRs have the same amino acid sequence. In certain embodiments, provided herein are methods of administering antibodies and antigen-binding fragments thereof that specifically bind to B7-H4 (e.g., human B7-H4) and comprise a combination of Kabat CDRs and Chothia CDRs.

[0130] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the IMGT numbering system as described in Lefranc MP, (1999) The Immunologist 7: 132-136 and Lefranc MP et al., (1999) Nucleic Acids Res 27: 209-212. According to the IMGT numbering scheme, VH-CDR1 is located at positions 26 to 35, VH-CDR2 is located at positions 51 to 57, VH-CDR3 is located at positions 93 to 102, VL-CDR1 is located at positions 27 to 32, VL-CDR2 is located at positions 50 to 52, and VL-CDR3 is located at positions 89 to 97. In a specific embodiment, provided herein are methods of administering antibodies and antigen-binding fragments thereof that specifically bind to B7-H4 (e.g., human B7-H4) and comprise the IMGT VH and VL CDRs of the 20502 antibody listed in Tables 3 and 4, e.g., as described in Lefranc MP (1999) supra and Lefranc MP et al., (1999) supra).

[0131] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to MacCallum RM et al., (1996) J Mol Biol 262:732-745. See also, for example, Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In a specific embodiment, provided herein are methods of administering an antibody or antigen-binding fragment thereof that specifically binds to B7-H4 (e.g., human B7-H4) and comprises the VH and VL CDRs of the 20502 antibody listed in Tables 3 and 4 as determined by the method of MacCallum RM et al.

[0132] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the AbM numbering scheme, which refers to the AbM hypervariable regions that represent a compromise between Kabat CDRs and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). In a specific embodiment, provided herein are methods of administering an antibody or antigen-binding fragment thereof that specifically binds to B7-H4 (e.g., human B7-H4) and comprises the VH and VL CDRs of the 20502 antibody listed in Tables 3 and 4 as determined by the AbM numbering scheme.

[0133] In specific aspects, provided herein are methods of administering an antibody comprising a heavy chain and a light chain.

[0134] Regarding light chains, in a specific embodiment, the light chains of the antibodies described herein are kappa light chains. The constant region of a human kappa light chain may comprise the following amino acid sequence:

[0135] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 23).

[0136] The constant region of the human kappa light chain can be encoded by the following nucleotide sequence:

[0137] CGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT(SEQ ID NO:24).

[0138] In a specific embodiment, the antibody that immunospecifically binds to a B7-H4 polypeptide (e.g., human B7-H4) for use in the methods described herein comprises a light chain wherein the amino acid sequence of the VL domain comprises a sequence listed in Table 4, and wherein the constant region of the light chain comprises the amino acid sequence of a human kappa light chain constant region.

[0139] In a specific embodiment, the antibody that immunospecifically binds to B7-H4 (e.g., human B7-H4) for use in the methods described herein comprises a heavy chain wherein the amino acid sequence of the VH domain comprises the amino acid sequence listed in Table 3, and wherein the constant region of the heavy chain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region.

[0140] The constant region of a human IgG1 heavy chain may comprise the following amino acid sequence:

[0141] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:25).

[0142] The constant region of the human IgG1 heavy chain can be encoded by the following nucleotide sequence:

[0143] GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA。(SEQ ID NO: 26).

[0144] In a specific embodiment, the antibodies that immunospecifically bind to B7-H4 (e.g., human B7-H4) used in the methods described herein comprise a VH domain and a VL domain comprising the amino acid sequences of any of the VH and VL domains described herein, and wherein the constant region comprises the amino acid sequence of a constant region of an IgG (e.g., human IgG) immunoglobulin molecule. In another specific embodiment, the antibodies that immunospecifically bind to B7-H4 (e.g., human B7-H4) used in the methods described herein comprise a VH domain and a VL domain comprising the amino acid sequences of any of the VH and VL domains described herein, and wherein the constant region comprises the amino acid sequence of a constant region of an IgG1 (e.g., human IgG1) immunoglobulin molecule.

[0145] It has been reported that antibodies with reduced fucose content have increased affinity for Fc receptors, such as FcγRIIIA. Therefore, in certain embodiments, the antibodies or antigen-binding fragments thereof used in the methods described herein have reduced fucose content or lack fucose (i.e., "no fucosylation"). Such antibodies or antigen-binding fragments thereof can be produced using techniques known to those skilled in the art. For example, they can be expressed in cells lacking or lacking fucosylation ability. In one specific example, a cell line with two alleles knocked out of the α1,6-fucosyltransferase gene (FUT8) can be used to produce antibodies or antigen-binding fragments thereof with reduced fucose content. The Lonza system is an example of a system that can be used to produce antibodies and antigen-binding fragments thereof with reduced fucose content. Alternatively, antibodies or antigen-binding fragments thereof with reduced or no fucose content can be produced, for example, by: (i) culturing cells under conditions that prevent or reduce fucosylation; (ii) removing fucose post-translationally (e.g., using a fucosidase); (iii) adding the desired carbohydrate post-translationally, for example, after recombinant expression of a non-glycosylated glycoprotein; or (iv) purifying the glycoprotein to select for non-fucosylated antibodies or antigen-binding fragments thereof. For methods for producing antibodies with no or reduced fucose content, see, for example, Longmore GD and Schachter H (1982) Carbohydr Res 100: 365-92 and Imai-Nishiya H et al., (2007) BMC Biotechnol. 7: 84.

[0146] In some embodiments, the afucosylated B7-H4 antibody or antigen-binding fragment thereof has enhanced ADCC activity in vitro compared to a fucosylated B7-H4 antibody or antigen-binding fragment thereof having the same amino acid sequence. In some embodiments, the afucosylated B7-H4 antibody or antigen-binding fragment thereof causes specific lysis that is at least 10, at least 15, at least 20, at least 25, at least 3, at least 35, at least 40, at least 45, at least 50, at least 60, at least 65, at least 70, or at least 75 percentage points higher than the specific lysis achieved using the fucosylated B7-H4 antibody. Specific lysis can be determined as described in Example 2 herein.

[0147] In some embodiments, the B7-H4 antibody or antigen-binding fragment thereof has enhanced affinity for FcγRIIIA compared to a fucosylated B7-H4 antibody or antigen-binding fragment thereof having the same amino acid sequence. In some embodiments, the afucosylated B7-H4 antibody or antigen-binding fragment thereof binds to FcγRIIIA with an affinity that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, at least 10-fold, at least 12-fold, at least 15-fold, at least 17-fold, or at least 20-fold greater than that of the fucosylated B7-H4 antibody or antigen-binding fragment thereof. In some embodiments, the affinity for FcγRIIIA is determined using surface plasmon resonance. In some embodiments, the FcγRIIIA is selected from FcγRIIIA(V158) and FcγRIIIA(F158). In some embodiments, the FcγRIIIA is FcγRIIIA(V158).

[0148] In some embodiments, the presence of fucose can be determined by methods including high performance liquid chromatography (HPLC), capillary electrophoresis, or MALDI-TOF mass spectrometry.

[0149] In specific embodiments, the antibody or antigen-binding fragment thereof (i) comprises the CDR sequences of 20502, the VH and VL sequences of 20502, or the heavy and light chain sequences of 20502, and (ii) is afucosylated.

[0150] In a specific embodiment, the composition comprises an antibody or antigen-binding fragment thereof that (i) comprises a CDR sequence of 20502, a VH and VL sequence of 20502, or a heavy chain and light chain sequence of 20502, and (ii) is afucosylated, e.g., wherein at least 95% of the antibodies in the composition are afucosylated, or wherein fucosylation is not detectable in the composition.

[0151] Engineered glycoforms can be used for a variety of purposes, including but not limited to enhancing or reducing effector function. Methods for generating engineered glycoforms in the antibodies or antigen-binding fragments thereof described herein include, but are not limited to, those disclosed in, for example: P et al., (1999) Nat Biotechnol 17:176-180; Davies J et al., (2001) Biotechnol Bioeng 74:288-294; Shields RL et al., (2002) J Biol Chem 277:26733-26740; Shinkawa T et al., (2003) J Biol Chem 278:3466-3473; Niwa R et al., (2004) Clin Cancer Res 1:6248-6255; Presta LG et al., (2002) Biochem Soc Trans 30:487-490; Kanda Y et al., (2007) Glycobiology 17:104-118; U.S. Patent Nos. 6,602,684; 6,946,292; and 7,214,775; U.S. Patent Publication Nos. US 2007 / 0248600; 2007 / 0178551; 2008 / 0060092; and 2006 / 0253928; International Publication Nos. WO 00 / 61739; WO 01 / 292246; WO 02 / 311140; and WO 02 / 30954; Potelligent TM Technology (Biowa, Inc. Princeton, NJ); and Glycosylation engineering technology (Glycartbiotechnology AG, Zurich, Switzerland). See also, for example, Ferrara C et al., (2006) Biotechnol Bioeng 93:851-861; International Publication Nos. WO 07 / 039818; WO 12 / 130831; WO 99 / 054342; WO 03 / 011878; and WO 04 / 065540.

[0152] In certain embodiments, any of the constant region mutations or modifications described herein can be introduced into one or both heavy chain constant regions of an antibody or antigen-binding fragment thereof described herein having two heavy chain constant regions.

[0153] In another specific embodiment, an antibody or antigen-binding fragment thereof described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises a VH domain comprising the VH CDR1, VL CDR2, and VL CDR3 amino acid sequences of the 20502 antibody listed in Table 1; (ii) the light chain comprises a VL domain comprising the VL CDR1, VH CDR2, and VH CDR3 amino acid sequences of the 20502 antibody listed in Table 2; (iii) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of a constant domain of a human IgG1 heavy chain; and (iv) the light chain further comprises a constant light chain domain comprising the amino acid sequence of a constant domain of a human kappa light chain.

[0154] In another specific embodiment, an antibody or antigen-binding fragment thereof described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises a VH domain comprising the amino acid sequence of the VH domain of the 20502 antibody listed in Table 3; (ii) the light chain comprises a VL domain comprising the amino acid sequence of the VL domain of the 20502 antibody listed in Table 4; (iii) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of a constant domain of a human IgG1 heavy chain; and (iv) the light chain further comprises a constant light chain domain comprising the amino acid sequence of a constant domain of a human kappa light chain.

[0155] In specific embodiments, antibodies or antigen-binding fragments thereof that immunospecifically bind to B7-H4 (e.g., human B7-H4) as described herein exhibit T cell checkpoint blocking activity. In specific embodiments, antibodies or antigen-binding fragments thereof that immunospecifically bind to B7-H4 (e.g., human B7-H4) as described herein increase interferon-γ (IFNγ) production in T cells. In specific embodiments, antibodies or antigen-binding fragments thereof that immunospecifically bind to B7-H4 (e.g., human B7-H4) as described herein increase T cell proliferation. In specific embodiments, antibodies or antigen-binding fragments thereof that immunospecifically bind to B7-H4 (e.g., human B7-H4) as described herein increase CD4+ T cell proliferation. In specific embodiments, antibodies or antigen-binding fragments thereof that immunospecifically bind to B7-H4 (e.g., human B7-H4) as described herein increase CD8+ T cell proliferation.

[0156] In specific embodiments, the antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to B7-H4 (e.g., human B7-H4) exhibit antibody-dependent cellular cytotoxicity (ADCC) activity. In specific embodiments, the antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to B7-H4 (e.g., human B7-H4) exhibit antibody-dependent cellular cytotoxicity (ADCC) activity on a cell line (e.g., SK-BR-3 cells) that has at least 300,000 cell surface B7-H4 molecules. In specific embodiments, the antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to B7-H4 (e.g., human B7-H4) exhibit antibody-dependent cellular cytotoxicity (ADCC) activity on a cell line (e.g., HCC1569 cells) that has at least 100,000 cell surface B7-H4 molecules. In specific embodiments, the antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to B7-H4 (e.g., human B7-H4) exhibit antibody-dependent cellular cytotoxicity (ADCC) activity on a cell line having at least 50,000 cell surface B7-H4 molecules (e.g., ZR-75-1 cells). In specific embodiments, the antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to B7-H4 (e.g., human B7-H4) exhibit antibody-dependent cellular cytotoxicity (ADCC) activity on a cell line having at least 30,000 cell surface B7-H4 molecules (e.g., MDA-MB-468 cells). In specific embodiments, the antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to B7-H4 (e.g., human B7-H4) exhibit antibody-dependent cellular cytotoxicity (ADCC) activity on a cell line having at least 15,000 cell surface B7-H4 molecules (e.g., HCC1964 cells).

[0157] In a specific aspect, an antigen-binding fragment as described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) is selected from the group consisting of: Fab, Fab', F(ab')2, and scFv, wherein the Fab, Fab', F(ab')2, or scFv comprises a heavy chain variable region sequence and a light chain variable region sequence of an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein. Fab, Fab', F(ab')2, or scFv can be produced by any technique known to those skilled in the art. In certain embodiments, the Fab, Fab', F(ab')2, or scFv further comprises a moiety that extends the half-life of the antibody in vivo. Such moieties are also referred to as "half-life extending moieties." Any moiety known to those skilled in the art for extending the half-life of a Fab, Fab', F(ab')2, or scFv in vivo can be used. For example, the half-life extending moiety may comprise an Fc region, a polymer, albumin, or an albumin-binding protein or compound. The polymer may include natural or synthetic, optionally substituted linear or branched polyalkylenes, polyalkenylenes, polyoxyalkylenes, polysaccharides, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, methoxypolyethylene glycol, lactose, amylose, dextran, glycogen or derivatives thereof. The substituent may include one or more hydroxyl groups, methyl groups or methoxy groups. In certain embodiments, Fab, Fab', F(ab')2 or scFv may be modified by adding one or more C-terminal amino acids for connecting a portion that extends the half-life. In certain embodiments, the portion that extends the half-life is polyethylene glycol or human serum albumin. In certain embodiments, Fab, Fab', F(ab')2 or scFv is fused to the Fc region.

[0158] 5.4 Pharmaceutical Compositions

[0159] Provided herein are methods for administering compositions comprising an anti-B7-H4 antibody or antigen-binding fragment thereof of the desired purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed. (See, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)). Compositions to be administered in vivo can be sterile. This is readily accomplished, for example, by filtration through a sterile filtration membrane.

[0160] In some embodiments, methods of administering a pharmaceutical composition are provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier. In specific embodiments, methods of administering a pharmaceutical composition are provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment, for example, wherein at least 80% of the antibodies in the composition are afucosylated. In specific embodiments, methods of administering a pharmaceutical composition are provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment, for example, wherein at least 85% of the antibodies in the composition are afucosylated. In specific embodiments, methods of administering a pharmaceutical composition are provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment, for example, wherein at least 90% of the antibodies in the composition are afucosylated. In specific embodiments, methods of administering a pharmaceutical composition are provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment, for example, wherein at least 95% of the antibodies in the composition are afucosylated. In a specific embodiment, a method of administering a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment, e.g., wherein at least 96% of the antibodies in the composition are afucosylated. In a specific embodiment, a method of administering a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment, e.g., wherein at least 97% of the antibodies in the composition are afucosylated. In a specific embodiment, a method of administering a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment, e.g., wherein at least 98% of the antibodies in the composition are afucosylated. In a specific embodiment, a method of administering a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment, e.g., wherein at least 99% of the antibodies in the composition are afucosylated. In specific embodiments, methods of administering a pharmaceutical composition are provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment, wherein fucose is not detectable in the composition.

[0161] In some embodiments, methods of administering a pharmaceutical composition are provided, wherein the pharmaceutical composition comprises (i) an isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, the antibody or antigen-binding fragment thereof comprising (a) the heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3, and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-10, respectively, (b) a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 11 and a variable light chain region comprising the amino acid sequence of SEQ ID NO: 12, or (c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a light chain comprising the amino acid sequence of SEQ ID NO: 22; and (ii) a pharmaceutically acceptable excipient.

[0162] Also provided herein are methods of administering a pharmaceutical composition, wherein the pharmaceutical composition comprises (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and comprises the heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3, and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-10, respectively, and (ii) a pharmaceutically acceptable excipient, wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the antibody or antigen-binding fragment thereof in the composition is afucosylated. In one embodiment, (i) the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 11 and a variable light chain region comprising the amino acid sequence of SEQ ID NO: 12, or (ii) the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a light chain comprising the amino acid sequence of SEQ ID NO: 22.

[0163] 5.5 Antibody Production and Polynucleotides

[0164] Antibodies and antigen-binding fragments thereof that immunospecifically bind to B7-H4 (e.g., human B7-H4) can be produced by any method known in the art for synthesizing antibodies and antigen-binding fragments thereof, such as by chemical synthesis or by recombinant expression techniques. Unless otherwise indicated, the methods described herein employ conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. Such techniques are described, for example, in the references cited herein and are fully described in the literature. See, for example, Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates); annual updates) Gait (editor) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (editor) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (editor) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0165] In certain aspects, provided herein are methods of administering an anti-B7-H4 antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising such an antibody or fragment, wherein the antibody or fragment is produced by recombinant expression of a polynucleotide comprising a nucleotide sequence in a host cell.

[0166] In certain aspects, the anti-B7-H4 antibodies or antigen-binding fragments administered according to the methods provided herein comprise a heavy chain variable region encoded by a polynucleotide comprising the nucleotide sequence shown in Table 9 (i.e., SEQ ID NO: 27). In certain aspects, the anti-B7-H4 antibodies or antigen-binding fragments administered according to the methods provided herein comprise a heavy chain variable region encoded by a polynucleotide comprising the nucleotide sequence shown in Table 9 (i.e., SEQ ID NO: 27) and a nucleotide sequence encoding a human gamma (γ) heavy chain constant region. In certain aspects, the anti-B7-H4 antibodies or antigen-binding fragments administered according to the methods provided herein comprise a heavy chain variable region encoded by a polynucleotide comprising the nucleotide sequence shown in Table 9 (i.e., SEQ ID NO: 27) and a heavy chain constant domain encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 26.

[0167] Table 9: Polynucleotide sequences encoding heavy chain variable regions

[0168]

[0169] In certain aspects, the anti-B7-H4 antibodies or antigen-binding fragments administered according to the methods provided herein comprise a light chain variable region encoded by a polynucleotide comprising the nucleotide sequence shown in Table 10 (i.e., SEQ ID NO: 28). In certain aspects, the anti-B7-H4 antibodies or antigen-binding fragments administered according to the methods provided herein comprise a light chain variable region encoded by a polynucleotide comprising the nucleotide sequence shown in Table 10 (i.e., SEQ ID NO: 28) and a nucleotide sequence encoding a human lambda light chain constant region. In certain aspects, the anti-B7-H4 antibodies or antigen-binding fragments administered according to the methods provided herein comprise a light chain variable region encoded by a polynucleotide comprising the nucleotide sequence shown in Table 10 (i.e., SEQ ID NO: 28) and a light chain constant domain encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 24.

[0170] Table 10: Polynucleotide sequences encoding light chain variable regions

[0171]

[0172] In certain aspects, the anti-B7-H4 antibody or antigen-binding fragment administered according to the methods provided herein comprises a variable heavy chain encoded by a polynucleotide comprising the nucleotide sequence encoding the variable heavy chain shown in Table 9 (i.e., SEQ ID NO: 27) and a variable light chain encoded by a polynucleotide comprising the nucleotide sequence encoding the variable light chain shown in Table 10 (i.e., SEQ ID NO: 28).

[0173] In certain aspects, the anti-B7-H4 antibody or antigen-binding fragment administered according to the methods provided herein comprises (i) a heavy chain encoded by a polynucleotide comprising a nucleotide sequence encoding a variable heavy chain as shown in Table 9 (i.e., SEQ ID NO: 27) and a nucleotide sequence encoding a human gamma (γ) heavy chain constant region, and (ii) a light chain encoded by a polynucleotide comprising a nucleotide sequence encoding a variable light chain as shown in Table 10 (i.e., SEQ ID NO: 28) and a nucleotide sequence encoding a human lambda light chain constant region.

[0174] In certain aspects, the anti-B7-H4 antibody or antigen-binding fragment administered according to the methods provided herein comprises (i) a heavy chain encoded by a polynucleotide comprising the nucleotide sequence encoding the variable heavy chain shown in Table 9 (i.e., SEQ ID NO: 27) and a nucleotide sequence encoding the heavy chain constant domain of SEQ ID NO: 26, and (ii) a light chain encoded by a polynucleotide comprising the nucleotide sequence encoding the variable light chain shown in Table 10 (i.e., SEQ ID NO: 28) and a nucleotide sequence encoding the light chain constant domain of SEQ ID NO: 24.

[0175] In certain aspects, the anti-B7-H4 antibodies or antigen-binding fragments administered according to the methods provided herein are encoded by optimized polynucleotides encoding the anti-B7-H4 antibodies, antigen-binding fragments thereof, or domains thereof, optimized, for example, by codon / RNA optimization, substitution with a heterologous signal sequence, and elimination of mRNA instability elements. Methods for generating optimized nucleic acids encoding anti-B7-H4 antibodies, antigen-binding fragments thereof, or domains thereof (e.g., heavy chain, light chain, VH domain, or VL domain) for recombinant expression by introducing codon changes (e.g., codon changes that encode the same amino acid due to the degeneracy of the genetic code) and / or eliminating inhibitory regions in mRNA can be performed, for example, by employing the optimization methods described in U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498.

[0176] Polynucleotides can be, for example, in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA. DNA can be double-stranded or single-stranded. If single-stranded, the DNA can be a coding strand or a non-coding (antisense) strand. In certain embodiments, the polynucleotide is a cDNA or DNA lacking one or more introns. In certain embodiments, the polynucleotide is a non-naturally occurring polynucleotide. In certain embodiments, the polynucleotide is recombinantly produced. In certain embodiments, the polynucleotide is isolated. In certain embodiments, the polynucleotide is substantially pure. In certain embodiments, the polynucleotide is purified from natural components.

[0177] In certain aspects, a vector (e.g., an expression vector) comprises a nucleotide sequence encoding an anti-B7-H4 antibody, antigen-binding fragment thereof, or domain thereof for recombinant expression in a host cell, preferably a mammalian cell. In certain aspects, a cell (e.g., a host cell) comprises such a vector for recombinant expression of an anti-B7-H4 antibody, or antigen-binding fragment thereof (e.g., a human or humanized antibody, or antigen-binding fragment thereof) described herein. Thus, methods for producing an antibody, or antigen-binding fragment thereof, described herein can comprise expressing such an antibody, or antigen-binding fragment thereof, in a host cell.

[0178] The expression vector can be transferred to cells (e.g., host cells) by conventional techniques, and the resulting cells can then be cultured by conventional techniques to produce the antibodies or antigen-binding fragments thereof described herein (e.g., antibodies or antigen-binding fragments comprising the six CDRs, VH, VL, VH and VL, heavy chains, light chains, or heavy and light chains of 20502) or domains thereof (e.g., VH, VL, VH and VL, heavy chains, or light chains of 20502).

[0179] In certain embodiments, The anti-B7-H4 antibodies or antigen-binding fragments thereof (e.g., antibodies or antigen-binding fragments thereof comprising the CDRs of 20502) administered according to the methods provided herein are produced in CHOK1SV cells.

[0180] In some embodiments, the anti-B7-H4 antibody or antigen-binding fragment thereof (e.g., an antibody or antigen-binding fragment thereof comprising the CDRs of 20502) administered according to the methods provided herein is produced in a host cell that lacks a functional α-1,6-fucosyltransferase gene (FUT8). In some embodiments, the host cell is a CHO cell.

[0181] In specific embodiments, the antibodies or antigen-binding fragments thereof administered according to the methods provided herein are isolated or purified. Typically, an isolated antibody or antigen-binding fragment thereof is one that is substantially free of other antibodies or antigen-binding fragments thereof that have a different antigenic specificity than the isolated antibody or antigen-binding fragment thereof. For example, in a specific embodiment, a preparation of an antibody or antigen-binding fragment thereof described herein is substantially free of cellular material and / or chemical precursors.

[0182] The following examples are offered by way of illustration and not by way of limitation.

[0183] 6. Examples

[0184] The examples in this section (ie, Section 6) are provided by way of illustration and not by way of limitation.

[0185] 6.1 Example 1: Assessing the Prevalence of B7-H4 Expression in Various Indications

[0186] B7-H4 mouse monoclonal antibody A57.1 (ATCC catalog number PTA-5180) was used to detect the presence of B7-H4 on archived samples, whole section pools, and tumor microarrays. Samples were treated with primary antibody and detected using a polymer detection system attached to DAB (Ventana Medical Systems).

[0187] B7-H4 was readily detected in the cell membrane and cytosol of tumor tissues harvested from patients with various cancers, including invasive ductal carcinoma, triple-negative breast cancer, ovarian cancer, non-small cell lung cancer, and endometrial cancer. Furthermore, B7-H4 was expressed at a high frequency in the indications listed in Table 11.

[0188] Table 11: B7-H4 Detection in Tumors

[0189]

[0190] B7-H4 is expressed in other cancers, such as breast cancer, kidney cancer (e.g., renal cell carcinoma), bladder cancer (e.g., urothelial cell carcinoma), pancreatic cancer, and thyroid cancer. See, e.g., Zhu, J., et al., Asian Pacific J. Cancer Prev. 14:3011-3015 (2011); Krambeck A, et al., PNAS 103:10391-10396 (2006); Fan, M., et al., Int. J. Clin. Exp. Pathol. 7:6768-6775 (2014); Xu, H., et al., Oncology Letters 11:1841-1846 (2016); and Liu, W., et al., Oncology Letters 8:2527-2534 (2014).

[0191] 6.2 Example 2: Afucosylated and Fucosylated 20502 Antibodies

[0192] Compared to fully fucosylated antibodies, antibodies with Fc regions with reduced fucose content in the glycan moiety can exhibit higher ADCC activity (Niwa R et al., Clinical Cancer Research 11(6):2327-36 (2005)). The B7-H4 antibody was produced in CHO-x cells (Yamane-Ohnuki N, et al., Biotechnology and Bioengineering 87(5):614-22 (2004)) used to produce normally fucosylated antibodies and in a CHO cell line (CHO-y cells) engineered to produce afucosylated antibody (supra).

[0193] Fucosylated and afucosylated 20502 antibodies were characterized by surface plasmon resonance (SPR). Briefly, anti-human Fab antibodies were immobilized on a carboxyl-derivatized SPR chip surface, and anti-B7-H4 antibodies were captured on the resulting surface at 5 μg / ml for 30 seconds. B7-H4 IgV-huIgG1 at various concentrations (0 nM, 3.7 nM, 11.1 nM, 33.3 nM, 100 nM, and 300 nM) was then flowed over the surface and allowed to bind to the anti-B7-H4 antibodies during the association phase, followed by a buffer wash during the dissociation phase.

[0194] B7-H4 IgV-huIgG1:

[0195] MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:29)

[0196] The data were fitted using a 1:1 binding model, and fucosylated and afucosylated 20502 showed similar binding to human B7-H4 protein. Therefore, glycosylation has no effect on binding.

[0197] The binding affinity of the Fc region of fucosylated 20502 (Ab-F) and afucosylated 20502 (Ab-A) to FcγRIIIa (V158) was also characterized by surface plasmon resonance (SPR). Briefly, protein A was covalently linked to a dextran chip using an amine coupling kit with 100mM ethylenediamine in 100mM sodium borate buffer (pH 8.0) as a blocking reagent. Ab-A or Ab-F were captured in a separate flow cell at 2 densities, and the protein A derivatization flow served as a reference control. FcγRIIIA (V158) was diluted in HBS-P+ running buffer and injected in duplicate at 6 concentrations (0nM, 1.37nM, 12.3nM, 37nM, 111nM, 333nM, and 1000nM). The association constant, dissociation constant, and affinity for Ab-A binding were calculated using the Biacore T200 evaluation software 1:1 binding model. The affinity constants for Ab-A and Ab-F binding were determined using the Biacore T200 evaluation software steady-state affinity model. The affinity of the afucosylated B7-H4 antibody for Fcγ receptor IIIA (V158) was 140-fold higher than that of the same antibody with a fucosylated Fc (Ab-F) (Table 12).

[0198] Table 12: Fcγ Receptor IIIa (FcγRIIIa) V158 Allele Binding

[0199] ka(1 / Ms) kd(1 / s) <![CDATA[K D (nM)]]> Ab-A 6.46E+05 9.54E-10 15 Ab-F N / A N / A 210

[0200] The T cell checkpoint blocking activity of fucosylated and afucosylated 20502 antibodies was also characterized. In these experiments, EasySep TM The Human T Cell Enrichment Kit enriches primary human T cells from PBMC. The enriched T cells are plated at 2x10 5 10 cells / mL were incubated with anti-CD3 / anti-CD28 Dynabeads at a ratio of one bead per cell at 37°C. After six days, the beads were magnetically removed, and the T cells were washed and plated at 1 x 10 6 T cells / mL were incubated with 10 U / mL IL-2 at 37°C. Four days later, T cells were washed and plated at 1x10 6 cells / mL and 2x10 6Artificial antigen-presenting cells (aAPCs) at a concentration of 10 cells / mL were incubated at 37°C in the presence of titrated doses of B7-H4 antibodies. aAPCs were treated with mitomycin C for one hour at 37°C and then washed extensively before being added to T cell co-cultures. 72 hours after co-culture of T cells, aAPCs, and B7-H4 antibodies, the plates were centrifuged, and the supernatants were harvested and assessed for IFNγ production by ELISA. IFNγ production was plotted against antibody concentration, and EC50 potency was calculated using nonlinear regression curve fitting (GraphPad Prism).

[0201] As measured by an increase in IFNγ production, the B7-H4 antibody showed potent T cell checkpoint blockade activity. Furthermore, there was no significant difference in potency between the afucosylated and fucosylated antibodies (Table 13).

[0202] Table 13: T cell checkpoint blockade efficacy

[0203]

[0204] In additional experiments, the ADCC activity of fucosylated and afucosylated 20502 antibodies was also characterized against target cell lines expressing B7-H4. Specifically, primary human PBMCs were cultured at 1x10 6 Cells / mL were used for cytokine activation. The next day, the cells were washed and incubated with SK-BR-3 target cells labeled with Calcein-AM at a 40:1 effector: target ratio. After 4 hours of incubation, target cell lysis was quantified using a fluorometer. Triton / X-treated samples served as maximum lysis control samples, while samples treated with culture medium alone served as background lysis control samples. Percent specific lysis (%) was calculated as follows: [1-((sample–culture medium control) / (maximum lysis–culture medium control))]x100. Percent specific lysis (%) was plotted against antibody concentration, and EC50 efficacy was calculated using nonlinear regression curve fitting (GraphPad Prism).

[0205] The B7-H4 antibody exhibited potent dose-dependent ADCC activity against the endogenous B7-H4 expressing breast cell line SK-BR-3. In addition, the afucosylated antibody exhibited significantly more potent ADCC activity compared to the fucosylated antibody (Table 14).

[0206] Table 14: ADCC activity

[0207]

[0208] 6.3 Example 3: Correlation between ADCC activity and receptor density

[0209] B7-H4 density was quantified on the surface of SK-BR-3, HCC1569, ZR-75-1, MDA-MB-48, and HCC1964 cells by FACS according to the manufacturer's instructions. 5 Incubate cells with 15 μg / mL B7-H4 antibody on ice for 25 minutes. TM Simply Cellular (QSC) microspheres (pre-coated with increasing concentrations of anti-mouse IgG capture antibody) were also incubated with 15 μg / mL B7-H4 antibody on ice for 25 minutes. After incubation, the cells and QSC microspheres were pelleted and washed, and samples were acquired on a flow cytometer. Data were analyzed using FlowJo software. The mean fluorescence intensity (MFI) was calculated and entered into A regression is automatically calculated in the spreadsheet, relating each bead's fluorescent channel value to its pre-assigned Antibody Binding Capacity (ABC) value. ABC values ​​are assigned once the MFI values ​​of the labeled cells are also added to the template.

[0210] The ADCC activity of B7-H4 antibodies against target cell lines expressing B7-H4 with different levels of B7-H4 cell surface density was evaluated. 4 SK-BR-3, HCC1569, ZR-75-1, MDA-MB-468, or HCC1964 target cells were incubated with dose-titrated B7-H4 antibodies at 4°C. After 25 minutes, single-use vials of Jurkat-huCD16 reporter cells from Promega were thawed and 7.5 x 10 4 Cells were added to the target cell / B7-H4 antibody mixture and incubated at 37°C. After 24 hours, samples were allowed to reach room temperature (RT) and incubated with Bio-Glo buffer. Substrate and luminescence were quantified on an EnVision multilabel reader. Data were plotted as luminescence versus antibody concentration, and EC50 potency was calculated using nonlinear regression curve fitting (GraphPad Prism).

[0211] B7-H4 antibody ADCC activity depends on B7-H4 cell surface density: as the number of cell surface molecules decreases, the amount of maximal ADCC activity also decreases. In addition, afucosylated antibodies exhibit improved ADCC activity compared to fucosylated antibodies, especially against target cells with lower B7-H4 cell surface density levels ( Figure 1 ).

[0212] 6.4 Example 4: In vivo anti-tumor efficacy

[0213] Unlike human tumors, mouse models do not endogenously express high levels of B7-H4 protein. To test afucosylated 20502 in mice, a syngeneic mouse cancer model was used that used cells engineered to express B7-H4 protein. Seven-week-old female BALB / c mice were purchased from Charles River Laboratories (Hollister, CA) and acclimated for up to three weeks before the start of the study. The murine colorectal cancer cell line CT26 was engineered to express a chimeric protein composed of the extracellular domain of murine B7-H4 and the transmembrane domain of murine B7H3. These tumor cells were plated at 1.0 x 10 6 Cells / 200 μL / mouse were subcutaneously implanted into the right flank of the mouse. Prior to inoculation, cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 2 mM L-glutamine for no more than three passages. Cells were grown at 37°C in a humidified atmosphere with 5% CO2. After reaching 80%-85% confluence, cells were harvested and plated at 5x10 cells / ml. 6 The cells were resuspended in a 1:1 mixture of serum-free RPMI1640 and Matrigel).

[0214] Mice were monitored for tumor growth twice a week after cell implantation. For tumor measurement, the length and width of each tumor were measured using calipers, and the volume was calculated according to the following formula: Tumor volume (mm 3 )=(width(mm)xlength(mm) 2 ) / 2. On the day of treatment initiation, all tumors were measured, outliers excluded, and mice were randomly assigned to treatment groups. For anti-B7-H4 treatment, afucosylated 20502 antibody was administered. As controls, mice were administered polyclonal human IgG (Bio X Cells, BE0092) or mouse IgG2a (Bio X Cells, BE0085). Antibodies were administered four times twice weekly via intravenous (iv) injection, starting on day 4 or 5 post-inoculation.

[0215] Continue to measure tumors at least twice a week until the tumor volume exceeds 10% of the animal's body weight or approximately 2000 mm 3 The changes in tumor size were shown by plotting individual tumors relative to the day the animals were inoculated with CT26 cells. P values ​​were calculated using unpaired two-tailed t-test analysis of the calculated tumor volumes on each day of the study.

[0216] The engineered CT26 model expressing B7-H4 protein showed significant dose-dependent tumor growth inhibition at all five dose levels ranging from 1 to 30 mg / kg ( Figure 2The most common effect in individual animals was tumor growth inhibition. However, treatment with afucosylated 20502 did result in complete tumor regression in 7 of 15 mice in the 30 mg / kg group, 6 of 15 mice in the 20 mg / kg group, and 5 of 15 mice in the 10 mg / kg group ( Figure 2 Compared to the negative control treatment group (human IgG), aglycosylated 20502 administered at 3 mg / kg or lower elicited minimal antitumor activity.

[0217] 6.5 Example 5: Nonclinical Pharmacokinetics

[0218] The pharmacokinetics (PK) and toxicokinetics (TK) of afucosylated 20502 were evaluated in mice, rats, and cynomolgus monkeys following weekly and / or repeated intravenous (IV) administration. The PK profile observed was consistent across all studies. In all species, afucosylated 20502 exhibited linear PK, with a dose-proportional increase in exposure (area under the serum concentration-time curve [AUC]) with increasing dose. After 4 weekly doses of 20502 between the first and last doses, weekly exposure (AUC 0-7天 ) increased approximately 2-fold; however, steady state was not reached. There were no significant sex differences in the serum afucosylated 20502 concentration-time profiles. In cynomolgus monkeys (across 2 different studies), the half-life estimated from recovered animals ranged from approximately 8.8 days to 12 days at dose levels ranging from 1 to 100 mg / kg. The estimated half-life in rats following administration of a single intravenous infusion of 40 mg / kg was approximately 13.2 days. The PK profile of afucosylated 20502 in animals supports a once every 3 weeks (Q3W) dosing regimen for IV infusion in humans.

[0219] 6.6 Example 6: Toxicology

[0220] Toxicology studies of afucosylated 20502 were conducted in rats and cynomolgus monkeys. These studies included a preliminary single-dose pharmacokinetic (PK) / tolerability study in rats, a preliminary repeat-dose toxicity study in cynomolgus monkeys, and an Investigational New Drug (IND)-enabled Good Laboratory Practice (GLP) repeat-dose toxicity study in rats and cynomolgus monkeys, as well as a GLP tissue cross-reactivity study using human, rat, and cynomolgus monkey tissues.

[0221] In a single-dose preliminary tolerability study in rats, animals received doses up to 40 mg / kg as a 30-minute intravenous (IV) infusion. Afucosylated 20502 had no effect on clinical observations, body weight, food consumption, clinical pathology (serum chemistry or hematology) assessments, overall observations, organ weights, or histopathology assessments.

[0222] In a preliminary repeated-dose toxicology study, cynomolgus monkeys received doses of up to 100 mg / kg of intravenous non-glycosylated 20502 four times weekly as a 30-minute intravenous infusion. All doses were well tolerated by cynomolgus monkeys. There were no unplanned mortalities related to the test article or changes attributable to the administration of fucosylated 20502 during the evaluation of clinical observations, body weights, clinical pathology, necropsy, organ weights, or histopathology parameters.

[0223] In a repeated dose GLP toxicology study, afucosylated 20502 was administered intravenously to rats and cynomolgus monkeys at dose levels of 1, 10, and 100 mg / kg / dose, four times weekly. The reversibility of toxicity was evaluated during a 6-week recovery period after the final administration. Evaluation parameters included ophthalmological examination, clinical observations, body temperature, body weight, food consumption, hematology, coagulation, clinical chemistry, urinalysis, organ weights, macroscopic and microscopic evaluations. In the cynomolgus monkey study, an electrocardiogram (ECG) was also evaluated to assess potential cardiotoxicity.

[0224] During evaluation in the GLP rat study, afucosylated 20502 was generally well tolerated, and no toxic effects were attributed to afucosylated 20502. The no observed adverse effect level (NOAEL) in Sprague Dawley rats was considered to be 100 mg / kg / dose.

[0225] In the GLP cynomolgus monkey study, afucosylated 20502 was generally well tolerated, and no adverse events (AEs) attributed to afucosylated 20502 were observed in any of the evaluated parameters. During the study, a higher incidence of diarrhea was observed in the higher dose groups at the end of the dosing period. Since the incidence was higher in affected animals at the mid- and high doses, and with onset later in the dosing period, this may be related to exposure to afucosylated 20502. There were no microscopic changes in the intestine in animals treated with afucosylated 20502, including those with diarrhea; therefore, this finding was considered non-adverse but possibly related to the test article. There was one death in the study. One animal in the mid-dose recovery group died on study day 35, 14 days after the last dose. Clinical observations, macroscopic, and microscopic evaluations were consistent with a diagnosis of intestinal volvulus. Intestinal volvulus occasionally occurs in cynomolgus monkeys, and this is believed to be a spontaneous symptom in these animals and not related to the test article. The NOAEL in cynomolgus monkeys is considered to be 100 mg / kg / dose.

[0226] In addition to in vivo toxicology studies, GLP-compliant tissue cross-reactivity studies were conducted to compare the binding of afucosylated 20502 to a panel of 36 tissues from rats, cynomolgus monkeys, and humans. The results showed that the binding pattern of afucosylated 20502 was similar across the three species and was restricted to the mammary epithelium.

[0227] Thus, afucosylated 20502 was well tolerated in cynomolgus monkeys and rats. The NOAEL in both species was found to be 100 mg / kg / dose, the highest dose tested when given as 4 weekly intravenous doses.

[0228] 6.7 Example 7: Phase 1a Dose Escalation and Exploration of Afucosylated 20502

[0229] A phase 1a, open-label, multicenter study of afucosylated 20502 was conducted in up to 34 patients with advanced solid tumors.

[0230] (A) Research design

[0231] Phase 1a includes a dose escalation phase and a dose exploration phase. Figure 3 The Phase 1a study protocol is provided in [ 14 ]. In the dose-escalation and dose-finding phases, afucosylated 20502 was administered as a 60-minute intravenous (IV) infusion every three weeks (Q3W) on Day 1 of each 21-day cycle. The dose of afucosylated 20502 was based on body weight on Day 1 of Cycle 1. After Cycle 1, the dose was recalculated at each infusion visit only if the patient's body weight changed by >10% from Day 1 of Cycle 1.

[0232] Phase 1a dose escalation includes an initial accelerated titration design, followed by a standard 3+3 dose escalation design greater than or equal to 1 mg / kg dose level until the maximum tolerated dose (MTD) and / or recommended dose (RD) for Phase 1b are determined. Up to 16 to 48 patients will participate in the dose escalation. Doses from 0.01 (or 0.005) to 20 mg / kg are administered according to the groups listed in Table 15, and the second dose of the patient must be at least 21 days after their first dose.

[0233] Table 15: Dosage Levels

[0234]

[0235] *If the MTD is exceeded at the first dose level of afucosylated 20502 (0.01 mg / kg), the dose will be reduced to 0.005 mg / kg.

[0236] During the Phase 1a dose escalation period, evaluation for dose-limiting toxicity (DLT) began on the first day of treatment after the start of infusion and continued for 21 days. A DLT was defined as any of the following, regardless of attribution (excluding those clearly due to the underlying disease or extrinsic causes): (i) non-hematologic toxicity of grade 3 or higher (except grade 3 nausea, vomiting, and diarrhea) occurring within the first 21 days of treatment, (ii) grade 3 nausea, vomiting, and diarrhea occurring within the first 21 days of treatment and persisting for at least 72 hours despite best supportive care, (iii) febrile neutropenia and / or documented infection with an absolute neutrophil count (ANC) of less than 1.0 × 10-10 cells / mL within the first 21 days of treatment. 9 / L, Grade 4 neutropenia lasting more than 7 days, Grade 4 thrombocytopenia (less than 25.0×10 9 / L) or grade 3 thrombocytopenia (less than 50.0–25.0 × 10 9 1 / L) associated with bleeding; (iv) aspartate aminotransferase / alanine aminotransferase (AST / ALT) exceeding 3 times the upper limit of normal (ULN) and concurrent total bilirubin exceeding 2 times the ULN, not related to liver involvement of cancer; (v) other clinically insignificant Grade 3 laboratory values ​​that do not resolve within 72 hours, or (vi) any Grade 4 laboratory value regardless of clinical sequelae.

[0237] The dosage level of 0.01, 0.03, 0.1 and 0.3 mg / kg is designed for accelerated titration, and at least 1 patient is recruited for each dosage level. After at least 1 patient completes 21 days of evaluation intervals, the dose is escalated to the next dosage level. If there is a single patient experience DLT or at least 2 patients experience moderate AE (at any dosage level) within the 21 days of evaluation intervals, other patients are recruited with the current dosage level, and the standard 3+3 dose escalation standard is applicable to the group and all subsequent administration groups. Moderate AE is defined as >= 2 grade AE, no matter how its attribution (except those events caused by potential disease or external causes). For this purpose, 2 grade laboratory values ​​are not considered as moderate AE, unless with clinical sequelae.

[0238] Intra-patient dose escalation will be allowed in patients enrolled at a dose level lower than the 1 mg / kg offered: (i) the patient does not experience a DLT; (ii) all other AEs have recovered to Grade 1 or lower prior to dose escalation; (iii) the patient may only escalate the dose by a maximum of 1 dose level every 21 days; and (iv) the patient cannot be dosed above the 1 mg / kg dose level unless the dose level has been cleared according to the standard 3+3 dose escalation design as described below.

[0239] The algorithm outlined in Table 16A below can be used for all standard 3+3 dose escalations.

[0240] Table 16A: Phase 1a Algorithm for 3+3 Dose Escalation Decisions

[0241]

[0242] Based on the evaluation of overall safety, tolerability, pharmacodynamics, pharmacokinetics and preliminary efficacy, the MTD and / or RD of afucosylated 20502 in Phase 1a were identified. The RD will take into account the toxicity observed during and after the DLT evaluation, as well as dose reductions and discontinuations due to toxicity that does not meet the DLT criteria. Therefore, the RD may or may not be the same as the identified MTD. For example, if the MTD is not reached, or if data from subsequent treatment cycles in Phase 1a provide additional information about the safety profile, the RD may be a dose that is different from, but not higher than, the MTD. The MTD will be at a dose level at which no more than 1 / 6 of the patients report a DLT. The RD will also be a dose at which no more than 1 / 6 of the patients report a DLT, but it may be lower than the MTD. In some embodiments, the MTD will be at a dose level at which no more than 1 / 3, 1 / 4 or 1 / 5 of the patients report a DLT. The RD will also be a dose at which no more than 1 / 3, 1 / 4 or 1 / 5 of the patients report a DLT, but it may be lower than the MTD.

[0243] The Phase 1a dose-finding cohort enrolled 3 more patients (up to 10 additional patients across all dose levels). During the Phase 1a dose-finding period, all patients were pre-screened using archived tumor tissue (or fresh biopsies if no archived tissue was available) to test for B7-H4 expression levels by immunohistochemistry (IHC). Archived tumor tissue (or fresh biopsies) were used for biomarker analysis as described herein. In addition, fresh biopsies were used during screening and post-processing for extended pharmacodynamic analysis.

[0244] In one embodiment, the suggested dose cohorts for Phase 1a monotherapy dose exploration are shown in Table 16B.

[0245] Table 16B: Proposed Dose Cohorts / Levels for Phase 1a Dose Exploration

[0246] Group dose plan 1 3 mg / kg Q3W 2 10 mg / kg Q3W 3 MTD / RD Q3W

[0247] Abbreviations: MTD = maximum tolerated dose; Q3W = every 3 weeks; RD = recommended dose.

[0248] In one embodiment, the recommended dose is 20 mg / kg.

[0249] (B) Subjects

[0250] Based on the following inclusion and exclusion criteria, a total of 12 to 24 patients were identified.

[0251] Phase 1a patients met all of the following inclusion criteria:

[0252] Histologically confirmed solid tumors, except primary central nervous system (CNS) tumors;

[0253] Unresectable locally advanced or metastatic disease;

[0254] Refractory or intolerant to existing therapies known to provide clinical benefit for the patient's condition; and

[0255] At least one measurable lesion at baseline according to RECIST v1.1 (Response Evaluation Criteria in Solid Tumors 1.1); tumor sites located in previously irradiated areas or areas receiving other locoregional therapy are not considered measurable unless disease progression has been demonstrated.

[0256] Exclusion criteria: Phase 1a patients had no history of anti-drug antibodies (ADA), severe allergic reactions, anaphylaxis, or other infusion-related reactions to prior biologics and no known hypersensitivity to any component of the afucosylated 20502 formulation.

[0257] (C) Results

[0258] The incidence of grade 3 and 4 adverse events, as well as clinical laboratory abnormalities defined as dose-limiting toxicities, were evaluated to show that afucosylated 20502 was safe and tolerable in patients with advanced solid tumors. The incidence of adverse events, clinical laboratory abnormalities, and ECG abnormalities were evaluated to determine the maximum tolerated dose and / or recommended dose of afucosylated 20502.

[0259] Pharmacokinetic parameters (AUC (area under the serum concentration-time curve), C max (maximum serum concentration), C min (minimum serum concentration), clearance (CL), t 1 / 2 (terminal half-life), V ss (distribution volume at steady state) and C 谷 The trough serum concentration at the end of the dosing interval was determined using a non-compartmental analysis based on serum afucosylated 20502 concentration-time data. Serum afucosylated 20502 concentrations were determined using an enzyme-linked immunosorbent assay (ELISA). The effect of immunogenicity (i.e., anti-drug antibody immune response to afucosylated 20502) on afucosylated 20502 exposure in patients with advanced solid tumors was assessed by measuring total anti-afucosylated 20502 antibodies from all patients.

[0260] The clinical benefit of afucosylated 20502 in patients with advanced solid tumors was also demonstrated. Tumor assessment included clinical examination and imaging (e.g., computed tomography (CT) scan or magnetic resonance imaging (MRI) with appropriate slice thickness according to RECIST v 1.1). Tumors were assessed at screening, every 9 weeks for the first 12 months, and every 12 weeks (+ / - 2 weeks) thereafter to show inhibition of tumor growth and tumor regression (e.g., complete tumor regression).

[0261] Overall response rate (ORR), duration of response (DOR) and progression-free survival (PFS) are also determined as the measurement values ​​of efficacy. ORR is defined as the total number of patients with confirmed response (complete response (CR) or partial response (PR) according to RECIST v.1.1) divided by the total number of patients who can evaluate the response. DOR is defined as the time from the subsequent confirmation of response (CR or PR) to the first observation of disease progression or death due to any cause. PFS is defined as the time from the first dose of the patient to the first observation of progressive disease or death due to any cause.

[0262] Pharmacodynamic biomarkers are also observed. Immune cell infiltrates in tumor biopsies before and during treatment can be analyzed. For example, changes in markers of tumor immune infiltrates (including but not limited to natural killer cells (NK), CD4, CD8 and / or other selected immune biomarkers) are assessed by IHC and / or ribonucleic acid (RNA) analysis. In addition, changes in cytokine levels (e.g., IL-2, IL-6, IL-10, TNF and / or interferon gamma (IFNγ)) are assessed by multiple analysis.

[0263] Patients received a range of dose levels of afucosylated 20502. Twenty-four patients with advanced solid tumors not selected for B7-H4 and a median of three (3) prior therapies were treated with the afucosylated 20502 antibody. In the dose escalation cohort, 18 patients received dose levels of 0.01 mg / kg to 20 mg / kg every three weeks (Q3W) in an accelerated titration followed by a 3+3 design. Patients were treated with a median of 3 (range = 1-11) doses of afucosylated 20502. The majority received 3 mg / kg (n=8) or 10 mg / kg (n=6) of afucosylated 20502. Seven (7) patients from the dose escalation cohort were retrospectively identified as B7-H4 positive. In a separate dose-finding cohort, six (6) B7-H4-positive patients (out of a total of 24 patients) were treated at 3 mg / kg or 10 mg / kg Q3W, with mandatory pre-treatment and on-treatment biopsies. No dose reduction was required, and no dose-limiting toxicities or treatment-related serious adverse events (SAEs) were observed in the 24 patients. Thus, afucosylated 20502 exhibited a favorable safety profile, and the data suggest that 20 mg / kg may be selected as the recommended dose.

[0264] 6.8 Example 8: Phase 1b Dose Expansion of Afucosylated 20502

[0265] A Phase 1b, open-label, multicenter study of afucosylated 20502 was conducted in up to 210 patients with specific solid tumor types with B7-H4 expression levels determined by immunohistochemistry (IHC). Specific solid tumor types were identified based on their high prevalence of B7-H4 expression and the limited availability of effective therapies in the unresectable and metastatic setting.

[0266] (A) Research design

[0267] Phase 1b is the dose expansion portion of the study. Figure 3 The Phase 1b study protocol is provided in . Enrollment in the Phase 1b dose expansion will begin after identification of the maximum tolerated dose (MTD) and / or recommended dose (RD) in Phase 1a.

[0268] Phase 1b includes tumor-specific cohorts of up to 30 patients each, as shown in Table 17. Phase 1b studies may have more or fewer cohorts than shown in Table 17, but not more than 7 cohorts.

[0269] Table 17: Phase 1b Expansion Cohorts and Tumor Types

[0270] Group Tumor type 1b1 Breast cancer 1b2 Ovarian cancer 1b3 Endometrial cancer 1b4 Urothelial carcinoma

[0271] Archival tumor tissue (or fresh biopsy tissue if archival tissue was not available) was used to test B7-H4 expression levels by immunohistochemistry (IHC) for prescreening all patients and for biomarker analysis. In addition, fresh biopsies collected during screening and post-treatment were used for extended pharmacodynamic analysis in a subset of patients (10 patients in each 30-patient cohort).

[0272] Afucosylated 20502 is administered as a 60-minute intravenous (IV) dose every three weeks (Q3W) on Day 1 of each 21-day cycle. The dose of afucosylated 20502 is based on body weight on Day 1 of Cycle 1. After Cycle 1, the dose will be recalculated at each infusion visit only if the patient's body weight changes by >10% from Day 1 of Cycle 1.

[0273] (B) Subjects

[0274] Up to 30 patients with breast, ovarian, endometrial, or urothelial cancer will participate. Additional tumor type-specific cohorts of up to 30 patients each may also participate.

[0275] Phase 1b patients met all of the following inclusion criteria:

[0276] All Phase 1a inclusion criteria (except histologically confirmed solid tumors and primary central nervous system (CNS) tumors);

[0277] Positive for B7-H4 expression in archival or fresh tumor samples as assessed by immunohistochemistry (IHC) assay;

[0278] For Group 1b1 – Breast Cancer

[0279] ο histologically or cytologically confirmed metastatic breast cancer;

[0280] o progressive disease on or after anthracycline and taxane chemotherapy or inability to tolerate anthracycline and taxane chemotherapy;

[0281] o Progressive disease on or after at least one line of systemic chemotherapy in the metastatic setting;

[0282] o Patients with estrogen receptor (ER) and / or progesterone receptor (PR)-positive disease (defined as ER and / or PR >1%) must be hormone-refractory (progressed after 3 consecutive endocrine therapies) or have symptomatic visceral disease; and

[0283] o Patients have HER2-negative disease;

[0284] For patients with triple-negative breast cancer (TNBC) in Cohort 1b1:

[0285] o histologically or cytologically confirmed metastatic TNBC; and

[0286] o At least two prior lines of systemic chemotherapy, at least one of which was administered in the metastatic setting;

[0287] For patients with hormone receptor-positive (HR+) breast cancer in Cohort 1b1:

[0288] o Histologically or cytologically confirmed metastatic HR+ breast cancer;

[0289] o Patients have received at least two prior lines of hormonal therapy; and

[0290] o Patients have received at least one prior line of systemic chemotherapy (in the adjuvant or metastatic setting)

[0291] For Group 1b2 – Ovarian cancer

[0292] o A diagnosis of histologically or cytologically confirmed recurrent epithelial ovarian, primary peritoneal, or fallopian tube cancer that is refractory to existing therapies known to provide clinical benefit; and

[0293] o Progressive disease on or after at least two prior treatment regimens, including at least one platinum-containing regimen, or inability to tolerate additional chemotherapy;

[0294] For Group 1b3 – Endometrial cancer

[0295] o histologically or cytologically confirmed recurrent or persistent endometrial cancer that is refractory to curative or established therapy; and

[0296] o Progressive disease on or after at least one prior systemic chemotherapy regimen, or inability to tolerate systemic chemotherapy;

[0297] For Group 1b4 – Urothelial carcinoma

[0298] o histologically or cytologically confirmed urothelial carcinoma; and

[0299] o Progressive disease on or after treatment with a platinum-containing regimen and a PD-1 / PD-L1-directed agent or intolerance to a platinum-containing regimen and a PD-1 / PD-L1-directed agent.

[0300] Phase 1b patients had no history of anti-drug antibodies (ADA), severe allergic reactions, anaphylaxis, or other infusion-related reactions to prior biologics and no known hypersensitivity to any component of the afucosylated 20502 formulation.

[0301] (C) Results

[0302] The incidence of adverse events, clinical laboratory abnormalities, and ECG abnormalities were evaluated to demonstrate the safety and tolerability of afucosylated 20502 in patients with B7-H4-positive advanced solid tumors.

[0303] Pharmacokinetic parameters (AUC, C max ,C min ,CL,t 1 / 2 ,V ss The volume of distribution at steady state was determined using a non-compartmental analysis based on serum afucosylated 20502 concentration-time data. Serum afucosylated 20502 concentrations were determined using an enzyme-linked immunosorbent assay (ELISA).

[0304] Pharmacodynamic biomarkers were also observed. For example, changes in markers of tumor immune infiltrates (including but not limited to natural killer (NK) cells, CD4, CD8, and / or other selected immune biomarkers) were assessed by IHC and / or RNA analysis. In addition, changes in cytokine levels (e.g., IL-2, IL-6, IL-10, TNF, and / or interferon gamma (IFNγ)) were assessed by multiplex analysis.

[0305] The effect of afucosylated 20502 exposure on immunogenicity (i.e., anti-drug antibody immune response to afucosylated 20502) in patients with B7-H4-positive advanced solid tumors can be assessed by measuring total anti-afucosylated 20502 antibodies from all patients.

[0306] A clinical benefit of afucosylated 20502 has also been demonstrated. Tumor assessment includes clinical examination and imaging (e.g., computed tomography (CT) scan or magnetic resonance imaging (MRI) with appropriate slice thickness according to RECIST v 1.1). Tumors are assessed at screening, every 9 weeks for the first 12 months, and every 12 weeks (+ / - 2 weeks) thereafter to show inhibition of tumor growth and tumor regression (e.g., complete tumor regression).

[0307] Overall survival (defined as the time from the patient's first dose to death due to any cause) was also determined as a measure of efficacy. Overall survival demonstrated a clinical benefit of afucosylated 20502 in patients with B7-H4-positive advanced solid tumors.

[0308] ***

[0309] The scope of the present invention is not limited by the specific embodiments described herein. Indeed, various modifications of the present invention, other than those described, will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0310] All references (e.g., publications or patents or patent applications) cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0311] Other embodiments are within the following claims. Sequence Listing <110> FIVE PRIME THERAPEUTICS, INC <120> B7-H4 Antibody Dosing Regimen <130> 3986.012PC02 / EKS / CLD / MS <150> US 62 / 802,100 <151> 2019-02-06 <150> US 62 / 633,527 <151> 2018-02-21 <160> 31 <170> PatentIn version 3.5 <210> 1 <211> 282 <212> PRT <213> Artificial Sequence <220> <223> Human B7-H4 <400> 1 Met Ala Ser Leu Gly Gln Ile Leu Phe Trp Ser Ile Ile Ser Ile Ile 1 5 10 15 Ile Ile Leu Ala Gly Ala Ile Ala Leu Ile Ile Gly Phe Gly Ile Ser 20 25 30 Gly Arg His Ser Ile Thr Val Thr Thr Val Ala Ser Ala Gly Asn Ile 35 40 45 Gly Glu Asp Gly Ile Leu Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu 50 55 60 Ser Asp Ile Val Ile Gln Trp Leu Lys Glu Gly Val Leu Gly Leu Val 65 70 75 80 His Glu Phe Lys Glu Gly Lys Asp Glu Leu Ser Glu Gln Asp Glu Met 85 90 95 Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val Ile Val Gly Asn 100 105 110 Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr 115 120 125 Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Gly Asn Ala Asn Leu Glu 130 135 140 Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Val Asn Val Asp Tyr Asn 145 150 155 160 Ala Ser Ser Glu Thr Leu Arg Cys Glu Ala Pro Arg Trp Phe Pro Gln 165 170 175 Pro Thr Val Val Trp Ala Ser Gln Val Asp Gln Gly Ala Asn Phe Ser 180 185 190 Glu Val Ser Asn Thr Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met 195 200 205 Lys Val Val Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser 210 215 220 Cys Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val 225 230 235 240 Thr Glu Ser Glu Ile Lys Arg Arg Ser His Leu Gln Leu Leu Asn Ser 245 250 255 Lys Ala Ser Leu Cys Val Ser Ser Phe Phe Ala Ile Ser Trp Ala Leu 260 265 270 Leu Pro Leu Ser Pro Tyr Leu Met Leu Lys 275 280 <210> 2 <211> 282 <212> PRT <213> Artificial Sequence <220> <223> Cynomolgus B7-H4 <400> 2 Met Ala Ser Leu Gly Gln Ile Leu Phe Trp Ser Ile Ile Ser Ile Ile 1 5 10 15 Phe Ile Leu Ala Gly Ala Ile Ala Leu Ile Ile Gly Phe Gly Ile Ser 20 25 30 Gly Arg His Ser Ile Thr Val Thr Thr Val Ala Ser Ala Gly Asn Ile 35 40 45 Gly Glu Asp Gly Ile Leu Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu 50 55 60 Ser Asp Ile Val Ile Gln Trp Leu Lys Glu Gly Val Ile Gly Leu Val 65 70 75 80 His Glu Phe Lys Glu Gly Lys Asp Glu Leu Ser Glu Gln Asp Glu Met 85 90 95 Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val Ile Val Gly Asn 100 105 110 Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr 115 120 125 Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Gly Asn Ala Asn Leu Glu 130 135 140 Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Val Asn Val Asp Tyr Asn 145 150 155 160 Ala Ser Ser Glu Thr Leu Arg Cys Glu Ala Pro Arg Trp Phe Pro Gln 165 170 175 Pro Thr Val Val Trp Ala Ser Gln Val Asp Gln Gly Ala Asn Phe Ser 180 185 190 Glu Val Ser Asn Thr Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met 195 200 205 Lys Val Val Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser 210 215 220 Cys Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val 225 230 235 240 Thr Glu Ser Glu Ile Lys Arg Arg Ser His Leu Gln Leu Leu Asn Ser 245 250 255 Lys Ala Ser Leu Cys Val Ser Ser Phe Leu Ala Ile Ser Trp Ala Leu 260 265 270 Leu Pro Leu Ala Pro Tyr Leu Met Leu Lys 275 280 <210> 3 <211> 283 <212> PRT <213> Artificial Sequence <220> <223> Murine B7 - H4 <400> 3 Met Ala Ser Leu Gly Gln Ile Ile Phe Trp Ser Ile Ile Asn Ile Ile 1 5 10 15 Ile Ile Leu Ala Gly Ala Ile Ala Leu Ile Ile Gly Phe Gly Ile Ser 20 25 30 Gly Lys His Phe Ile Thr Val Thr Thr Phe Thr Ser Ala Gly Asn Ile 35 40 45 Gly Glu Asp Gly Thr Leu Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu 50 55 60 Asn Gly Ile Val Ile Gln Trp Leu Lys Glu Gly Ile Lys Gly Leu Val 65 70 75 80 His Glu Phe Lys Glu Gly Lys Asp Asp Leu Ser Gln Gln His Glu Met 85 90 95 Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val Val Val Gly Asn 100 105 110 Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr 115 120 125 Thr Cys Tyr Ile Arg Thr Ser Lys Gly Lys Gly Asn Ala Asn Leu Glu 130 135 140 Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Ile Asn Val Asp Tyr Asn 145 150 155 160 Ala Ser Ser Glu Ser Leu Arg Cys Glu Ala Pro Arg Trp Phe Pro Gln 165 170 175 Pro Thr Val Ala Trp Ala Ser Gln Val Asp Gln Gly Ala Asn Phe Ser 180 185 190 Glu Val Ser Asn Thr Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met 195 200 205 Lys Val Val Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser 210 215 220 Cys Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val 225 230 235 240 Thr Asp Ser Glu Val Lys Arg Arg Ser Gln Leu Gln Leu Leu Asn Ser 245 250 255 Gly Pro Ser Pro Cys Val Phe Ser Ser Ala Phe Val Ala Gly Trp Ala 260 265 270 Leu Leu Ser Leu Ser Cys Cys Leu Met Leu Arg 275 280 <210> 4 <211> 282 <212> PRT <213> Artificial Sequence <220> <223> Rat B7-H4 <400> 4 Met Ala Ser Leu Gly Gln Ile Ile Phe Trp Ser Ile Ile Asn Val Ile 1 5 10 15 Ile Ile Leu Ala Gly Ala Ile Val Leu Ile Ile Gly Phe Gly Ile Ser 20 25 30 Gly Lys His Phe Ile Thr Val Thr Thr Phe Thr Ser Ala Gly Asn Ile 35 40 45 Gly Glu Asp Gly Thr Leu Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu 50 55 60 Asn Gly Ile Val Ile Gln Trp Leu Lys Glu Gly Ile Lys Gly Leu Val 65 70 75 80 His Glu Phe Lys Glu Gly Lys Asp Asp Leu Ser Gln Gln His Glu Met 85 90 95 Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val Val Val Gly Asn 100 105 110 Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr 115 120 125 Thr Cys Tyr Ile His Thr Ser Lys Gly Lys Gly Asn Ala Asn Leu Glu 130 135 140 Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Ile Asn Val Asp Tyr Asn 145 150 155 160 Ala Ser Ser Glu Ser Leu Arg Cys Glu Ala Pro Arg Trp Phe Pro Gln 165 170 175 Pro Thr Val Ala Trp Ala Ser Gln Val Asp Gln Gly Ala Asn Phe Ser 180 185 190 Glu Val Ser Asn Thr Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met 195 200 205 Lys Val Val Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser 210 215 220 Cys Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val 225 230 235 240 Thr Asp Ser Glu Val Lys Arg Arg Ser Gln Leu Glu Leu Leu Asn Ser 245 250 255 Gly Pro Ser Pro Cys Val Ser Ser Val Ser Ala Ala Gly Trp Ala Leu 260 265 270 Leu Ser Leu Ser Cys Cys Leu Met Leu Arg 275 280 <210> 5 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 <400> 5 Gly Ser Ile Lys Ser Gly Ser Tyr Tyr Trp Gly 1 5 10 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 <400> 6 Asn Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Arg Ser 1 5 10 15 <210> 7 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 <400> 7 Ala Arg Glu Gly Ser Tyr Pro Asn Gln Phe Asp Pro 1 5 10 <210> 8 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 <400> 8 Arg Ala Ser Gln Ser Val Ser Ser Asn Leu Ala 1 5 10 <210> 9 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 <400> 9 Gly Ala Ser Thr Arg Ala Thr 1 5 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 <400> 10 Gln Gln Tyr His Ser Phe Pro Phe Thr 1 5 <210> 11 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH 20502 <400> 11 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Lys Ser Gly 20 25 30 Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Asn Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Glu Gly Ser Tyr Pro Asn Gln Phe Asp Pro Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 12 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL 20502 <400> 12 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80​​​​​​​​​​​​​​​​​​​​​​​Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly 20 25 <210> 14 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> VH FR2 <400> 14 Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5 10 <210> 15 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> VH FR3 <400> 15 Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 20 25 30 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VH FR4 <400> 16 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 17 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> VL FR1 <400> 17 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VL FR4 <400> 20 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> twenty one <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Full-length heavy chain amino acid sequence 20502 <400> twenty one Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Lys Ser Gly 20 25 30 Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Asn Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Glu Gly Ser Tyr Pro Asn Gln Phe Asp Pro Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 22 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Full - length light chain amino acid sequence 20502 <400> 22 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Ser Phe Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 23 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Constant region of human κ light chain <400> 23 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80<o001223>Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 24 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Constant region of human κ light chain nucleotide <400> 24 cggaccgtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 It should be noted that there is a small error in the original text. In the line with ID=19, it should be " " instead of "<o001223>". This has been corrected in the translation.tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg t 321 <210> 25 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Constant region of human IgG1 heavy chain <400> 25 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 26 <211> 990 <212> DNA <213> Artificial Sequence <220> <223> Constant region of human IgG1 heavy chain nucleotide sequence <400> 26 gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gggtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960 cagaagagcc tctccctgtc tccgggtaaa 990 <210> 27 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide sequence encoding the heavy chain variable region 20502 <400> 27 cagctgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcaaa agtggtagtt actactgggg ctggatccgc 120 cagcccccag ggaaggggct ggagtggatt gggaacatct attatagtgg gagcacctac 180 tacaacccgt ccctcagaag tcgagtcacc atatccgtag acacgtccaa gaaccagttc 240 tccctgaagc tgagttctgt gaccgccgca gacacggcgg tgtactactg cgccagagaa 300 ggatcttacc ccaatcagtt tgatccatgg ggacagggta cattggtcac cgtctcctca 360 <210> 28 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide sequence encoding the variable region of the light chain 20502 <400> 28 gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcaacttag cctggtacca gcagaaacct 120 ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg tatcccagcc 180 aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240 gaagattttg cagtttatta ctgtcagcag taccactcct tccctttcac ttttggcgga 300 gggaccaagg ttgagatcaa a 321 <210> 29 <211> 385 <212> PRT <213> Artificial Sequence <220> <223> B7-H4 IgV-huIgG1 <400> 29 Met Ala Ser Leu Gly Gln Ile Leu Phe Trp Ser Ile Ile Ser Ile Ile 1 5 10 15 Ile Ile Leu Ala Gly Ala Ile Ala Leu Ile Ile Gly Phe Gly Ile Ser 20 25 30 Gly Arg His Ser Ile Thr Val Thr Thr Val Ala Ser Ala Gly Asn Ile 35 40 45 Gly Glu Asp Gly Ile Leu Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu 50 55 60 Ser Asp Ile Val Ile Gln Trp Leu Lys Glu Gly Val Leu Gly Leu Val 65 70 75 80 His Glu Phe Lys Glu Gly Lys Asp Glu Leu Ser Glu Gln Asp Glu Met 85 90 95 Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val Ile Val Gly Asn 100 105 110 Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr 115 120 125 Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Gly Asn Ala Asn Leu Glu 130 135 140 Tyr Lys Thr Gly Ala Phe Ser Gly Ser Glu Pro Lys Ser Ser Asp Lys 145 150 155 160 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 165 170 175 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 180 185 190 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 195 200 205 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 210 215 220 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 225 230 235 240 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 245 250 255 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 260 265 270 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 275 280 285 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 290 295 300 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 305 310 315 320 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 325 330 335 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 340 345 350 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 355 360 365 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 370 375 380 Lys 385 <210> 30 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> hPD-1 sequence <400> 30 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 31 <211> 290 <212> PRT <213> Artificial Sequence <220> <223> hPD-L1 <400> 31 Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu 35 40 45 Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile 50 55 60 Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser 65 70 75 80 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn 85 90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val 115 120 125 Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val 130 135 140 Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160 Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser 165 170 175 Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180 185 190 Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195 200 205 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu 210 215 220 Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His 225 230 235 240 Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr 245 250 255 Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys 260 265 270 Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu 275 280 285 Glu Thr 290

Claims

1. Use of an antibody or antigen-binding fragment thereof in the preparation of a medicament for treating a B7-H4-expressing solid tumor in a human subject, wherein the antibody or antigen-binding fragment thereof specifically binds to human B7-H4 and comprises: a heavy chain variable region (VH) complementarity determining region (CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 5, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 6, a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 7, a light chain variable region (VL) CDR1 consisting of the amino acid sequence of SEQ ID NO: 8, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 9, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 10, wherein the B7-H4-expressing solid tumor is selected from ovarian cancer, endometrial cancer, urothelial carcinoma, and hormone receptor (HR)-positive breast cancer, wherein the medicament is for administration at about 0.005 mg / kg to about 20 mg / kg of the antibody or antigen-binding fragment thereof.

2. Use of a pharmaceutical composition for the preparation of a medicament for treating a B7-H4-expressing solid tumor in a human subject, wherein the pharmaceutical composition comprises: (i) an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to human B7-H4 and comprises: a VH CDR1 consisting of the amino acid sequence of SEQ ID NO: 5, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 6, a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 7, a VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 8, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 9, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 10; and (ii) a pharmaceutically acceptable excipient, wherein the solid tumor expressing B7-H4 is selected from ovarian cancer, endometrial cancer, urothelial carcinoma and hormone receptor (HR)-positive breast cancer, wherein at least 95% of the antibodies or antigen-binding fragments thereof in the composition are afucosylated, and wherein the medicament is for administration at about 0.005 to about 20 mg / kg of the antibody or antigen-binding fragment thereof.

3. The use of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is administered intravenously.

4. The use of claim 1 or 2, wherein B7-H4 has been detected in the solid tumor using immunohistochemistry (IHC) prior to administration.

5. The use according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises: a VH comprising the amino acid sequence set forth in SEQ ID NO: 11; and / or a VL comprising the amino acid sequence set forth in SEQ ID NO:

12.

6. The use according to claim 1 or 2, wherein the antibody or antigen-binding fragment comprises a heavy chain constant region and / or a light chain constant region. The use according to claim 6 , wherein the heavy chain constant region is a human immunoglobulin IgG1 heavy chain constant region, and / or wherein the light chain constant region is a human immunoglobulin IgGκ light chain constant region.

8. The use of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 25; and / or a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO:

23.

9. The method of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 21; and / or a light chain comprising the amino acid sequence set forth in SEQ ID NO:

22.

10. The use according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof.

11. The use according to claim 1, wherein the antibody or antigen-binding fragment thereof is afucosylated.

12. The method of claim 1 or claim 2, wherein the antibody or antigen-binding fragment thereof is a full-length antibody.

13. The use of claim 1 or claim 2, wherein the antibody or antigen-binding fragment thereof is an antigen-binding fragment.

14. The use according to claim 13, wherein the antigen-binding fragment comprises Fab, Fab', F(ab')2, single-chain Fv (scFv), disulfide-linked Fv, IgGΔCH2, miniantibody, F(ab')3, tetravalent antibody, trivalent antibody, bivalent antibody, Fcab, mAb 2 , (scFv)2 or scFv-Fc.

15. The use of claim 2, wherein fucosylation is not detectable in the composition.

16. The use of claim 1 or claim 2, wherein the solid tumor is unresectable, locally advanced, or metastatic.

17. The use of claim 1 or claim 2, wherein the subject has not received prior therapy with a PD-1 / PD-L1 antagonist.

18. Use of an antibody or antigen-binding fragment thereof in the preparation of a medicament for treating a B7-H4-expressing solid tumor in a human subject, wherein the antibody or antigen-binding fragment thereof specifically binds to human B7-H4 and comprises: a VH comprising the amino acid sequence set forth in SEQ ID NO: 11; and a VL comprising the amino acid sequence set forth in SEQ ID NO: 12, wherein the B7-H4-expressing solid tumor is selected from ovarian cancer, endometrial cancer, urothelial carcinoma, and hormone receptor (HR)-positive breast cancer, wherein the medicament is for administration to the subject at a dose of about 20 mg / kg of the antibody or antigen-binding fragment thereof approximately once every three weeks.

19. Use of a pharmaceutical composition for the preparation of a medicament for treating a B7-H4-expressing solid tumor in a human subject, wherein the pharmaceutical composition comprises: (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and comprises: a VH comprising the amino acid sequence set forth in SEQ ID NO: 11; and a VL comprising the amino acid sequence set forth in SEQ ID NO: 12; and (ii) a pharmaceutically acceptable excipient, wherein the solid tumor expressing B7-H4 is selected from ovarian cancer, endometrial cancer, urothelial carcinoma and hormone receptor (HR)-positive breast cancer, wherein at least 95% of the antibodies or antigen-binding fragments thereof in the composition are afucosylated, and wherein the medicament is for intravenous administration to the subject at about 20 mg / kg of the antibody or antigen-binding fragment thereof approximately once every three weeks.

20. The use of claim 18 or 19, wherein the antibody comprises: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 21; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 22.

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