HK40057716B - Combination therapy for cancer - Google Patents

Description

Combination therapy for cancer

Technical Field

This disclosure relates to a method of treating diseases such as cancer by administering an antibody that specifically binds to human B7-H4 in combination with a PD-1/PD-L1 antagonist such as pembrolizumab. Advantageous dosing regimens are provided.

Background Technology

B7-H4 (also known as B7x, B7-S1, and VTCN1) is an immunomodulatory molecule homologous to other members of the B7 family, including PD-L1. It is a type I transmembrane protein composed of IgV and IgC extracellular domains. Although B7-H4 expression is relatively limited at the protein level in healthy tissues, it 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 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.

Given the expression and function of B7-H4, antibodies that specifically bind to B7-H4 are being developed for therapies involving the regulation of B7-H4 activity, such as for the treatment of cancer.

PD-1 is a key immune checkpoint receptor expressed by activated T cells and B cells and mediates immunosuppression. PD-1 is a member of the CD28 receptor family, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands for PD-1, programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2), have been identified. They are expressed on antigen-presenting cells and in many cancers and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1. Inhibition of the PD-1/PD-L1 interaction, for example through inhibition by anti-PD-1 or anti-PD-L1 antibodies, mediates potent antitumor activity.

Therefore, an effective dosing regimen is needed to administer antibodies that bind to B7-H4 and inhibitors that interact with PD-1/PD-L1.

Summary of the Invention

This article provides a method for administering an anti-B7-H4 antibody and its antigen-binding fragment in combination with a PD-1/PD-L1 antagonist using a therapeutically effective dosage regimen. The anti-B7-H4 antibody and its antigen-binding fragment may be a 20502 antibody or its antigen-binding fragment; or an antibody or antigen-binding fragment containing the heavy and light chain variable regions (CDRs) of the 20502 antibody; or an antibody or antigen-binding fragment containing the heavy and light chain variable regions of the 20502 antibody, including the unfucosylated form of any of the above. The PD-1/PD-L1 antagonist may be an anti-PD-1 antibody, such as pembrolizumab, or an antibody or antigen-binding fragment containing the heavy and light chain variable regions (CDRs) of pembrolizumab; or an antibody or antigen-binding fragment containing the heavy and light chain variable regions of pembrolizumab.

In some aspects, a method of treating a solid tumor in a human subject includes administering to the subject (a) about 0.1 to about 20 mg/kg of an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment specifically binding to human B7-H4 and comprising 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; and (b) about 200 mg of pembrolizumab. In some embodiments, (a) and (b) are administered simultaneously or sequentially.

In some aspects, a method of treating a solid tumor in a human subject includes administering to the subject (a) a pharmaceutical composition comprising (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 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; and (ii) a pharmaceutically acceptable excipient, wherein at least 95% of the antibody or antigen-binding fragment thereof in the composition is unfucosylated, and wherein about 0.1 to about 20 mg/kg of the antibody or antigen-binding fragment thereof is administered; and (b) a pharmaceutical composition comprising pembrolizumab, wherein about 200 mg of pembrolizumab is administered. In some embodiments, (a) and (b) are administered simultaneously or sequentially.

In some aspects, the CDR of the antibody or its antigen-binding fragment is a CDR defined by Kabat, a CDR defined by Chothia, or a CDR defined by AbM. In some aspects, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and CDR3 sequences each contain the amino acid sequences shown in SEQ ID NO:5-10.

In some aspects, the subject is administered about 20 mg/kg of anti-B7-H4 antibody or an antigen-binding fragment thereof. In some aspects, the subject is administered about 10 mg/kg of anti-B7-H4 antibody or an antigen-binding fragment thereof. In some aspects, the subject is administered about 3 mg/kg of anti-B7-H4 antibody or an antigen-binding fragment thereof. In some aspects, the subject is administered about 1 mg/kg of anti-B7-H4 antibody or an antigen-binding fragment thereof. In some aspects, the subject is administered about 0.3 mg/kg of anti-B7-H4 antibody or an antigen-binding fragment thereof. In some aspects, the subject is administered about 0.1 mg/kg of anti-B7-H4 antibody or an antigen-binding fragment thereof.

In some respects, the anti-B7-H4 antibody or its antigen-binding fragment and/or pembrolizumab are administered approximately every three weeks.

In some respects, the anti-B7-H4 antibody or its antigen-binding fragment and/or pembrolizumab are administered intravenously.

In some respects, B7-H4 had been detected in the solid tumors using immunohistochemistry (IHC) prior to application.

In some aspects, the anti-B7-H4 antibody or its antigen-binding fragment comprises VH and/or VL, wherein the VH comprises the amino acid sequence listed in SEQ ID NO:11, and the VL comprises the amino acid sequence listed in SEQ ID NO:12. In some aspects, the anti-B7-H4 antibody or its antigen-binding fragment comprises a heavy chain constant region and/or a light chain constant region. In some aspects, the heavy chain constant region is the human immunoglobulin IgG ₁ heavy chain constant region, and/or the light chain constant region is the human immunoglobulin IgG κ light chain constant region. In some aspects, the antibody or its antigen-binding fragment comprises a heavy chain constant region and/or a light chain constant region, wherein the heavy chain constant region comprises the amino acid sequence listed in SEQ ID NO:25, and the light chain constant region comprises the amino acid sequence listed in SEQ ID NO:23. In some aspects, the anti-B7-H4 antibody or its antigen-binding fragment comprises a heavy chain and/or a light chain, wherein the heavy chain comprises the amino acid sequence listed in SEQ ID NO:21, and the light chain comprises the amino acid sequence listed in SEQ ID NO:22.

In some respects, the antibody or its antigen-binding fragment is a human antibody or its antigen-binding fragment.

In some respects, the anti-B7-H4 antibody or its antigen-binding fragment is unfucosylated.

In some aspects, the antibody or its antigen-binding fragment is a full-length antibody. In some aspects, the antibody or its antigen-binding fragment is an antigen-binding fragment. In some aspects, the antigen-binding fragment comprises Fab, Fab', F(ab') ₂ , single-chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, intracellular antibody, IgGΔCH2, microantibody, F(ab') ₃ , tetravalent antibody, trivalent antibody, bivalent antibody, single-domain antibody, DVD-Ig, Fcab, ^mAb2 , (scFv) ₂ , or scFv-Fc.

In some respects, fucosylation is undetectable in the composition containing the anti-B7-H4 antibody.

In some respects, the solid tumor expresses B7-H4.

In some respects, the solid tumor is unresectable, locally advanced, or metastatic.

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 carcinoma. 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 some aspects, the subject has not received prior therapy with a PD-1/PD-L1 antagonist.

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

In some respects, the administration of the anti-B7-H4 antibody or its antigen-binding fragment and the anti-PD-1 antibody or its antigen-binding fragment produce a synergistic effect.

In some aspects, a method of treating a solid tumor in a human subject includes administering to the subject (i) about 20 mg/kg of an anti-B7-H4 antibody, said antibody specifically binding to human B7-H4 and comprising a VH having the amino acid sequence of SEQ ID NO:11 and a VL having the amino acid sequence of SEQ ID NO:12; and (ii) about 200 mg of an anti-PD-1 antibody, said antibody comprising a VH having the amino acid sequence of SEQ ID NO:32 and a VL having the amino acid sequence of SEQ ID NO:33, wherein said anti-B7-H4 antibody and said anti-PD-1 antibody are administered intravenously about every three weeks.

In some aspects, a method of treating a solid tumor in a human subject includes administering to the subject (a) a pharmaceutical composition comprising (i) an anti-B7-H4 antibody that specifically binds to human B7-H4 and comprises a VH having the amino acid sequence of SEQ ID NO:11 and a VL having the amino acid sequence of SEQ ID NO:12, and (ii) a pharmaceutically acceptable excipient, wherein at least 95% of the anti-B7-H4 antibody in the composition is unfucosylated, and wherein about 20 mg/kg of the antibody is administered; and (b) a pharmaceutical composition comprising an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a VH having the amino acid sequence of SEQ ID NO:32 and a VL having the amino acid sequence of SEQ ID NO:33; and a pharmaceutically acceptable excipient, wherein about 200 mg of the antibody or the antigen-binding fragment thereof is administered, wherein the anti-B7-H4 antibody and the anti-PD-1 antibody are administered intravenously approximately every three weeks.

In some aspects, the anti-B7-H4 antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence listed in SEQ ID NO:21 and the light chain comprising the amino acid sequence listed in SEQ ID NO:22. In some aspects, the anti-PD-1 antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence listed in SEQ ID NO:30 and the light chain comprising the amino acid sequence listed in SEQ ID NO:31.

In some respects, a solid tumor is breast cancer or ovarian cancer, optionally referring to triple-negative breast cancer.

Attached Figure Description

Figures 1A, 1B, and 1C illustrate the in vivo antitumor efficacy of the anti-B7-H4 antibody combined with the anti-PD-1 antibody. (See Example 4.)

Figure 2 illustrates the dose-dependent antitumor activity of the anti-B7-H4 antibody. (See Example 4.)

Figure 3 shows that the anti-B7-H4 antibody and the anti-PD1 antibody are combined in a synergistic manner, even when the dosage of the anti-B7-H4 antibody is not effective as a monotherapy. (See Example 4.)

Detailed Implementation

This document provides a method for administering an antibody (e.g., a monoclonal antibody) specifically binding to B7-H4 (e.g., human B7-H4) and its antigen-binding fragment in combination with a PD-1/PD-L1 antagonist (e.g., pembrolizumab). The anti-B7-H4 antibody and its antigen-binding fragment may be administered in combination with a PD-1/PD-L1 antagonist (e.g., pembrolizumab), for example, to treat a subject with a solid tumor. In a specific embodiment, approximately 20 mg/kg, approximately 10 mg/kg, approximately 3 mg/kg, approximately 1 mg/kg, approximately 0.3 mg/kg, or approximately 0.1 mg/kg of the antibody or its antigen-binding fragment are combined with approximately 200 mg of pembrolizumab and administered to the subject, for example, wherein the administration is performed approximately every three weeks.

4.1 Terminology

As used herein, the term "B7-H4" refers to a mammalian B7-H4 polypeptide, including but not limited to natural B7-H4 polypeptides and B7-H4 polypeptide isoforms. "B7-H4" encompasses full-length, unprocessed B7-H4 polypeptides as well as B7-H4 polypeptides produced through intracellular processing. As used herein, the term "human B7-H4" refers to a polypeptide containing 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.

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

The term "antibody fragment" refers to a portion of a complete antibody. "Antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to the portion of a complete antibody that binds to an antigen. An antigen-binding fragment may contain an antigen recognition site of the complete antibody (e.g., a complementarity-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 generated.

The terms “anti-B7-H4 antibody,” “B7-H4 antibody,” and “antibody bound to B7-H4” refer to antibodies that specifically bind to B7-H4 with sufficient affinity, enabling them to be used as diagnostic and/or therapeutic agents targeting B7-H4. As used herein, the terms “specifically bind,” “immunely specifically bind,” “immunely specifically recognize,” and “specifically recognize” are similar terms in the context of antibodies or their antigen-binding fragments. These terms indicate that the antibody or its antigen-binding fragment binds to the epitope via its antigen-binding domain, and that binding requires a degree of complementarity between the antigen-binding domain and the epitope. Therefore, antibodies that "specifically bind" 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 to a less than approximately 10% extent that the antibody binds to unrelated non-B7-H4 proteins (e.g., other members of the B7 protein family, such as PD-L1), as measured, for example, by radioimmunoassay (RIA). In one specific embodiment, antibodies or antigen-binding fragments thereof that specifically bind to human, cynomolgus monkey, mouse, and rat B7-H4 are provided herein.

"Monoclonal" antibodies or their antigen-binding fragments refer to a group of homogeneous antibodies or antigen-binding fragments that participate in the highly specific binding of a single antigenic determinant or epitope. This contrasts with polyclonal antibodies, which typically consist of different antibodies targeting different antigenic determinants. The term "monoclonal antibody" or its antigen-binding fragment encompasses full-length and complete monoclonal antibodies, as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins containing antibody moieties, and any other modified immunoglobulin molecules containing antigen recognition sites. Furthermore, "monoclonal antibody" or its antigen-binding fragment refers to any number of such antibodies and their antigen-binding fragments prepared in any manner, including but not limited to hybridoma, phage selection, recombinant expression, and transgenic animals.

As used herein, the terms “variable region” or “variable domain” are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of the light or heavy chain, typically about 110 to 120 or 110 to 125 amino acids from the amino terminus of the mature heavy chain and about 90 to 115 amino acids from the mature light chain, which differ in sequence between antibodies and are responsible for the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated in those regions called complementarity-determining regions (CDRs), while more conserved regions within a variable domain are called frame regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for antibody-antigen interactions and specificity. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region comprises a rodent or mouse CDR and a human frame region (FR). In certain embodiments, the variable region is a primate (e.g., a non-human primate) variable region. In some implementations, the variable region includes the rodent or rat CDR and the primate (e.g., non-human primate) frame region (FR).

The terms “VL” and “VL domain” are used interchangeably to refer to the variable region of the light chain of an antibody.

The terms “VH” and “VH domain” are used interchangeably to refer to the variable region of the heavy chain of an antibody.

The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the variable regions of the heavy and light chains of antibodies or their antigen-binding fragments. In some respects, CDRs 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, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within the antibody heavy chain molecule are typically located at amino acid positions 31 to 35 (which may optionally contain one or two additional amino acids after 35 (referred to as 35A and 35B in the Kabat numbering scheme)) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, the CDRs within the antibody light chain molecule are typically located at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In one specific embodiment, the CDRs of the antibody described herein have been determined according to the Kabat numbering scheme.

Conversely, Chothia refers to the location of the structural loop (Chothia and Lesk, J.Mol.Biol.196:901-917 (1987)). When using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the loop length (this is because the Kabat numbering scheme places the insert at H35A and H35B; if neither 35A nor 35B is present, 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 region represents a compromise between the Kabat CDR and the Chothia structural loop and is used through OxfordMolecular's AbM antibody modeling software.

As used herein, the terms “constant region” and “constant domain” are interchangeable and have the same meaning in the art. A constant region is a part of an antibody that does not directly participate in antibody-antigen binding but can exhibit a variety of effector functions, such as the antibody portion interacting with the Fc receptor, or the carboxyl-terminal portion of the light chain and/or heavy chain. The constant region of an immunoglobulin molecule typically has a more conserved amino acid sequence than the variable domain of an immunoglobulin. In some respects, antibody or antigen-binding fragments contain a constant region or a portion thereof sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

As used herein, based on the amino acid sequence of constant structural domains, the term "heavy chain" in relation to antibody use can refer to any different type, such as α(α), δ(δ), ε(ε), γ(γ), and μ(μ), which produce antibodies of the IgA, IgD, IgE, IgG, and IgM classes, including subclasses of IgG, such as _IgG1 , _IgG2 , _IgG3 , and _IgG4 . The amino acid sequence of the heavy chain is well known in the art. In a specific embodiment, the heavy chain is the human heavy chain.

As used herein, based on the amino acid sequence of a constant domain, the term "light chain" can refer to any different type, such as κ(κ) or λ(λ), in relation to antibody use. Light chain amino acid sequences are well known in the art. In this particular embodiment, the light chain is a human light chain.

The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof whose amino acid sequence is derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to the variable regions of antibodies or antigen-binding fragments 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 antibodies or antigen-binding fragments thereof derived from another species (usually human) to avoid triggering an immune response in said species.

The term "humanized antibody" or its antigen-binding fragment refers to a non-human (e.g., mouse) antibody or its antigen-binding fragment that is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof containing minimal non-human (e.g., mouse) sequence. Typically, a humanized antibody or its antigen-binding fragment is a human immunoglobulin in which residues from the complementarity-determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) with the desired specificity, affinity, and capability ("CDR transplantation") (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). In some cases, certain Fv framework region (FR) residues of a human immunoglobulin are replaced by corresponding residues from an antibody or fragment of a non-human species with the desired specificity, affinity, and capability. Humanized antibodies or their antigen-binding fragments can be further modified by substituting additional residues within the Fv framework region and/or non-human CDR residues to improve and optimize the specificity, affinity, and/or ability of the antibody or its antigen-binding fragment. Generally, humanized antibodies or their antigen-binding fragments will contain a variable domain containing all or substantially all CDR regions corresponding to non-human immunoglobulins, and all or substantially all FR regions being those of the common sequence of human immunoglobulins. Humanized antibodies or their antigen-binding fragments may also contain 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 generating humanized antibodies are described in U.S. Patent 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 implementations, "humanized antibody" is an antibody with surface remodeling.

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 complete or full-length antibodies and fragments thereof.

"Fucosylated-free" antibodies or their antigen-binding fragments, or "fucosyl-deficient" antibodies or their antigen-binding fragments, refer to IgG1 or IgG3 isotype antibodies or their antigen-binding fragments that lack fucosyl in their constant-region glycosylation. Glycosylation of human IgG1 or IgG3 occurs at Asn297 because the glycosylation of the core fucosylated biantennary complex oligosaccharide terminates at at most two Gal residues. In some embodiments, fucosylated-free antibodies lack fucosyl at Asn297. Depending on the amount of terminal Gal residues, these structures are named G0, G1 (a 1,6 or a 1,3), or G2 glycan residues. See, for example, Raju, T.S., BioProcess Int. 1:44-53 (2003). CHO-type glycosylation of antibody Fc is described, for example, Routier, F.FL, Glycoconjugate J. 14:201-207 (1997).

Methods for measuring fucose include any methods known in the art. For the purposes of this document, fucose is detected by the method described in Example 1 of WO2015/017600, which is incorporated herein by reference in its entirety. Briefly, glycan analysis is performed by releasing glycans from an antibody (e.g., by enzymatic release), labeling the glycans with anthranilic acid (2-AA), and then purifying the labeled glycans. The glycans are separated using normal-phase HPLC with fluorescence detection, and the relative abundance of each glycan in the antibody is measured. The glycans can be definitively identified as lacking or containing fucose by mass spectrometry. In some embodiments, fucose is undetectable in compositions comprising multiple fucosylated antibodies or antigen-binding fragments thereof. In some embodiments, the fucosylated antibody or antigen-binding fragment thereof has enhanced affinity for FcγRIIIA. In some embodiments, the fucosylated antibody or antigen-binding fragment thereof has enhanced affinity for FcγRIIIA (V158). In some implementations, the fucosylated antibody or its antigen-binding fragment has enhanced affinity for FcγRIIIA (F158).

"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 its antigen-binding fragment) and its binding partner (e.g., an antigen). Unless otherwise stated, 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 its antigen-binding fragment and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant ( _KD ). Affinity can be measured and/or represented in a variety of ways known in the art, including, but not limited to, the equilibrium dissociation constant ( _KD ) and the equilibrium association constant ( _KA ). _KD is calculated as the quotient of _koff / _kon , while _KA is calculated as the quotient of _kon / _koff . _kon refers to, for example, the association rate constant of an antibody or its antigen-binding fragment with an antigen, and _koff refers to, for example, the dissociation of an antibody or its antigen-binding fragment with an antigen. _kon and _koff can be determined by techniques known to those skilled in the art, such as or KinExA.

As used herein, "epitope" is a term in the art and refers to a localized region of an antigen to which an antibody or its antigen-binding fragment can specifically bind. An epitope can be, for example, a continuous amino acid sequence of a polypeptide (linear or continuous epitope), or an epitope can be, for example, derived from two or more discontinuous regions of one or more polypeptides (conformal, nonlinear, discontinuous, or non-continuous epitopes). In some embodiments, the epitope to which the antibody or its antigen-binding fragment specifically binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assays, hydrogen/deuterium exchange in binding mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any method known in the art (e.g., Giegé 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/its antigen-binding fragment: Antigen crystals can be studied using well-known X-ray diffraction techniques and computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, for example, Meth Enzymol (1985), vols. 114 and 115, edited by Wyckoff HW et al.; U.S. 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(Pt10):1316-1323) were improved. Mutagenesis mapping studies can be performed using any method known to those skilled in the art. For descriptions of mutagenesis techniques, including alanine scanning mutagenesis, see, for example, Champe M et al. (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085.

The terms “programmed cell death protein 1” and “PD-1” refer to immunosuppressive receptors 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 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 being PD-L2), both of which downregulate T cell activation and cytokine secretion upon binding to PD-1. As used herein, the term “PD-L1” includes human PD-L1 (hPD-L1), naturally occurring variants and isoforms of hPD-1, and species homologs of hPD-L1.

The term "PD-1/PD-L1 antagonist" refers to a portion 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 bound to PD-1, such as nivolumab (OPDIVO) and pembrolizumab (KEYTRUDA); PD-L1 antagonists, such as antibodies bound to PD-L1 (e.g., atezolizumab (TECENTRIQ), durvalumab, and avelumab); fusion proteins, such as AMP-224; and peptides, such as AUR-012.

"Pembrolizumab" refers to a humanized anti-PD-1 monoclonal antibody, which is the active ingredient in a commercially available pharmaceutical formulation sold by Merck & Co.

"Isolated" peptides, antibodies, polynucleotides, carriers, cells, or compositions are peptides, antibodies, polynucleotides, carriers, cells, or compositions that do not exist in nature. Isolated peptides, antibodies, polynucleotides, carriers, cells, or compositions include those that have been purified to the point that they no longer exist in the form they are found in nature. In some embodiments, the isolated antibodies, polynucleotides, carriers, cells, or compositions are substantially pure. As used herein, "substantially pure" means 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.

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, may contain modified amino acids, and may be non-amino acid-discontinuous. The term also covers amino acid polymers that have been modified naturally or through intervention; for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeled component. Also included within the definition are, for example, one or more analogues containing amino acids (including, for example, non-natural amino acids), and other modified polypeptides known in the art. It should be understood that because the polypeptides of the present invention are antibody-based, in some embodiments, the polypeptides may be present as single-chain or associated chains.

As used herein, the term "host cell" can be any type of cell, such as primary cells, cultured cells, or cells derived from a cell line. In a specific embodiment, the term "host cell" refers to a cell transfected with a nucleic acid molecule and its progeny or potential progeny. The progeny of such cells may differ from the parent cell transfected with the nucleic acid molecule, for example, due to mutations or environmental influences that can occur during passage or integration of the nucleic acid molecule into the host cell genome.

The term "pharmaceutical formulation" refers to a formulation in a form that allows the bioactivity of the active ingredient to be effective and does not contain any other components that would have unacceptable toxicity to a subject to whom the formulation will be administered. The formulation may be sterile.

As used herein, the terms “administer,” “administering,” “administration,” etc., refer to methods (e.g., intravenous administration) used to deliver a drug, such as an anti-B7-H4 antibody or its antigen-binding fragment, to a desired site of biological action. Administration techniques that can be used with the agents and methods described herein can be 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.

As used herein, the terms "subject" and "patient" are used interchangeably. A subject may 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). In some embodiments, the subject is a cynomolgus monkey. In some embodiments, the subject is a human.

The term "therapeutic effective dose" refers to a drug that is effective in treating a subject's disease or condition, such as the amount of an anti-B7-H4 antibody or its antigen-binding fragment. In the case of cancer, a therapeutically effective dose of a drug may reduce the number of cancer cells; reduce tumor size or burden; inhibit cancer cell infiltration into surrounding organs to a certain extent; inhibit tumor metastasis to a certain extent; inhibit tumor growth to a certain extent; alleviate one or more cancer-related symptoms to a certain 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, a reduction in stable disease (SD), progressive disease (PD), time to progression (TTP), or any combination thereof. To the extent that the drug can prevent the growth and/or kill existing cancer cells, it may be cytoseptic and/or cytotoxic.

Terms such as “treatment,” “curative,” “treatment,” “relief,” and “relief” refer to therapeutic measures that can cure, slow down, alleviate, and stop the progression of a pathological disease or condition. Therefore, those requiring treatment include those already diagnosed with or suspected of having the stated condition. In some embodiments, a subject’s cancer is successfully “treated” according to the method of the invention if the patient exhibits one or more of the following: a reduction or complete absence of cancer cells; a reduction in tumor size; inhibition or absence of cancer cell infiltration into surrounding organs, including, for example, cancer spread to soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; relief of one or more symptoms associated with the specific cancer; a reduction in morbidity and mortality; an improvement in quality of life; a reduction in tumorigenicity, tumorigenic frequency, or tumorigenic capacity of the tumor; a reduction in the number or frequency of cancer stem cells in the 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), progressive disease (PD), time to progression (TTP), or any combination thereof.

The terms “cancer” and “cancerous” refer to or describe a physiological disorder in mammals in which a population of cells is characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, gynecological cancers (e.g., breast cancer (including triple-negative breast cancer, hormone receptor (HR)-positive 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 carcinoma). Cancer can be “B7-H4-expressing cancer” or “B7-H4-positive cancer”. Such terms refer to cancers containing cells that express B7-H4. Cancer can be a solid tumor that expresses B7-H4. Cancer can be a primary tumor or can be an advanced or metastatic cancer.

"Refractory" cancer is cancer that continues to progress even after anti-tumor treatments (such as chemotherapy) are administered to the cancer patient.

"Recurrent" cancer is cancer that recurs at the initial site or a distant location after an initial treatment has been initiated.

As demonstrated herein, administration of anti-B7-H4 antibody or its antigen-binding fragment and anti-PD-1 antibody or its antigen-binding fragment can provide a “synergistic” or “synergistic effect,” meaning that the combined effect of the active ingredients is greater than the sum of the effects of using the active ingredients alone. Synergistic effects can be achieved when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined unit dose formulation; (2) delivered sequentially, alternately, or in parallel as separate formulations; or (3) via some other regimen. In the case of delivery in alternating therapies, synergistic effects can be achieved when the compounds are administered or delivered sequentially (e.g., via separate syringes for different injections).

As used in this disclosure and claims, unless the context clearly indicates otherwise, the singular forms “a/an” and “the” include the plural forms.

It should be understood that while the term "comprises" is used to describe embodiments herein, other similar embodiments described in relation to "consisting of" and/or "primarily consisting of" are also provided. In this disclosure, "comprises," "comprising," "containing," and "having" may have the meanings they are given under U.S. Patent Law and may mean "includes," "including," etc.; "consisting essentially of" or "consists essentially of" also have the meanings they are given under U.S. Patent Law, and the terms are open-ended, thus allowing for extensions beyond the described existence, provided that the essential or novel features described are not altered by extension to the described existence, but excluding prior art embodiments.

Unless explicitly fixed or obvious from the context, the term “or” is to be understood as included as used herein. The term “and/or” as used herein in phrases such as “A and/or B” is intended to include “A and B”, “A or B”, “A”, and “B”. Similarly, 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).

As used herein, the terms “about” and “approximately” when used to modify numerical or numerical ranges indicate that deviations of 5% to 10% above and below the stated value or range are still within the expected meaning of the stated value or range.

Any composition or method provided herein may be combined with one or more of any other compositions and methods provided herein.

4.2 Methods of treating cancer

In one aspect, this document provides a method for treating cancer in a human subject, the method comprising administering to a subject in need (i) an anti-B7-H4 antibody or an antigen-binding fragment thereof or a pharmaceutical composition thereof as described herein, in combination with (ii) a PD-1/PD-L1 inhibitor or a pharmaceutical composition thereof as described herein, wherein (i) and (ii) are administered simultaneously or sequentially. For “simultaneous” administration, in some embodiments, the agents in (i) and (ii) are administered as separate formulations on the same day, one after the other; or in other embodiments, the agents in (i) and (ii) are mixed together prior to administration and thus administered as a mixture. For example, in some embodiments, the agents in (i) and (ii) may be packaged and stored in the same vial (i.e., a fixed-dose formulation), or alternatively, vials containing each individual agent may be mixed together just before administration. For “sequential” administration, the agents in (i) and (ii) are administered as separate formulations on different days. In various embodiments, the agents may be administered in vivo via multiple routes, including but not limited to intravenous routes.

Combining anti-B7-H4 antibodies or their antigen-binding fragments with PD-1/PD-L1 inhibitors can be synergistic.

One PD-1 inhibitor is pembrolizumab. The table below lists the heavy and light chain sequences of pembrolizumab. Against the background of the heavy and light chain sequences, the CDR sequence is shown in bold, and the variable region sequence is underlined.

pembrolizumab sequence

On the one hand, PD-1 inhibitors are antibody or antigen-binding fragments containing the heavy and light chain variable regions (CDRs) of pembrolizumab; or antibody or antigen-binding fragments containing the heavy and light chain variable regions of pembrolizumab.

In one aspect, a method of treating cancer in a human subject includes administering to the subject in need (i) the anti-B7-H4 antibody or its antigen-binding fragment thereof as described herein, or its pharmaceutical composition as described herein, wherein about 0.005 to about 20 mg/kg of the anti-B7-H4 antibody or its antigen-binding fragment thereof is administered, and (ii) the pembrolizumab or its pharmaceutical composition as described herein, wherein about 200 mg of pembrolizumab is administered, and wherein (i) and (ii) are administered simultaneously or sequentially at a frequency of, for example, about once every three weeks.

In one aspect, a method of treating cancer in a human subject includes administering to a subject in need (i) the anti-B7-H4 antibody described herein or its antigen-binding fragment or its pharmaceutical composition described herein, wherein about 0.1 mg/kg of the anti-B7-H4 antibody or its antigen-binding fragment is administered, and (ii) the pembrolizumab described herein or its pharmaceutical composition, wherein about 200 mg of pembrolizumab is administered, and wherein (i) and (ii) are administered simultaneously or sequentially, for example, at a frequency of about once every three weeks. In another aspect, a method of treating cancer in a human subject includes administering to a subject in need (i) the anti-B7-H4 antibody described herein or its antigen-binding fragment or its pharmaceutical composition described herein, wherein about 0.3 mg/kg of the anti-B7-H4 antibody or its antigen-binding fragment is administered, and (ii) the pembrolizumab described herein or its pharmaceutical composition, wherein about 200 mg of pembrolizumab is administered, and wherein (i) and (ii) are administered simultaneously or sequentially, for example, at a frequency of about once every three weeks. In one aspect, a method of treating cancer in a human subject includes administering to a subject in need (i) the anti-B7-H4 antibody described herein or its antigen-binding fragment or its pharmaceutical composition described herein, wherein about 1 mg/kg of the anti-B7-H4 antibody or its antigen-binding fragment is administered, and (ii) the pembrolizumab described herein or its pharmaceutical composition, wherein about 200 mg of pembrolizumab is administered, and wherein (i) and (ii) are administered simultaneously or sequentially, for example, at a frequency of about once every three weeks. In another aspect, a method of treating cancer in a human subject includes administering to a subject in need (i) the anti-B7-H4 antibody described herein or its antigen-binding fragment or its pharmaceutical composition described herein, wherein about 3 mg/kg of the anti-B7-H4 antibody or its antigen-binding fragment is administered, and (ii) the pembrolizumab described herein or its pharmaceutical composition, wherein about 200 mg of pembrolizumab is administered, and wherein (i) and (ii) are administered simultaneously or sequentially, for example, at a frequency of about once every three weeks. In one aspect, a method of treating cancer in a human subject includes administering to a subject in need (i) the anti-B7-H4 antibody described herein or its antigen-binding fragment or its pharmaceutical composition described herein, wherein about 10 mg/kg of the anti-B7-H4 antibody or its antigen-binding fragment is administered, and (ii) the pembrolizumab described herein or its pharmaceutical composition, wherein about 200 mg of pembrolizumab is administered, and wherein (i) and (ii) are administered simultaneously or sequentially, for example, at a frequency of about once every three weeks. In another aspect, a method of treating cancer in a human subject includes administering to a subject in need (i) the anti-B7-H4 antibody described herein or its antigen-binding fragment or its pharmaceutical composition described herein, wherein about 20 mg/kg of the anti-B7-H4 antibody or its antigen-binding fragment is administered, and (ii) the pembrolizumab described herein or its pharmaceutical composition, wherein about 200 mg of pembrolizumab is administered, and wherein (i) and (ii) are administered simultaneously or sequentially, for example, at a frequency of about once every three weeks.

In one aspect, a method of treating cancer in a human subject includes administering to a subject in need (i) the anti-B7-H4 antibody described herein or its antigen-binding fragment or its pharmaceutical composition described herein, wherein about 0.1 mg/kg of the anti-B7-H4 antibody or its antigen-binding fragment is administered, and (ii) the pembrolizumab described herein or its pharmaceutical composition, wherein about 200 mg of pembrolizumab is administered, and wherein (i) and (ii) are administered simultaneously or sequentially at a frequency of once every three weeks. In another aspect, a method of treating cancer in a human subject includes administering to a subject in need (i) the anti-B7-H4 antibody described herein or its antigen-binding fragment or its pharmaceutical composition described herein, wherein about 0.3 mg/kg of the anti-B7-H4 antibody or its antigen-binding fragment is administered, and (ii) the pembrolizumab described herein or its pharmaceutical composition, wherein about 200 mg of pembrolizumab is administered, and wherein (i) and (ii) are administered simultaneously or sequentially at a frequency of once every three weeks. In one aspect, a method of treating cancer in a human subject includes administering to a subject in need (i) the anti-B7-H4 antibody described herein or its antigen-binding fragment or its pharmaceutical composition described herein, wherein about 1 mg/kg of the anti-B7-H4 antibody or its antigen-binding fragment is administered, and (ii) the pembrolizumab described herein or its pharmaceutical composition, wherein about 200 mg of pembrolizumab is administered, and wherein (i) and (ii) are administered simultaneously or sequentially at a frequency of once every three weeks. In another aspect, a method of treating cancer in a human subject includes administering to a subject in need (i) the anti-B7-H4 antibody described herein or its antigen-binding fragment or its pharmaceutical composition described herein, wherein about 3 mg/kg of the anti-B7-H4 antibody or its antigen-binding fragment is administered, and (ii) the pembrolizumab described herein or its pharmaceutical composition, wherein about 200 mg of pembrolizumab is administered, and wherein (i) and (ii) are administered simultaneously or sequentially at a frequency of once every three weeks. In one aspect, a method of treating cancer in a human subject includes administering to a subject in need (i) the anti-B7-H4 antibody described herein or its antigen-binding fragment or its pharmaceutical composition described herein, wherein about 10 mg/kg of the anti-B7-H4 antibody or its antigen-binding fragment is administered, and (ii) the pembrolizumab described herein or its pharmaceutical composition, wherein about 200 mg of pembrolizumab is administered, and wherein (i) and (ii) are administered simultaneously or sequentially at a frequency of once every three weeks. In another aspect, a method of treating cancer in a human subject includes administering to a subject in need (i) the anti-B7-H4 antibody described herein or its antigen-binding fragment or its pharmaceutical composition described herein, wherein about 20 mg/kg of the anti-B7-H4 antibody or its antigen-binding fragment is administered, and (ii) the pembrolizumab described herein or its pharmaceutical composition, wherein about 200 mg of pembrolizumab is administered, and wherein (i) and (ii) are administered simultaneously or sequentially at a frequency of once every three weeks.

In some embodiments of the methods provided herein, the anti-B7-H4 antibody or its antigen-binding fragment, or the pharmaceutical composition comprising the anti-B7-H4 antibody or its antigen-binding fragment, is administered intravenously. In some embodiments of the methods provided herein, pembrolizumab or a pharmaceutical composition thereof is administered intravenously.

In some embodiments, this document provides methods for treating cancers selected from the group consisting of: breast cancer (e.g., advanced breast cancer, triple-negative breast cancer, hormone receptor (HR)-positive breast cancer, or ductal carcinoma), endometrial cancer, ovarian cancer, urothelial carcinoma, non-small cell lung cancer (e.g., squamous cell carcinoma), head and neck squamous cell carcinoma (HNSCC), Hodgkin lymphoma (e.g., classical Hodgkin lymphoma), melanoma, pancreatic cancer, thyroid cancer, kidney cancer (e.g., renal cell carcinoma), bladder cancer (e.g., urothelial carcinoma), gastric cancer, cervical cancer, and high microsatellite instability carcinoma. In one embodiment, this document provides methods for treating advanced breast cancer (including triple-negative breast cancer, hormone receptor (HR)-positive), ovarian cancer, endometrial cancer, or urothelial carcinoma. In one embodiment, this document provides methods for treating breast cancer. In one embodiment, the breast cancer is triple-negative breast cancer. In one embodiment, this document provides methods for treating hormone receptor (HR)-positive breast cancer. In one embodiment, this document provides methods for treating ovarian cancer. In one embodiment, this document provides methods for treating endometrial cancer. In one embodiment, this document provides methods for treating urothelial carcinoma. In one embodiment, this document provides a method for treating gastrointestinal cancer. In one embodiment, this document provides a method for treating gynecological cancer. In one embodiment, this document provides a method for treating head and neck cancer. In one embodiment, this document provides a method for treating genitourinary system cancers. In one embodiment, this document provides a method in which the subject has not received prior therapy with a PD-1/PD-L1 antagonist. In some embodiments, such methods include administering to a patient in need (e.g., a human patient) an anti-B7-H4 antibody or an antigen-binding fragment thereof provided herein, or a pharmaceutical composition comprising an anti-B7-H4 antibody or an antigen-binding fragment thereof provided herein, in combination with a PD-1/PD-L1 inhibitor or a pharmaceutical composition thereof provided herein.

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

Biological samples 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. Biological samples can also be blood samples in which circulating tumor cells (or “CTCs”) express B7-H4 and are detected.

The intent of determining the expression level of B7-H4 protein includes directly (e.g., by measuring or estimating absolute protein levels) or relatively (e.g., by comparing with protein levels in a second biological sample) qualitatively or quantitatively measuring or estimating the level of B7-H4 protein in a first biological sample. The expression level of the B7-H4 peptide in the first biological sample can be measured or estimated and compared with a standard B7-H4 protein level, which is determined either by a second biological sample that has not been affected by the disease or by averaging levels from a population of samples that have not been affected by the disease. As will be understood in the art, once the “standard” B7-H4 peptide level is known, it can be repeatedly used as a standard for comparison.

In another embodiment, the anti-B7-H4 antibody described herein, or its antigen-binding fragment or pharmaceutical composition thereof, is administered to a patient diagnosed with cancer (e.g., a human patient) to increase the proliferation of T cells, CD4 ⁺ T cells, or CD8 ⁺ T cells in the patient. In such an embodiment, a PD-1/PD-L1 antagonist described herein, such as pembrolizumab, is administered to the patient to block the binding of PD-1 to PD-L1 and PD-L2 and to activate T cells. In another embodiment, the anti-B7-H4 antibody, or its antigen-binding fragment or pharmaceutical composition thereof, is administered to a patient diagnosed with cancer (e.g., a human patient) to increase the production of interferon-γ (IFNγ) in the patient. In such an embodiment, a PD-1/PD-L1 antagonist described herein, such as pembrolizumab, is administered to the patient to block the binding of PD-1 to PD-L1 and PD-L2 and to activate T cells. In another embodiment, an anti-B7-H4 antibody or its antigen-binding fragment or pharmaceutical composition is administered to a patient diagnosed with cancer (e.g., a human patient) to block the inhibitory activity of B7-H4 against T cells in the patient. In such an embodiment, a PD-1/PD-L1 antagonist described herein, such as pembrolizumab, is administered to the patient to block the binding of PD-1 to PD-L1 and PD-L2 and to activate T cells. In another embodiment, an anti-B7-H4 antibody or its antigen-binding fragment or pharmaceutical composition is administered to a patient diagnosed with cancer (e.g., a human patient) to reduce B7-H4-expressing cancer cells in the patient. In such an embodiment, a PD-1/PD-L1 antagonist described herein, such as pembrolizumab, is administered to the patient to block the binding of PD-1 to PD-L1 and PD-L2 and to activate T cells.

In some embodiments, the present invention relates to an anti-B7-H4 antibody or an antigen-binding fragment thereof or a pharmaceutical composition thereof, for use in combination with a PD-1/PD-L1 antagonist such as pembrolizumab or a pharmaceutical composition thereof as a medicine, wherein the medicine is used to administer about 0.1 mg/kg to about 20 mg/kg (e.g., 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 its antigen-binding fragment, and about 200 mg of pembrolizumab. In such embodiments, the antibody or its antigen-binding fragment and pembrolizumab may be co-formulated or separately formulated for simultaneous or sequential administration. In some aspects, the present invention relates to an anti-B7-H4 antibody or an antigen-binding fragment thereof or a pharmaceutical composition thereof provided herein, for use in a method of treating cancer in combination with a PD-1/PD-L1 antagonist such as pembrolizumab or a pharmaceutical composition thereof, wherein the antibody or an antigen-binding fragment thereof is administered at about 0.1 mg/kg to about 20 mg/kg (e.g., 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), and about 200 mg of pembrolizumab is administered, wherein the administration is sequential or simultaneous. In some aspects, the present invention relates to an anti-B7-H4 antibody or antigen-binding fragment or pharmaceutical composition thereof provided herein, for use in combination with a PD-1/PD-L1 antagonist such as pembrolizumab for the treatment of a subject with cancer, said method comprising administering to the subject about 0.1 mg/kg to about 20 mg/kg (e.g., 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 or pharmaceutical composition thereof provided herein, and about 200 mg of the pembrolizumab or pharmaceutical composition thereof provided herein, wherein the administration is sequential or simultaneous.

In some embodiments, the present invention relates to an anti-B7-H4 antibody or its antigen-binding fragment or pharmaceutical composition provided herein, for use in combination with a PD-1/PD-L1 antagonist such as pembrolizumab or a pharmaceutical composition thereof as a medicine, wherein the medicine is used to administer 0.1 mg/kg to 20 mg/kg (e.g., 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 its antigen-binding fragment, and 200 mg of pembrolizumab. In such embodiments, the antibody or its antigen-binding fragment and pembrolizumab may be co-formulated or separately formulated for simultaneous or sequential administration. In some aspects, the present invention relates to an anti-B7-H4 antibody or an antigen-binding fragment thereof or a pharmaceutical composition thereof provided herein, for use in combination with a PD-1/PD-L1 antagonist such as pembrolizumab or a pharmaceutical composition thereof for the treatment of cancer, wherein 0.1 mg/kg to 20 mg/kg of said antibody or an antigen-binding fragment thereof (e.g., 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg or 20 mg/kg) is administered, and 200 mg of pembrolizumab is administered, wherein the administration is sequential or simultaneous. In some aspects, the present invention relates to an anti-B7-H4 antibody or antigen-binding fragment thereof or pharmaceutical composition provided herein for use in combination with a PD-1/PD-L1 antagonist such as pembrolizumab for the treatment of a subject with cancer, the method comprising administering to the subject 0.1 mg/kg to 20 mg/kg (e.g., 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 or pharmaceutical composition provided herein, and administering 200 mg of the pembrolizumab or pharmaceutical composition thereof provided herein, wherein the administration is sequential or simultaneous.

In some respects, the amino acids of human PD-1 are:

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEV PTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL(SEQ IDNO:40).

In some respects, the amino acid sequence of human PD-L1 is:

MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHE LTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET(SEQ IDNO:41).

4.3 B7-H4 antibody and its antigen-binding fragment

This document provides a method for treating cancer in a human subject, the method comprising administering to the subject an antibody (e.g., a monoclonal antibody, such as a chimeric, humanized, or human antibody) specifically binding to B7-H4 (e.g., human B7-H4) in combination with an antigen-binding fragment thereof, in combination with a PD-1/PD-L1 antagonist (such as pembrolizumab). 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, mouse, and rat B7-H4 are known in the art and are also provided herein, represented by SEQ ID NO: 1-4, respectively.

Human B7-H4:

MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKT GAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKASLCVSSFFAISWALLPLSPYLMLK(SEQ ID NO:1)

Crab-eating macaques B7-H4:

MASLGQILFWSIISIIFILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVIGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKT GAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKASLCVSSFLAISWALLPLAPYLMLK(SEQ ID NO:2)

Rodent B7-H4

MASLGQIIFWSIINIIIILAGAIALIIGFGISGKHFITVTTFTSAGNIGEDGTLSCTFEPDIKLNGIVIQWLKEGIKGLVHEFKEGKDDLSQQHEMFRGRTAVFADQVVVGNASLRLKNVQLTDAGTYTCYIRTSKGKGNANLEYKTG AFSMPEINVDYNASSESLRCEAPRWFPQPTVASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTDSEVKRRSQLQLLNSGPSPCVFSSAFVAGWALLSLSCCLMLR(SEQ ID NO:3)

Rat B7-H4

MASLGQIIFWSIINVIIILAGAIVLIIGFGISGKHFITVTTFTSAGNIGEDGTLSCTFEPDIKLNGIVIQWLKEGIKGLVHEFKEGKDDLSQQHEMFRGRTAVFADQVVVGNASLRLKNVQLTDAGTYTCYIHTSKGKGNANLEYKT GAFSMPEINVDYNASSESLRCEAPRWFPQPTVASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTDSEVKRRSQLELLNSGPSPCVSSVSAAGWALLSLSCCLMLR(SEQ ID NO:4)

In some embodiments, the antibody or its antigen-binding fragment used in the methods described herein specifically binds to human B7-H4. In some embodiments, the antibody or its antigen-binding fragment used in the methods described herein specifically binds to human and cynomolgus monkey B7-H4. In some embodiments, the antibody or its antigen-binding fragment used in the methods described herein specifically binds to human, mouse, and rat B7-H4. In some embodiments, the antibody or its antigen-binding fragment used in the methods described herein specifically binds to human, cynomolgus monkey, mouse, and rat B7-H4.

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 some embodiments, an antibody or antigen-binding fragment thereof used in the methods described herein specifically binds to the IgV domain of human B7-H4. Therefore, this document provides a method for administering an antibody or antigen-binding fragment thereof comprising a polypeptide specifically bound to amino acids 35-146 of SEQ ID NO: 1.

In some embodiments, the antibody or its antigen-binding fragment used in the methods described herein specifically binds to human B7-H4 and comprises six CDRs of the 20502 antibody as listed in Tables 1 and 2. “20502” refers to the 20502 antibody described herein.

Table 1. VH CDR amino acid sequence ¹

The VH CDR in Table ¹ is determined based on Kabat.

Table 2. VL CDR amino acid sequence ²

The VL CDR in Table ² is determined based on Kabat.

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

Table 3: Variable Heavy Chain (VH) Amino Acid Sequence

In some embodiments, the antibody or its antigen-binding fragment used in the methods described herein specifically binds to human B7-H4 and comprises a VL containing the 20502 antibody listed in Table 4.

Table 4: Variable Light Chain (VL) Amino Acid Sequences

In some embodiments, the antibody or its antigen-binding fragment used in the methods described herein specifically binds to human B7-H4 and includes the VH and VL of the 20502 antibody listed in Tables 3 and 4.

In some embodiments, the antibody or its antigen-binding fragment used in the methods described herein specifically binds to human B7-H4 and includes the VH frame region of the 20502 antibody listed in Table 5.

Table 5. VH FR amino acid sequence ³

The VH framework regions described in ^Table 5 are defined by the Kabat numbering system based on CDRs. Therefore, the VH CDRs are determined by Kabat, and the framework regions are the amino acid residues surrounding the CDRs in the variable region, in the form of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

In some embodiments, the antibody or its antigen-binding fragment used in the methods described herein specifically binds to human B7-H4 and includes the VL frame region of the 20502 antibody listed in Table 6.

Table 6. VL FR amino acid sequence ⁴

The VL framework regions described in ^Table 6 are defined by the Kabat numbering system based on CDRs. Therefore, VL CDRs are determined by Kabat, and the framework regions are the amino acid residues surrounding the CDRs in the variable region, in the form of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

In some embodiments, the antibody or its antigen-binding fragment used in the methods described herein specifically binds to human B7-H4 and includes the four VH frame regions and four VL frame regions of the 20502 antibody listed in Tables 5 and 6.

In some embodiments, the antibody or its antigen-binding fragment used in the methods described herein specifically binds to human B7-H4 and includes the heavy chain sequence of the 20502 antibody listed in Table 7.

Table 7: Full-length heavy chain amino acid sequence

In some embodiments, the antibody or its antigen-binding fragment used in the methods described herein specifically binds to human B7-H4 and includes the light chain sequence of the 20502 antibody listed in Table 8.

Table 8: Full-length light chain amino acid sequence

In some embodiments, the antibody or antigen-binding fragment used in the methods described herein specifically binds to human B7-H4 and includes the heavy chain and light chain sequences of the 20502 antibody listed in Tables 7 and 8.

In some respects, antibodies or antigen-binding fragments thereof used in the methods described herein are described by their individual VL domain, or their individual VH domain, 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, describing the humanization of mouse anti-αvβ3 antibodies by identifying complementary light or heavy chains, respectively, from human light or heavy chain libraries, thereby producing humanized antibody variants with affinity as high as or higher 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, describing a method for generating antibodies that specifically bind to specific antigens by using a specific VL domain (or VH domain) and screening for complementary VH domains or (VL domains) in a library. The screening, conducted by ELISA, yielded 14 new mating agents targeting the specific VH domain and 13 new mating agents targeting the specific VL domain, which are strong binding agents 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.

In some respects, the CDR of an antibody or its antigen-binding fragment can be determined according to the Chothia numbering scheme, which refers to the location of the immunoglobulin structural loop (see, for example, 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. Patent No. 7,709,226). Typically, when using the Kabat numbering convention, the Chothia CDR-H1 ring is located at positions 26 to 32, 33, or 34 of the heavy chain amino acids; the Chothia CDR-H2 ring is located at positions 52 to 56 of the heavy chain amino acids; and the Chothia CDR-H3 ring is located at positions 95 to 102 of the heavy chain amino acids. The Chothia CDR-L1 ring is located at positions 24 to 34 of the light chain amino acids; the Chothia CDR-L2 ring is located at positions 50 to 56 of the light chain amino acids; and the Chothia CDR-L3 ring is located at positions 89 to 97 of the light chain amino acids. When using the Kabat numbering convention, the end of the Chothia CDR-H1 ring varies between H32 and H34 depending on the length of the ring (this is because the Kabat numbering scheme places the insert at H35A and H35B; if neither 35A nor 35B exists, the ring ends at 32; if only 35A exists, the ring ends at 33; if both 35A and 35B exist, the ring ends at 34).

In some aspects, this document provides methods for administering antibodies and their antigen-binding fragments that specifically bind to B7-H4 (e.g., human B7-H4) and comprise Chothia VH and VL CDRs of the 20502 antibody listed in Tables 3 and 4. In some embodiments, this document provides methods for administering antibodies or their antigen-binding fragments that specifically bind to B7-H4 (e.g., human B7-H4) and comprise one or more CDRs, wherein Chothia and Kabat CDRs have the same amino acid sequence. In some embodiments, this document provides methods for administering antibodies and their antigen-binding fragments that specifically bind to B7-H4 (e.g., human B7-H4) and comprise a combination of Kabat CDR and Chothia CDR.

In some respects, the CDR of an antibody or its antigen-binding fragment can be determined according to the IMGT numbering system described in Lefranc M-P, (1999) The Immunologist 7:132-136 and Lefranc M-P 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 at positions 51 to 57, VH-CDR3 at positions 93 to 102, VL-CDR1 at positions 27 to 32, VL-CDR2 at positions 50 to 52, and VL-CDR3 at positions 89 to 97. In one particular embodiment, this document provides a method for administering an antibody and its antigen-binding fragment thereof, said antibody and its antigen-binding fragment specifically binding to B7-H4 (e.g., human B7-H4) and comprising IMGT VH and VL CDR of the 20502 antibody listed in Tables 3 and 4, for example, as described above by Lefranc M-P (1999) and Lefranc M-P et al. (1999).

In some respects, the CDR of an antibody or its antigen-binding fragment 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 particular embodiment, this document provides a method for administering an antibody or its antigen-binding fragment that specifically binds to B7-H4 (e.g., human B7-H4) and comprises the VH and VL CDRs of antibody 20502 as determined by the method in MacCallum RM et al.

In some respects, the CDR of an antibody or its antigen-binding fragment can be determined according to the AbM numbering scheme, which refers to the AbM hypervariable region representing a compromise between the Kabat CDR and the Chothia structural loop, and is used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). In one particular embodiment, this document provides a method for administering an antibody or its antigen-binding fragment that specifically binds to B7-H4 (e.g., human B7-H4) and comprises the VH and VL CDRs of antibody 20502 as determined by the AbM numbering scheme listed in Tables 3 and 4.

In a specific respect, this article provides a method for administering antibodies comprising heavy and light chains.

Regarding the light chain, in one specific embodiment, the light chain of the antibody described herein is a κ light chain. The constant region of the human κ light chain may contain the following amino acid sequence:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:23).

The constant region of the human κ light chain can be encoded by the following nucleotide sequence:

CGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 24).

In one particular embodiment, the antibody used in the methods described herein to specifically bind to a B7-H4 polypeptide (e.g., human B7-H4) comprises a light chain, wherein the amino acid sequence of the VL domain comprises the sequences listed in Table 4, and wherein the constant region of the light chain comprises the amino acid sequence of the constant region of the human κ light chain.

In one particular embodiment, the antibody used in the methods described herein to specifically bind to B7-H4 (e.g., human B7-H4) comprises a heavy chain, wherein the amino acid sequence of the VH domain comprises the amino acid sequences listed in Table 3, and wherein the constant region of the heavy chain comprises the amino acid sequence of the constant region of the human γ(γ) heavy chain.

The constant region of the human IgG ₁ heavy chain may contain the following amino acid sequence:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:25).

The constant region of the human _IgG1 heavy chain can be encoded by the following nucleotide sequence:

GCCTCCACCAAGGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCT GCAACGTGAATCACAAGCCCAGCAACACCAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCT GACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA. (SEQ ID NO:26)

In one embodiment, the antibody used in the methods described herein to specifically bind to B7-H4 (e.g., human B7-H4) comprises a VH domain and a VL domain, the VH domain and VL domain comprising the amino acid sequences of any VH and VL domains described herein, and wherein the constant region comprises the amino acid sequence of the constant region of an IgG (e.g., human IgG) immunoglobulin molecule. In another embodiment, the antibody used in the methods described herein to specifically bind to B7-H4 (e.g., human B7-H4) comprises a VH domain and a VL domain, the VH domain and VL domain comprising the amino acid sequences of any VH and VL domains described herein, and wherein the constant region comprises the amino acid sequence of the constant region of an IgG ₁ (e.g., human IgG ₁ ) immunoglobulin molecule.

It has been reported that antibodies with reduced fucosylation content have increased affinity for Fc receptors, such as FcγRIIIA. Therefore, in some embodiments, the antibodies or antigen-binding fragments thereof used in the methods described herein have reduced fucosylation content or lack fucosylation (i.e., “unfucosylated”). Such antibodies or antigen-binding fragments thereof can be generated using techniques known to those skilled in the art. For example, they can be expressed in cells lacking or devoid of fucosylation capacity. In one specific example, a cell line with two allele knockouts of the α1,6-fucosyltransferase gene (FUT8) can be used to generate antibodies with reduced fucosylation content or antigen-binding fragments thereof. The Lonza system is an example of such a system that can be used to generate antibodies with reduced fucosylation content and antigen-binding fragments thereof. Alternatively, antibodies or their antigen-binding fragments with reduced or no fucose content can be generated, for example, by: (i) culturing cells under conditions that prevent or reduce fucosylation; (ii) removing fucose post-translationally (e.g., with 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 unfucosylated antibodies or their antigen-binding fragments. For methods for generating 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.

In some embodiments, the fucosylated B7-H4 antibody or its antigen-binding fragment exhibits enhanced ADCC activity in vitro compared to a fucosylated B7-H4 antibody or its antigen-binding fragment having the same amino acid sequence. In some embodiments, the fucosylated B7-H4 antibody or its antigen-binding fragment causes at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, or 75 percentage points higher specificity cleavage compared to that induced by the use of a fucosylated B7-H4 antibody. Specificity cleavage can be determined as described in Example 2 herein.

In some embodiments, the B7-H4 antibody or its antigen-binding fragment has enhanced affinity for FcγRIIIA compared to a fucosylated B7-H4 antibody or its antigen-binding fragment having the same amino acid sequence. In some embodiments, the unfucosylated B7-H4 antibody or its antigen-binding fragment binds to FcγRIIIA with an affinity 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 higher than that of the fucosylated B7-H4 antibody or its antigen-binding fragment. In some embodiments, surface plasmon resonance is used to determine the affinity for FcγRIIIA. In some embodiments, FcγRIIIA is selected from FcγRIIIA(V158) and FcγRIIIA(F158). In some embodiments, FcγRIIIA is FcγRIIIA(V158).

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

In a specific implementation, the antibody or its antigen-binding fragment (i) comprises the CDR sequence of 20502, the VH and VL sequences of 20502, or the heavy and light chain sequences of 20502, and (ii) is not fucosylated.

In a specific embodiment, the composition comprises an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof (i) comprising a CDR sequence of 20502, a VH and VL sequence of 20502, or a heavy chain and a light chain sequence of 20502, and (ii) being free of fucosylation, for example, wherein at least 95% of said antibody in the composition is free of fucosylation, or wherein fucosylation is undetectable in the composition.

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 described herein include, but are not limited to, those disclosed, such as those in: 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) ClinCancer 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 Publications Nos. US 2007/0248600; 2007/0178551; 2008/0060092; and 2006/0253928; International Publications Nos. WO 00/61739; WO 01/292246; WO 02/311140; and WO 02/30954; Potillegent ^™ 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.

In some implementations, any constant region mutation or modification described herein may be introduced into one or both heavy chain constant regions of the antibody or its antigen-binding fragment described herein that have two heavy chain constant regions.

In another specific embodiment, the antibody or antigen-binding fragment thereof that specifically binds to B7-H4 (e.g., human B7-H4) as described herein comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises a VH domain comprising the amino acid sequences of VH CDR1, VL CDR2, and VL CDR3 of the 20502 antibody listed in Table 1 (SEQ ID NO: 5, 6, and 7, respectively); (ii) the light chain comprises a VL domain comprising the amino acid sequences of VLCDR1, VH CDR2, and VH CDR3 of the 20502 antibody listed in Table 2 (SEQ ID NO: 8, 9, and 10, respectively); (iii) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of a constant domain of the human IgG ₁ heavy chain; and (iv) the light chain further comprises a constant light chain domain comprising the amino acid sequence of a constant domain of the human κ light chain.

In another specific embodiment, the antibody or antigen-binding fragment thereof that specifically binds to B7-H4 (e.g., human B7-H4) as described herein 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 (SEQ ID NO: 11); (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 (SEQ ID NO: 12); (iii) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of the human IgG ₁ heavy chain; and (iv) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of the human κ light chain.

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

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

In one specific aspect, the antigen-binding fragment that specifically binds to B7-H4 (e.g., human B7-H4) as described herein is selected from the group consisting of Fab, Fab', F(ab') ₂ , and scFv, wherein said 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 an antigen-binding fragment thereof as described herein. Fab, Fab', F(ab')2, or scFv can be generated by any technique known to those skilled in the art. In some embodiments, Fab, Fab', F(ab') ₂ , or scFv further comprises a portion that extends the half-life of the antibody in vivo. This portion is also referred to as the "half-life extension portion". Any portion known to those skilled in the art for extending the half-life of Fab, Fab', F(ab') ₂ , or scFv in vivo can be used. For example, the half-life extension portion may include 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 polyalkylene, polyolefin, polyoxyethylene, polysaccharide, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, methoxy polyethylene glycol, lactose, amylose, dextran, glycogen, or derivatives thereof. Substituents may include one or more hydroxyl, methyl, or methoxy groups. In some embodiments, Fab, Fab', F(ab') ₂ , or scFv may be modified by adding one or more C-terminal amino acids for linking the half-life extension moiety. In some embodiments, the half-life extension moiety is polyethylene glycol or human serum albumin. In some embodiments, Fab, Fab', F(ab') ₂ , or scFv is fused to the Fc region.

4.4 Pharmaceutical Compositions

This article provides a method of administering a composition comprising an anti-B7-H4 antibody or its antigen-binding fragment of desired purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). The acceptable carrier, excipient, or stabilizer is non-toxic to the recipient at the dose and concentration used. (See, for example, Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th edition (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th edition, Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd edition, Pharmaceutical Press (2000)). The composition intended for in vivo administration can be sterile. This is easily achieved through filtration, for example, using a sterile filter membrane.

In some embodiments, a method of administering a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises a fucosylated anti-B7-H4 antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier. In a specific embodiment, a method of administering a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises a fucosylated anti-B7-H4 antibody or an antigen-binding fragment, for example, wherein at least 80% of the antibody in the composition is fucosylated. In a specific embodiment, a method of administering a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises a fucosylated anti-B7-H4 antibody or an antigen-binding fragment, for example, wherein at least 85% of the antibody in the composition is fucosylated. In a specific embodiment, a method of administering a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises a fucosylated anti-B7-H4 antibody or an antigen-binding fragment, for example, wherein at least 90% of the antibody in the composition is fucosylated. In a specific embodiment, a method of administering a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises a fucosylated anti-B7-H4 antibody or an antigen-binding fragment, for example, wherein at least 95% of the antibody in the composition is fucosylated. In specific embodiments, a method of administering a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises a fucosylated anti-B7-H4 antibody or an antigen-binding fragment, for example, wherein at least 96% of the antibody in the composition is fucosylated. In specific embodiments, a method of administering a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises a fucosylated anti-B7-H4 antibody or an antigen-binding fragment, for example, wherein at least 97% of the antibody in the composition is fucosylated. In specific embodiments, a method of administering a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises a fucosylated anti-B7-H4 antibody or an antigen-binding fragment, for example, wherein at least 98% of the antibody in the composition is fucosylated. In specific embodiments, a method of administering a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises a fucosylated anti-B7-H4 antibody or an antigen-binding fragment, for example, wherein at least 99% of the antibody in the composition is fucosylated. In a specific embodiment, a method of administering a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises a fucosylated anti-B7-H4 antibody or an antigen-binding fragment, wherein fucose is undetectable in the composition.

In some embodiments, a method of administering a pharmaceutical composition is 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 comprising (a) sequences of complementary determinant regions (CDR) 1, VH CDR2, VH CDR3 and light chain variable regions (VL) CDR1, CDR2 and CDR3, respectively, of SEQ ID NO:5-10; (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.

This document also provides a method of administering a pharmaceutical composition comprising (i) an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof specifically binding to human B7-H4 and comprising 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 NO: 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 said antibody or antigen-binding fragment thereof in the composition are free of fucosylation. In one embodiment, (i) the antibody or its antigen-binding fragment comprises a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 11 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 12, or (ii) the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 21 and a light chain containing the amino acid sequence of SEQ ID NO: 22.

This document also provides a method for administering an additional pharmaceutical composition in addition to a pharmaceutical composition comprising an anti-B7-H4 antibody or an antigen-binding fragment thereof. In such embodiments, the additional pharmaceutical composition comprises a PD-1/PD-L1 antagonist, such as pembrolizumab. In such embodiments, the additional pharmaceutical composition comprising pembrolizumab is provided as a 50 mg lyophilized powder in a single-dose vial for reconstitution, or as a 100 mg/4 ml (25 mg/ml) solution in a single-dose vial. In such embodiments, an amount of the additional pharmaceutical composition is provided to deliver 200 mg of pembrolizumab to a patient in a given administration. The additional pharmaceutical composition may be administered simultaneously or sequentially with the pharmaceutical composition comprising an anti-B7-H4 antibody or an antigen-binding fragment thereof.

4.5 Antibody production and polynucleotides

Antibodies that specifically bind to B7-H4 (e.g., human B7-H4) and their antigen-binding fragments can be produced by any method known in the art for synthesizing antibodies and their antigen-binding fragments, such as by chemical synthesis or by recombinant expression techniques. Unless otherwise stated, the methods described herein employ conventional techniques from molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the scope of this art. These techniques are described, for example, in the references cited herein and are fully illustrated in the literature. See, e.g., Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al. Human,Current Protocols in Molecular Biology,John Wiley&Sons(1987and annualupdates);Current Protocols in Immunology,John Wiley&Sons(19 87and annual updates) Gait (editor) (1984) Oligonucleotide Synthesis: A Practical Approach, IRLPress; Eckstein (editor) (1991) Oligonucleotides and A nalogues: A Practical Approach, IRL Press; Birren B et al., (Editors) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

In some respects, this article provides a method for administering an anti-B7-H4 antibody or an antigen-binding fragment thereof, or a pharmaceutical composition comprising such an antibody or fragment, wherein the antibody or fragment is generated by recombinant expression of a polynucleotide comprising a nucleotide sequence in a host cell.

In some aspects, the anti-B7-H4 antibody or antigen-binding fragment administered according to the methods provided herein comprises a heavy chain variable region encoded by a polynucleotide comprising the nucleotide sequence shown in Table 9 (i.e., SEQ ID NO: 27). In some aspects, the anti-B7-H4 antibody or antigen-binding fragment administered according to the methods provided herein comprises 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 γ (γ) heavy chain constant region. In some aspects, the anti-B7-H4 antibody or antigen-binding fragment administered according to the methods provided herein comprises 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.

Table 9: Polynucleotide sequences encoding the variable region of the heavy chain

In some aspects, the anti-B7-H4 antibody or antigen-binding fragment administered according to the method provided herein comprises a light chain variable region encoded by a polynucleotide comprising the nucleotide sequence shown in Table 10 (i.e., SEQ ID NO: 28). In some aspects, the anti-B7-H4 antibody or antigen-binding fragment administered according to the method provided herein comprises 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 λ light chain constant region. In some aspects, the anti-B7-H4 antibody or antigen-binding fragment administered according to the method provided herein comprises 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.

Table 10: Polynucleotide sequences encoding the variable region of the light chain

In some respects, 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).

In some respects, 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 human γ(γ) heavy chain constant region, 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 human λ light chain constant region.

In some respects, 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.

In some respects, the anti-B7-H4 antibody or antigen-binding fragment administered according to the methods provided herein is encoded by an optimized polynucleotide encoding the anti-B7-H4 antibody or its antigen-binding fragment or its domain, said optimization being, for example, through codon/RNA optimization, substitution with a heterologous signal sequence, and elimination of unstable elements in the mRNA. The method of generating optimized nucleic acids encoding the anti-B7-H4 antibody or its antigen-binding fragment or its domain (e.g., heavy chain, light chain, VH domain, or VL domain) for recombinant expression by introducing codon changes (e.g., codon changes encoding the same amino acid due to the degeneracy of the genetic code) and/or eliminating repressive regions in the mRNA can therefore be carried out using, for example, 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.

Polynucleotides can be in the form of RNA or DNA, for example. 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 some embodiments, the polynucleotide is cDNA or DNA lacking one or more introns. In some embodiments, the polynucleotide is a non-naturally occurring polynucleotide. In some embodiments, the polynucleotide is recombinant-derived. In some embodiments, the polynucleotide is isolated. In some embodiments, the polynucleotide is substantially pure. In some embodiments, the polynucleotide is purified from a natural component.

In some aspects, the vector (e.g., an expression vector) comprises a nucleotide sequence encoding an anti-B7-H4 antibody and its antigen-binding fragment or a domain thereof for recombinant expression in a host cell, preferably a mammalian cell. In some aspects, the cell (e.g., a host cell) comprises such a vector for recombinant expression of the anti-B7-H4 antibody or its antigen-binding fragment (e.g., a human or humanized antibody or its antigen-binding fragment). Therefore, methods for generating the antibody or its antigen-binding fragment described herein may include expressing such antibody or its antigen-binding fragment in a host cell.

Expression vectors can be transferred to cells (e.g., host cells) using conventional techniques, and the resulting cells can then be cultured using conventional techniques to produce antibodies or antigen-binding fragments thereof (e.g., antibodies or antigen-binding fragments comprising the six CDRs, VH, VL, VH and VL, heavy chain, light chain, or heavy chain and light chain of 20502) or their domains (e.g., VH, VL, VH and VL, heavy chain, or light chain of 20502).

In some embodiments, an anti-B7-H4 antibody or an antigen-binding fragment thereof (e.g., an antibody containing a CDR of 20502 or an antigen-binding fragment thereof) is generated in CHOK1SV cells according to the methods provided herein.

In some embodiments, an anti-B7-H4 antibody or an antigen-binding fragment thereof (e.g., an antibody containing a CDR of 20502 or an antigen-binding fragment thereof) is generated in a host cell lacking a functional α-1,6-fucosyltransferase gene (FUT8). In some embodiments, the host cell is a CHO cell.

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

The following examples are provided by way of illustration rather than limitation.

5. Examples

The embodiments in this section are provided illustratively and not restrictively.

5.1 Example 1: Assessment of the prevalence of B7-H4 expression across multiple indications

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

B7-H4 is readily detected in cell membranes and cytosols within tumor tissues harvested from various cancer patients, including invasive ductal carcinoma, triple-negative breast cancer, ovarian cancer, non-small cell lung cancer, and endometrial cancer. Furthermore, it is also frequently expressed in the indications listed in Table 11.

Table 11: Detection of B7-H4 in tumors

B7-H4 is expressed in other cancers, such as breast cancer, kidney cancer (e.g., renal cell carcinoma), bladder cancer (e.g., urothelial carcinoma), pancreatic cancer, and thyroid cancer. See, for example, 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).

5.2 Example 2: Fucosylated and Fucosylated 20502 Antibodies

Antibodies with a reduced fucose content in the glycan moiety exhibit higher ADCC activity compared to fully fucosylated antibodies (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 conventionally fucosylated antibodies and in CHO cell lines engineered to produce fucosylation-free antibodies (CHO-y cells) (ibid.).

Fucosylated and unfucosylated 20502 antibodies were characterized by surface plasmon resonance (SPR). Briefly, anti-human Fab antibody was immobilized on a carboxyl-derived SPR chip surface, and anti-B7-H4 antibody was captured on the resulting surface at 5 μg/ml for 30 seconds. Various concentrations (0 nM, 3.7 nM, 11.1 nM, 33.3 nM, 100 nM, and 300 nM) of B7-H4 IgV-huIgG1 were then flowed through the surface, binding to the anti-B7-H4 antibody during the association phase, followed by buffer washing during the dissociation phase.

B7-H4 IgV-huIgG1:

MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:29)

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

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

Table 12: Allele binding of Fcγ receptor IIIa (FcγRIIIa) V158

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

The T-cell checkpoint blocking activity of fucosylated and non-fucosylated 20502 antibodies was also characterized. In these experiments, primary human T cells were enriched from PBMCs using the EasySep™ Human T Cell Enrichment Kit according to the manufacturer's instructions. The enriched T cells were incubated at 37°C with anti-CD3/anti-CD28 Dynabeads at a ratio of one bead per cell at 2 x ^10⁵ cells/mL. After six days, the beads were magnetically removed, and the T cells were washed and incubated at 37°C with ¹⁰ U/mL IL-2 at 1 x 10⁶ cells/mL. After four days, the T cells were washed and incubated at 37°C with artificial antigen-presenting cells (aAPCs) at a concentration of ² x ^10⁶ cells/mL in the presence of B7-H4 antibody dose titration. aAPC was treated with mitomycin C at 37°C for one hour, and then thoroughly washed before being added to the T cell co-culture. After 72 hours of co-culturing of T cells, aAPC, and B7-H4 antibody, the plate was centrifuged, and the supernatant was harvested and IFNγ production was assessed by ELISA. IFNγ production was plotted against antibody concentration, and EC50 potency was calculated using a nonlinear regression curve fitting (GraphPad Prism).

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

Table 13: T-cell checkpoint blockade efficacy

In another experiment, the ADCC activity of B7-H4-expressing target cell lines against fucosylated and non-fucosylated 20502 antibodies was characterized. Specifically, primary human PBMCs were activated with 200 IU/mL IL-2 at 37°C at a concentration of 1 x ^10⁶ cells/mL. 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, the lysis of target cells was quantified using a fluorometer. Triton/X-treated samples served as maximum lysis controls, while samples treated with culture medium alone served as background lysis controls. The percentage of specific lysis (%) was calculated as follows: [1 - ((sample – culture medium control) / (maximum lysis – culture medium control))] x 100. The percentage of specific lysis (%) was plotted against antibody concentration, and EC50 potency was calculated using a nonlinear regression curve fitting (GraphPad Prism).

B7-H4 antibodies exhibited potent dose-dependent ADCC activity against the SK-BR-3 mammary cell line expressing endogenous B7-H4. Furthermore, the non-fucosylated antibodies showed significantly stronger ADCC activity compared to fucosylated antibodies (Table 14).

Table 14: ADCC Activity

5.3 Example 3: Correlation between ADCC activity and receptor density

B7-H4 density was quantified on the surface of SK-BR-3, HCC1569, ZR-75-1, MDA-MB-48, and HCC1964 cells using FACS according to the manufacturer's instructions. Specifically, ¹ x 10⁵ cells were incubated on ice for 25 min with 15 μg/mL B7-H4 antibody. In parallel, one drop of Quantum ^™ Simply Cellular (QSC) microspheres (pre-coated with escalating concentrations of anti-mouse IgG capture antibody) was also incubated on ice for 25 min with 15 μg/mL B7-H4 antibody. After incubation, the cells and QSC microspheres were precipitated and washed, and samples were collected using flow cytometry. Data were analyzed using FlowJo software. The mean fluorescence intensity (MFI) was calculated and entered into a spreadsheet. A regression was automatically calculated that correlated the fluorescence channel value of each bead with its pre-assigned antibody binding capacity (ABC) value. ABC values were assigned once the MFI values of the labeled cells were also added to the template.

The ADCC activity of the B7-H4 antibody against B7-H4-expressing target cell lines with varying B7-H4 cell surface densities was evaluated. Specifically, ¹ x 10⁴ SK-BR-3, HCC1569, ZR-75-1, MDA-MB-468, or HCC1964 target cells were co-incubated with dose-titrated B7-H4 antibody at 4°C. After 25 min, single-use vials of Jurkat-huCD16 reporter cells from Promega were thawed, and 7.5 x ^10⁴ cells were added to the target cell/B7-H4 antibody mixture and incubated at 37°C. After 24 h, the samples were brought to room temperature (RT) and incubated with Bio-Glo buffer. Substrate and luminescence were quantified using an EnVision multilabel reader. Data were plotted as luminescence versus antibody concentration, and EC50 potency was calculated using nonlinear regression curve fitting (GraphPad Prism).

B7-H4 antibody ADCC activity depends on B7-H4 cell surface density: the amount of maximum ADCC activity decreases as the number of cell surface molecules decreases. Furthermore, non-fucosylated antibodies exhibit improved ADCC activity compared to fucosylated antibodies, particularly against target cells with lower B7-H4 cell surface density levels (Figure 1).

5.4 Example 4: In vivo antitumor efficacy in combination with anti-PD-1 antibody

Methods: Eight-week-old female BALB/c mice were purchased from Charles River Laboratories (Hollister, CA) and acclimatized for up to two weeks before the start of the study. The mouse mammary gland cell line 4T1 was engineered to express a chimeric protein containing the extracellular domain of mouse B7-H4 and the transmembrane domain of mouse B7H3. Tumor cells were orally implanted into the mammary fat pads of mice at a dose of 0.5 x ^10⁵ cells/50 μl/mouse. Before inoculation, cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) for no more than three passages. Cells were grown in a humid environment with 5% CO₂ at 37°C. After reaching 80-85% confluence, cells were harvested and resuspended in serum-free RPMI 1640, and injected into the mammary fat pads of each mouse ventrally.

Tumor growth in mice was monitored twice weekly after cell implantation. The length and width of each tumor were measured using calipers, and the volume was calculated using the formula: Tumor volume (mm3) = (width (mm) × length (mm)2) / 2. On the day treatment began, all tumors were measured, outliers were excluded, and mice were randomly assigned to treatment groups. For anti-B7-H4 treatment, a 20502 mouse alternative called 20502-msIgG2a-F was used, which contains the 20502 variable region fused with fucosylated mouse IgG2a. As a control, mice were administered msIgG2a (anti-HEL). Starting from day 11 post-inoculation, 20502-msIgG2a-F or msIgG2a was administered four times weekly via intravenous (iv) injection. Starting from day 11 post-inoculation, anti-PD-1 (a modified version of RMP1-14 (Bio X cells) containing the Fc-silenced msIgG2a domain) was administered twice weekly via intraperitoneal (ip) injection. Continue to measure the tumor at least twice a week until the tumor volume exceeds 10% of the animal's body weight or approximately 2000 ^mm³ .

Results: Changes in tumor size, mean tumor volume, and survival percentage are shown in Figures 1A, 1B, and 1C, respectively. Treatment with 20502-msIgG2a-F or anti-PD-1 significantly reduced tumor growth compared to the msIgG2a control (p<0.05). Co-administration of 20502-msIgG2a-F and anti-PD-1 significantly enhanced tumor growth inhibition compared to either monotherapy (p<0.05). Furthermore, the combination therapy resulted in complete tumor regression in 5 out of 12 mice. P-values were calculated using a one-way ANOVA analysis of the calculated tumor volume on each day of the study, and multiple comparisons were made between groups.

In other experiments, 20502-msIgG2a-F as a monotherapy showed dose-dependent antitumor activity in engineered 4T1 models at low doses of 1 mg/kg and 3 mg/kg (Figure 2). Combining 0.3 mg/kg, 3 mg/kg, or 30 mg/kg doses of 20502-msIgG2a-F with an anti-PD-1 antibody (5 mg/kg) in engineered 4T1 models and similar engineered B16 (melanoma) models produced synergistic antitumor activity at almost all tested doses of 20502-msIgG2a-F (Figure 3). (PD-1 antibody was administered three times on the same day to the 4T1 model with the first three doses of 20502-msIgG2a-F, and twice on the same day to the B16 model with the second and fourth doses of 20502-msIgG2a-F). This synergistic effect was particularly unexpected at the 0.3 mg/kg dose, as 20502-msIgG2a-F alone at this dose did not significantly affect tumor volume (Figure 2). Furthermore, the efficacy (survival percentage) observed when 20502-msIgG2a-F was combined with anti-PD1 at a dose of 0.3 mg/kg was surprisingly comparable to, or even better than, the efficacy observed when combined with anti-PD1 at doses of 3 mg/kg or 30 mg/kg. Therefore, these results indicate that the synergistic combination of anti-PD1 and anti-B7-H4 antibodies at doses where the latter is ineffective as monotherapy offers advantages not only in efficacy but also in safety.

5.5 Example 5: Non-clinical pharmacokinetics

The pharmacokinetics (PK) and toxicokinetics (TK) of fucosylated 20502 were evaluated in mice, rats, and cynomolgus monkeys following weekly and/or repeated intravenous (IV) administration. The PK characteristics observed were consistent across all studies. In all species, fucosylated 20502 exhibited linear PK, with exposure increasing proportionally to the dose (area under the serum concentration-time curve [AUC]). Weekly exposure (AUC _{0–7 days} ) increased approximately two-fold after four weekly administrations of 20502 between the first and last doses; however, a steady-state was not reached. No significant sex differences were observed in the serum fucosylated 20502 concentration-time curves. In cynomolgus monkeys (spanning two different studies), the estimated half-life from recovery animals ranged from approximately 8.8 to 12 days at dose levels ranging from 1 to 100 mg/kg. The estimated half-life in rats following a single intravenous infusion of 40 mg/kg was approximately 13.2 days. The PK characteristics of fucosylated 20502 in animals support IV infusion in humans at a 3-week (Q3W) dosing regimen.

5.6 Example 6: Toxicology

Toxicological studies of fucosylated 20502 were conducted in rats and cynomolgus monkeys. These studies included preliminary single-dose pharmacokinetic (PK)/tolerance studies in rats, preliminary repeated-dose toxicity studies in cynomolgus monkeys, repeated-dose toxicity studies of investigational new drug (IND) achieved with good laboratory practice (GLP) in rats and cynomolgus monkeys, and GLP tissue cross-reactivity studies using human, rat, and cynomolgus monkey tissues.

In a preliminary single-dose tolerance study in rats, animals received doses up to 40 mg/kg as intravenous (IV) infusions over 30 minutes. Fucosylated 20502 had no effect on clinical outcomes, body weight, food consumption, clinicopathological (serological or hematological) assessments, overall outcomes, organ weight, or histopathological assessments.

In a preliminary repeated-dose toxicology study, cynomolgus monkeys received intravenous doses of up to 100 mg/kg of fucosylated 20502 four times weekly as 30-minute intravenous infusions. All doses were well tolerated by the monkeys. No unplanned mortality related to the test article or changes attributable to fucosylated 20502 administration were observed during assessments of clinical outcomes, body weight, clinicopathology, necropsy, organ weight, or histopathological parameters.

In repeated-dose GLP toxicology studies, fucosylated 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 assessed during a 6-week recovery period following the final administration. Assessment parameters included ophthalmological examination, clinical observations, body temperature, body weight, food consumption, hematology, coagulation, clinical chemistry, urinalysis, organ weight, and macroscopic and microscopic assessments. In the cynomolgus monkey study, electrocardiogram (ECG) was also evaluated to assess potential cardiotoxicity.

During the evaluation of GLP rat studies, fucosylated 20502 was generally well tolerated, and no toxic effects were attributed to it. The No Adverse Effect Level (NOAEL) in Sprague Dawley rats was considered to be 100 mg/kg/dose.

In the GLP cynomolgus monkey study, fucosylated 20502 was generally well tolerated, and no adverse events (AEs) attributable to fucosylated 20502 were observed in any of the assessed parameters. A higher incidence of diarrhea was observed at the end of the dosing period in the higher-dose groups during the study. This may be related to fucosylated 20502 exposure, given the higher morbidity in affected animals at medium and high doses and those experiencing diarrhea later in the dosing period. No microscopic changes were observed in the intestines of animals treated with fucosylated 20502 (including those with diarrhea); therefore, this finding is considered non-adverse but may be related to the test article. One death occurred during the study. One animal in the medium-dose recovery group died on day 35 of the study, 14 days after the last dose. Clinical observations, macroscopic and microscopic assessments were consistent with the diagnosis of intestinal volvulus. Intestinal volvulus occurs occasionally in cynomolgus monkeys, and this is considered a spontaneous symptom in these animals and is unrelated to the test article. The NOAEL in cynomolgus monkeys is considered to be 100 mg/kg/dose.

In addition to in vivo toxicology studies, a GLP compliance tissue cross-reactivity study was conducted to compare the binding of fucosylated 20502 to a group of 36 tissues from rats, cynomolgus monkeys, and humans. The results showed that the binding pattern of fucosylated 20502 was similar in the three species and was limited to mammary epithelium.

Therefore, fucosylated 20502 was well tolerated in cynomolgus monkeys and rats. The NOAEL in both species was considered to be 100 mg/kg/dose, which was the highest dose tested when administered intravenously four times a week.

5.7 Example 7: Study on the phase 1a dose escalation/safety introduction and phase 1b dose expansion of the combination of fucosylated 20502 and pembrolizumab.

For convenience, in this embodiment, the unfucosylated 20502 is referred to as A-20502.

Title: Phase 1a/1b study of the combination of anti-B7-H4 antibody and pembrolizumab (anti-PD1 antibody) in patients with advanced solid tumors

Phase 1a goals and endpoints:

Phase 1b goals and endpoints:

In some embodiments, pembrolizumab for injection is provided in a sterile, preservative-free, white to off-white lyophilized powder in single-dose vials. Each vial can be reconstituted and diluted for intravenous infusion. Each 2 mL of such reconstituted solution contains 50 mg of pembrolizumab, formulated with L-histidine (3.1 mg), polysorbate 80 (0.4 mg), and sucrose (140 mg). Hydrochloric acid/sodium hydroxide may be included to adjust the pH to 5.5. The lyophilized powder is reconstituted by adding 2.3 mL of sterile water for injection (USP) along the wall of the vial rather than directly onto the lyophilized powder (resulting in a concentration of 25 mg/mL). The vial is slowly rotated (without shaking) for up to 5 minutes to remove air bubbles.

In other embodiments, pembrolizumab can be administered as a single injection of a 100 mg/4 mL (25 mg/mL) solution in a vial. The injection is a sterile, preservative-free, clear to slightly milky white, colorless to slightly yellow solution that requires dilution for intravenous infusion. Each vial contains 100 mg of pembrolizumab in a 4 mL solution. Each 1 mL solution contains 25 mg of pembrolizumab, prepared in the following solutions: L-histidine (1.55 mg), polysorbate 80 (0.2 mg), sucrose (70 mg), and water for injection (USP).

Study Design: This is a phase 1a/1b open-label, multicenter study designed to evaluate the dosing, safety, tolerability, pharmacokinetics (PK), pharmacodynamics, and preliminary efficacy of the combination of A-20502 and pembrolizumab in advanced solid tumors.

This study includes a phase 1a dose escalation/safety introduction (phase 1a dose escalation) and a phase 1b dose extension (phase 1b dose extension) for the combination of A-20502 and pembrolizumab.

Pre-screening of archived tumor tissue (or fresh biopsy tissue obtained if no archived tissue is available) can be performed, and the expression level of B7-H4 (a transmembrane protein of the B7 family, also known as B7S1, B7x, or VTCN1) can be tested by immunohistochemistry (IHC) performed in a central laboratory for patients in phase 1a dose exploration and phase 1b dose expansion, as well as for biomarker analysis.

In some implementations, archived tumor tissue and/or fresh biopsy tissue are provided as follows:

Phase 1a dose escalation:

• Provide archival tumor tissue (or fresh biopsy tissue if no archival tissue is available) for retrospective biomarker analysis of the patient.

Phase 1a dose exploration:

• Archived tumor tissue is used to assess B7-H4 expression levels via an IHC test performed in the central laboratory for pre-screening and for biomarker analysis; if archived tissue is unavailable, fresh biopsy tissue will be used for this test.

Fresh biopsy tissue will be used during screening and post-processing.

Phase 1b dose expansion:

• Archived tumor tissue is used to assess B7-H4 expression levels via an IHC test performed in a central laboratory for pre-screening and for biomarker analysis; if archived tissue is unavailable, fresh biopsy tissue is used for this test.

• During screening and post-processing, fresh biopsy tissue will be used for expanded pharmacodynamic analysis for a subset of patients (e.g., at least 10 patients in every 30 patients in the cohort).

Further details are provided below for each study phase of the Phase 1a dose escalation and Phase 1b dose expansion sections of this summary.

Phase 1a Safety Introduction (A-20502 in combination with pembrolizumab): At least 3 patients will be recruited to participate in A-20502 as a monotherapy in combination with 200 mg pembrolizumab Q3W at the maximum tolerated dose (MTD) and/or recommended dose (RD) and to evaluate dose-limiting toxicities (DLT). An additional 10 patients in total may be treated with A-20502 in combination with pembrolizumab at RD. If necessary, the dose of A-20502 may be reduced according to the reduction algorithm described below. The proposed dose levels are:

·1aC1: A-20502(RD) + pembrolizumab 200mg IV Q3W

·1aC2: A-20502 (10mg/kg) + pembrolizumab 200mg IV Q3W

·1aC3: A-20502 (3mg/kg) + pembrolizumab 200mg IV Q3W

Abbreviations: IV = intravenous; Q3W = once every 3 weeks; RD = recommended dose.

In some implementations, the RD is 20 mg/kg. In a phase 1a/1b monotherapy study in patients with advanced solid tumors, A-20502 was well tolerated at doses up to 20 mg/kg without dose-limiting toxicities. Based on pharmacokinetic studies in patients in the phase 1a/1b monotherapy study, the trough concentration of A-20502 observed at the RD of 20 mg/kg is expected to achieve ≥95% receptor saturation for both B7-H4 and FcγIIIa in terms of affinity. A-20502 achieves dose-proportional exposure at doses ≥0.3 mg/kg, with a half-life of 1 to 2 weeks. Serum concentrations of A-20502 at 20 mg/kg were similar over time across different tumor types (breast, ovarian, and endometrial), regardless of pembrolizumab administration.

Other dose levels (e.g., 0.1, 0.3, 1, or 20 mg/kg) can be used in combination studies, based on factors such as safety, tolerability, objective response, pharmacokinetics, and pharmacodynamics, as well as estimates of effective exposure inferred from nonclinical data. For example, lower or intermediate doses of A-20502 based on safety data can be used. The DLT criteria for the combination of A-20502 and pembrolizumab are as follows:

DLT during the 1a dose escalation period is defined as any of the following events, regardless of attribution (excluding those clearly caused by an underlying disease or external factors):

• Any grade 3 or higher non-hematologic toxicity (excluding grade 3 nausea, vomiting, and diarrhea) occurring within 21 days of treatment.

• Grade 3 nausea, vomiting, and diarrhea lasting >72 hours within the first 21 days of treatment, despite best supportive care.

• Recorded infection with high fever and/or an absolute neutropenia (ANC) <1.0 × ^10⁹ /L within the first 21 days of treatment, grade 4 neutropenia lasting longer than 7 days, grade 4 thrombocytopenia (<25.0 × ^10⁹ /L), or grade 3 thrombocytopenia (<50.0–25.0 × ^10⁹ /L) with bleeding.

• An aspartate aminotransferase/alanine aminotransferase (AST/ALT) ratio >3 × upper limit of normal (ULN) and concurrent total bilirubin >2 × ULN are not associated with liver involvement in cancer.

• Any Level 4 experimental value, without considering clinical sequelae

• Other Level 3 experimental values that are not clinically significant according to the Investigator and Sponsor Agreement and have not resolved within 72 hours.

Specific DLT Considerations for the Combination of A-20502 and Pembrolizumab

Because pembrolizumab is a known immune checkpoint inhibitor, and one of the proposed mechanisms of action for A-20502 is immune checkpoint blockade, immune-related adverse events (irAEs) are expected with this combination. irAEs are defined as clinically significant AEs of unknown etiology related to investigational drug exposure and consistent with immune-mediated mechanisms. Against this backdrop, the first occurrence of the following irAEs will not be considered DLT, as they hold promise for treatment with immunotherapy:

• Grade 3 tumor flare (defined as localized pain, irritation, or rash at the site of a known or suspected tumor).

Grade 3 rash

• Grade 3 immune-related adverse events (irAEs) that resolve to Grade 1 or lower within 14 days.

• Transient (resolved within 6 hours of onset) Grade 3 infusion-related adverse events

A second occurrence of these events in the same patient or in different patients (excluding grade 3 tumour) will be considered DLT.

The DLT assessment interval begins on day 1 of treatment after the infusion begins and continues for 21 days. The algorithm outlined in the table below will be applied to dosing decisions. In response to DLT, the dose of A-20502 may be reduced as needed. The MTD and/or RD of A-20502 in combination with pembrolizumab will be the dose at which DLT occurs in patients ≤1/3–6.

Algorithm for dose reduction decisions regarding the safety of A-20502 when combined with pembrolizumab in Phase 1a:

1b period portfolio expansion:

Phase 1b may consist of a cohort prospectively identified by IHC as having B7-H4 expression. In some implementations, there are two cohorts of A-20502 combined with pembrolizumab in selected tumor types:

• Cohort 1bC1: TNBC (A-20502 combined with pembrolizumab)

• Cohort 1bC2: Ovarian cancer (A-20502 combined with pembrolizumab)

In another implementation, the phase 1b combination has an initial cohort, and other cohorts, such as TNBC, can subsequently recruit in the phase 1b trial:

• Cohort 1bC1: Ovarian cancer (A-20502 in combination with pembrolizumab)

In some implementations, up to three additional combined queues can be added.

Based on newly available clinical and translational data from the study, additional cohorts of tumor types to be treated with A-20502 in combination with pembrolizumab can be opened during the trial (e.g., 30 patients or fewer in any single cohort).

Dosage: In some implementations, A-20502 will be administered as a single dose via intravenous infusion over 60 minutes (±5 minutes) on day 1 of each 21-day cycle, Q3W. The dose of A-20502 will be based on the patient's body weight on day 1 of cycle 1 (C1D1). After cycle 1, the A-20502 dose will be recalculated at each infusion follow-up visit as long as the patient's body weight has changed by >10% relative to day 1 of cycle 1.

Pembrolizumab will be administered at a dose of 200 mg via an IV infusion over 30 minutes (±5 minutes) after the completion of the IV infusion at A-20502, starting on day 1 of cycle 1 and repeated on day 1 of each 21-day cycle Q3W.

There is no pre-specified maximum dose for the A-20502 and pembrolizumab combination. Patients may continue receiving both drugs in combination, according to their study-specified cohort/dosage, until the investigator assesses disease progression or the patient meets any other protocol-defined withdrawal criteria. If a patient discontinues one of the two drugs, the patient may continue receiving the other drug alone.

If the benefit/risk assessment favors the continued administration of the investigational treatment (e.g., if the patient continues to experience clinical benefits and tolerate the treatment as assessed by the investigator), then treatment beyond disease progression may be administered to patients with progressive disease in accordance with the Response Evaluation Criteria in SolidTumors version 1.1 (RECIST v1.1).

Study duration: The study duration for individual patients includes screening (up to 28 days), treatment, and end-of-treatment (EOT) follow-up, which includes follow-up visits at approximately 28 (±7) days and 100 (±7) days after the last dose. Because all patients are eligible to receive treatment before disease progression, the actual treatment duration for each individual patient will vary depending on the expected time to progression for their respective tumor type.

In addition, patients recruited in the Phase 1b dose expansion will be followed up for up to 2 years of survival (LTFU, including scans and survival status, Q12W).

Number of patients: The estimated number of patients planned for this study is as follows, but this number may be adjusted accordingly.

Phase 1a may recruit 6 to 22 patients or 10 to 22 patients who will receive the combination of A-20502 and pembrolizumab in a safety-introducing trial. Another one or two cohorts, such as 30 or fewer patients, will be recruited to evaluate the A-20502 and pembrolizumab combination.

Qualification criteria:

Inclusion criteria: Inclusion criteria for period 1a

The inclusion criteria for recruiting patients who have progressed to phase 1a are as follows:

1) Histologically confirmed solid tumors, excluding primary central nervous system (CNS) tumors.

2) Unresectable, locally advanced, or metastatic disease.

3) Be able to understand and sign an informed consent form (ICF) approved by the Institutional Review Board/Independent Ethics Committee (IRB/IEC) before any research-specific evaluation.

4) Existing therapies known to provide clinical benefit to the patient’s condition are refractory or intolerable.

5) According to RECIST v1.1, all patients must have at least one measurable lesion at baseline; tumor sites located in previously irradiated areas or areas receiving other local area therapies are not considered measurable unless lesion progression has been confirmed.

6) Sufficient washout period for prior anticancer therapy (i.e., ≥5 half-lives or 4 weeks from the last dose, whichever is shorter).

7) If there is a file tumor tissue and the patient agrees to provide the file tumor for retrospective biomarker analysis, or if there is no file tissue, the patient must undergo a fresh tumor biopsy during screening (a biopsy is required for patients in the Phase 1a dose exploration portion).

8) Be ≥18 years old when signing the ICF.

9) The Eastern Cooperative Oncology Group (ECOG) performance status score is 0 or 1.

10) Researchers believe that life expectancy is at least 3 months.

11) Willing and able to comply with all research procedures.

12) Previous radiation therapy must be completed at least 2 weeks before the first dose of the study drug.

13) The previous radiopharmaceutical (e.g., strontium, samarium) must be completed at least 8 weeks before the first dose of the study drug.

14) Previous surgeries requiring general anesthesia must be completed at least one week prior to the administration of the first dose of the study drug. Surgeries requiring local/epidural anesthesia must be completed at least 72 hours prior to the administration of the first dose of the study drug. The patient must recover from any surgery.

15) Screening laboratory values must meet the following conditions:

hematology

a. Neutrophils ≥1200 cells/μL

b. Platelet count ≥75× ^10³ /μL

c. Hemoglobin (Hb) ≥ 9.0 g/dL

kidney:

Serum creatinine <1.5×ULN or creatinine clearance (CrCl) ≥40 mL/min (using the Cockcroft/Gault formula)

liver:

d. AST and ALT ≤ 3 × ULN (AST and ALT < 5 × ULN are permissible in patients with liver metastases)

e. Bilirubin <1.5×ULN (except for patients with Gilbert's syndrome, who must have total bilirubin <3 mg/dL)

16) Negative serum β-human chorionic gonadotropin (β-hCG) pregnancy test ≤96 hours before treatment on day 1 of cycle 1 (only for women of fertility).

17) In sexually active patients (women and men of childbearing potential), the willingness to use two effective methods of contraception, one of which must be a physical barrier method (condom, diaphragm, or cervical/vacuum cap), is required for six months following the last dose of A-20502. Other effective forms of contraception include:

◆Permanent sterilization (hysterectomy and/or bilateral oophorectomy, or bilateral tubal ligation or vasectomy) must be performed at least 6 months prior to screening.

◆Women of childbearing age who have been on stable oral contraceptive therapy or have an intrauterine device or implanted device for at least 90 days prior to the study, or who have adopted abstinence from sexual activity as a lifestyle choice.

The inclusion criteria for Phase 1b are as follows:

18) All inclusion criteria for period 1a (exception: inclusion criterion #1).

19) B7-H4 expression is positive in archived or fresh tumor samples, as assessed by an accompanying validated central laboratory IHC assay.

20) A history of other malignant tumors is permitted, provided that there has been definitive treatment within the past 2 years and there is no sign of recurrence (exceptions: non-melanoma skin cancer, lobular carcinoma in situ, and cervical cancer in situ are permitted if there has been definitive treatment within the past 2 years).

Additional cohort-specific inclusion criteria for phase 1b

Cohort 1bC1 Ovarian Cancer:

• Diagnosis of recurrent epithelial ovarian cancer, primary peritoneal cancer, or fallopian tube cancer that is histologically or cytologically confirmed and refractory to existing therapies known to offer clinical benefit.

• Disease that has progressed during or after at least two prior treatment regimens, including at least one platinum-based regimen, or for which additional chemotherapy is intolerable.

• No prior treatment with anti-PD1 or PD-L1 targeted drugs

Queue 1bC2 TNBC :

• Metastatic TNBC confirmed by histology or cytology

• At least two previous-line systemic chemotherapy therapies, at least one of which was administered in a metastatic setting.

• No prior treatment with anti-PD1 or PD-L1 targeted drugs

Eligibility criteria: Exclusion criteria (Phase 1a and Phase 1b)

Patients meeting any of the following criteria may be excluded:

1) Systemic immunosuppressive medications, such as steroids or absorbed topical steroids (prednisone at a dose >10 mg/day or an equivalent daily dose), must be discontinued at least 2 weeks prior to the first dose of the study drug. Short-term high-dose steroids, sustained low-dose steroids (prednisone <10 mg/day), inhaled, intranasal, intraocular, and intra-articular steroid injections are permitted.

2) During the screening, the New York Heart Association (NYHA) indicated a decline in heart function > grade 2.

3) Uncontrolled or severe heart disease, such as unstable angina.

4) During screening, according to institutional guidelines, the heart rate-corrected QT interval (QTc) is >450 msec for men or >470 msec for women.

5) History of anti-drug antibodies (ADA), severe allergy, anaphylactic reaction or other infusion-related reactions to previous biological reagents.

6) Any component of the study product (IP) formulation and/or known hypersensitivity to pembrolizumab.

7) Vaccines administered within 4 weeks prior to the first dose of the investigational drug (e.g., human papillomavirus [HPV] vaccine). Inactivated seasonal influenza vaccines can be given to patients without restriction before and during treatment. Influenza vaccines containing live virus or other clinically designated vaccines for infectious diseases (i.e., pneumothorax, chickenpox, etc.) may be permitted, but must be discussed with the sponsor’s medical monitor, and a clean-up period for the investigational drug may be required before and after vaccination.

8) Patients with an unresolved infection or a history of chronic, acute, clinically significant infection (viral, bacterial, fungal, or other) that the investigator believes may exclude them from exposure to biological agents or may pose a safety risk to them.

9) Patients with abnormal serum chemistry values that the investigator considers clinically significant. This includes patients exhibiting clinical signs and symptoms related to their abnormal serum chemistry values, as well as patients with asymptomatic serum chemistry values that the investigator considers clinically significant (e.g., hypokalemia or hyponatremia).

10) Any uncontrolled medical condition or mental illness that the researcher believes poses a safety risk to the patient or interferes with participation in the study or the interpretation of individual patient outcomes.

11) Pregnancy or breastfeeding.

12) Active, known, or suspected autoimmune diseases requiring treatment within the past 2 years. Patients with type 1 diabetes, hypothyroidism requiring only hormone replacement, skin conditions not requiring systemic treatment (such as vitiligo, psoriasis, or alopecia), or conditions not expected to recur without external triggers are permitted to be recruited.

13) A known history of a positive human immunodeficiency virus (HIV) 1 or 2 test or a known history of acquired immunodeficiency syndrome (AIDS).

14) Positive hepatitis B surface antigen (HBsAg) test, or detectable hepatitis C virus RNA (HCV RNA) indicating acute or chronic infection.

15) Persistent adverse events resulting from prior treatment > grade 1 (excluding grade 2 alopecia or peripheral neuropathy) based on the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE).

16) Symptomatic interstitial lung disease or inflammatory pneumonia.

17) Untreated or active CNS or leptomeningeal metastases. Patients are eligible if they have been treated for metastases and have neurologically recovered to baseline or are neurologically stable (excluding residual signs or symptoms related to CNS treatment) at least 2 weeks prior to the first dose of the study drug.

18) Evidence of coagulopathy or bleeding diathesis. Allow the patient to receive a stable therapeutic dose of anticoagulants.

19) Complete the transfusion of blood or platelets within 72 hours prior to the first dose of the study drug.

20) Any uncontrolled inflammatory gastrointestinal (GI) disease, including Crohn's disease and ulcerative colitis.

Testing and observation: Safety assessment includes vital signs, weight, physical examination, ECOG score, laboratory tests (hematology, serology and urinalysis), electrocardiogram (ECG), and monitoring for adverse events (AEs) and concomitant medications.

Archived tumor tissue and consent forms provided for archived tumors (or consent to fresh tumor biopsy during screening) will be used for biomarker analysis to explore the relationship between baseline target levels, tumor immunophenotype, and pharmacodynamic response.

Tumor tissue was collected before and during treatment from all patients in the Phase 1a dose exploration and a subset of patients in the Phase 1b dose expansion (up to 15 patients in every 30-patient cohort) for expanded pharmacodynamic analysis.

Efficacy assessment may include radiographic imaging every 6 weeks. Response will be assessed according to RECIST v1.1.

Statistical methods

Efficacy Analysis

ORR can be aggregated by frequency and percentage at the 90% confidence interval (CI) for each dose/cohort. Duration of response (DOR) for complete response (CR) and partial response (PR) patients can be aggregated by the number of responders, the number and percentage of events/censoring, and the Kaplan-Meier estimate of median DOR at the 95% CI. Progression-free survival (PFS) for treated patients can be aggregated by the number and percentage of PFS patients at each dose/cohort. PFS can also be aggregated using the Kaplan-Meier method at the 95% CI. ORR, DOR, and PFS can be determined using RECIST v1.1.

Pharmacokinetic analysis

Individual and mean (±SD) serum A-20502 concentration-time data can be tabulated and plotted by dose level/cohort. PK parameters can be tabulated and summarized by dose level/cohort, where appropriate and applicable. The effect of immunogenicity on A-20502 exposure can be assessed, tabulated, and summarized by dose level/cohort, where data allows. Individual and mean (±SD) _Cmax and _Cvalence of pembrolizumab concentration-time data can be tabulated and plotted by cohort. If data are available, PK parameters, such as cumulative ratio and steady-state arrival, can be tabulated and summarized by dose level/cohort.

Immunogenicity analysis

Baseline ADA-positive subjects are defined as subjects who have an ADA-positive sample at baseline. ADA-positive subjects are those who have at least one ADA-positive sample relative to baseline after treatment initiation. The frequency distributions of baseline ADA-positive subjects and ADA-positive subjects after treatment initiation can be summarized separately for A-20502 and pembrolizumab.

Pharmacodynamic analysis

The significance of selected pharmacodynamic biomarkers in tumor and peripheral blood samples before and during treatment was evaluated.

***

The scope of this invention is not limited to the specific embodiments described herein. In fact, various modifications to the invention will become apparent to those skilled in the art, in addition to those described, based on the foregoing description and drawings. Such modifications are intended to fall within the scope of the appended claims.

All references cited herein (e.g., publications, patents, or patent applications) are incorporated herein in their entirety by reference, and for all purposes, the extent of such reference is as if each individual reference (e.g., publications, patents, or patent applications) were specifically and individually incorporated herein in their entirety by reference for all purposes.

Other embodiments are defined in the following claims.

sequence list

<110> Five Prime Therapeutics, Inc.

<120> Combination therapy for cancer

<130> 3986.018PC03

<150> US 62/854,494

<151> 2019-05-30

<150> US 62/802,091

<151> 2019-02-06

<150> US 62/745,464

<151> 2018-10-15

<160> 41

<170> PatentIn version 3.5

<210>  1

<211>  282

<212> PRT

<213> Homo sapiens

<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> Crab-eating macaque 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> Rodents 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 amino acids

<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 amino acids

<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

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

100 105

<210> 13

<211>  26

<212> PRT

<213> Artificial Sequence

<220>

<223> VH FR1

<400> 13

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

20

<210> 18

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> VL FR2

<400> 18

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

1 5 10 15

<210> 19

<211> 32

<212> PRT

<213> Artificial Sequence

<220>

<223> VL FR3

<400> 19

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys

20 25 30

<210>  20

<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>  21

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Full-length heavy chain amino acids

<400>  21

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 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 acids

<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

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

<400>  24

cggaccgtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300

agcttcaacaggggagagtgt 321

<210> 25

<211> 330

<212> PRT

<213> Artificial Sequence

<220>

<223> 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> Heavy-chain constant structural domain

<400> 26

gcctccacca aggggcccatc 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 cgcggggagga 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> Nucleotides encoding variable heavy chains

<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> Nucleotide sequence encoding variable light chain

<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> 447

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-PD-1 antibody heavy chain

<400> 30

Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 31

<211>  218

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-PD-1 antibody light chain

<400> 31

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 32

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-PD-1 antibody VH amino acid sequence

<400> 32

Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 33

<211>  111

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-PD-1 antibody VL amino acid sequence

<400> 33

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 34

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-PD-1 antibody VH-CDR1

<400> 34

Asn Tyr Tyr Met Tyr

1 5

<210> 35

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-PD-1 antibody VH-CDR2

<400> 35

Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe Lys

1 5 10 15

Asn

<210> 36

<211>  11

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-PD-1 antibody VH-CDR3

<400> 36

Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr

1 5 10

<210> 37

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-PD-1 antibody VL-CDR1

<400> 37

Arg Ala Ser Lys Gly Val Ser Thr Ser Gly Tyr Ser Tyr Leu His

1 5 10 15

<210> 38

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-PD-1 antibody VL-CDR2

<400> 38

Leu Ala Ser Tyr Leu Glu Ser

1 5

<210> 39

<211>  9

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-PD-1 antibody VL-CDR3

<400> 39

Gln His Ser Arg Asp Leu Pro Leu Thr

1 5

<210> 40

<211>  288

<212> PRT

<213> Homo sapiens

<400>  40

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 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> 41

<211>  290

<212> PRT

<213> Homo sapiens

<400>  41

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 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 (54)

1. Use of an anti-B7-H4 antibody or its antigen-binding fragment and an anti-PD-1 antibody or its antigen-binding fragment in the manufacture of a medicament for treating a solid tumor in a human subject, wherein: (i) Administering about 0.1 to about 20 mg/kg of the said anti-B7-H4 antibody or its antigen-binding fragment, wherein the anti-B7-H4 antibody or its antigen-binding fragment specifically binds to human B7-H4 and comprises the following amino acid sequences: heavy chain variable region (VH) complementarity-determining region (CDR) 1 as shown in SEQ ID NO:5, VH CDR2 as shown in SEQ ID NO:6, VH CDR3 as shown in SEQ ID NO:7, light chain variable region (VL) CDR1 as shown in SEQ ID NO:8, VL CDR2 as shown in SEQ ID NO:9, and VLCDR3 as shown in SEQ ID NO:10; and (ii) Administer about 200 mg of the anti-PD-1 antibody or its antigen-binding fragment thereof, wherein the anti-PD-1 antibody or its antigen-binding fragment comprises the amino acid sequence VH CDR1 as shown in SEQ ID NO:34, the amino acid sequence VH CDR2 as shown in SEQ ID NO:35, the amino acid sequence VH CDR3 as shown in SEQ ID NO:36, the amino acid sequence VL CDR1 as shown in SEQ ID NO:37, the amino acid sequence VL CDR2 as shown in SEQ ID NO:38, and the amino acid sequence VL CDR3 as shown in SEQ ID NO:39. 2. (a) the use in manufacturing a pharmaceutical composition comprising an anti-B7-H4 antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable excipient, and (b) the use in manufacturing a pharmaceutical composition comprising an anti-PD-1 antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable excipient, said pharmaceutical composition for treating a solid tumor in a human subject, wherein: (a) The anti-B7-H4 antibody or its antigen-binding fragment specifically binds to human B7-H4 and comprises the amino acid sequences as shown in SEQ ID NO:5 (VH CDR1), SEQ ID NO:6 (VH CDR2), SEQ ID NO:7 (VH CDR3), SEQ ID NO:8 (VL CDR1), SEQ ID NO:9 (VL CDR2), and SEQ ID NO:10 (VL CDR3). At least 95% of the anti-B7-H4 antibody or its antigen-binding fragment in the composition is unfucosylated, and Administer about 0.1 to about 20 mg/kg of the antibody or its antigen-binding fragment; and (b) The anti-PD-1 antibody or its antigen-binding fragment comprises the amino acid sequence VHCDR1 as shown in SEQ ID NO:34, VHCDR2 as shown in SEQ ID NO:35, VHCDR3 as shown in SEQ ID NO:36, VLCDR1 as shown in SEQ ID NO:37, VLCDR2 as shown in SEQ ID NO:38, and VLCDR3 as shown in SEQ ID NO:39, and about 200 mg of the anti-PD-1 antibody or antigen-binding fragment is administered. 3. The use as described in claim 1 or 2, wherein the subject is administered about 20 mg/kg of the anti-B7-H4 antibody or its antigen-binding fragment. 4. The use as described in claim 1 or 2, wherein the subject is administered about 10 mg/kg of the anti-B7-H4 antibody or its antigen-binding fragment. 5. The use as described in claim 1 or 2, wherein the subject is administered about 3 mg/kg of the anti-B7-H4 antibody or its antigen-binding fragment. 6. The use as described in claim 1 or 2, wherein the subject is administered about 1 mg/kg of the anti-B7-H4 antibody or its antigen-binding fragment. 7. The use as described in claim 1 or 2, wherein the subject is administered about 0.3 mg/kg of the anti-B7-H4 antibody or its antigen-binding fragment. 8. The use as described in claim 1 or 2, wherein the subject is administered about 0.1 mg/kg of the anti-B7-H4 antibody or its antigen-binding fragment. 9. The use as described in claim 1 or 2, wherein the anti-B7-H4 antibody or its antigen-binding fragment and the anti-PD-1 antibody or its antigen-binding fragment are administered simultaneously. 10. The use as claimed in claim 9, wherein the anti-B7-H4 antibody or its antigen-binding fragment and the anti-PD-1 antibody or its antigen-binding fragment are administered as separate formulations on the same day. 11. The use as described in claim 1 or 2, wherein the anti-B7-H4 antibody or its antigen-binding fragment and the anti-PD-1 antibody or its antigen-binding fragment are administered sequentially. 12. The use as claimed in claim 11, wherein the anti-PD-1 antibody or its antigen-binding fragment is administered after administration of the anti-B7-H4 antibody or its antigen-binding fragment. 13. The use as described in claim 1 or 2, wherein the anti-B7-H4 antibody or its antigen-binding fragment is administered approximately every three weeks. 14. The use as described in claim 1 or 2, wherein the anti-PD-1 antibody or its antigen-binding fragment is administered approximately every three weeks. 15. The use as described in claim 1 or 2, wherein the anti-B7-H4 antibody or its antigen-binding fragment and the anti-PD-1 antibody or its antigen-binding fragment are each administered approximately every three weeks. 16. The use as described in claim 1 or 2, wherein the anti-B7-H4 antibody or its antigen-binding fragment is administered intravenously. 17. The use as described in claim 1 or 2, wherein the anti-PD-1 antibody or its antigen-binding fragment is administered intravenously. 18. The use as described in claim 1 or 2, wherein B7-H4 has been detected in the solid tumor by immunohistochemistry (IHC) prior to application. 19. The use as claimed in claim 1 or 2, wherein the anti-B7-H4 antibody or its antigen-binding fragment comprises the amino acid sequence VH as shown in SEQ ID NO:11 and/or the amino acid sequence VL as shown in SEQ ID NO:12. 20. The use as described in claim 1 or 2, wherein the anti-B7-H4 antibody or antigen-binding fragment comprises a heavy chain constant region and/or a light chain constant region. 21. The use as claimed in claim 20, wherein the heavy chain constant region is the human immunoglobulin IgG ₁ heavy chain constant region, and/or wherein the light chain constant region is the human immunoglobulin IgG κ light chain constant region. 22. The use as claimed in claim 1 or 2, wherein the anti-B7-H4 antibody or its antigen-binding fragment comprises a heavy chain constant region as shown in SEQ ID NO:25 and/or a light chain constant region as shown in SEQ ID NO:23. 23. The use as described in claim 1 or 2, wherein the anti-B7-H4 antibody or its antigen-binding fragment comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:21 and/or a light chain with an amino acid sequence as shown in SEQ ID NO:22. 24. The use as described in claim 1 or 2, wherein the anti-B7-H4 antibody or its antigen-binding fragment is a human antibody or its antigen-binding fragment. 25. The use as claimed in claim 1, wherein the anti-B7-H4 antibody or its antigen-binding fragment is unfucosylated. 26. The use as described in claim 1 or 2, wherein the anti-B7-H4 antibody or its antigen-binding fragment is a full-length antibody. 27. The use as described in claim 1 or 2, wherein the anti-B7-H4 antibody or its antigen-binding fragment is an antigen-binding fragment. 28. The use as claimed in claim 27, wherein the antigen-binding fragment comprises Fab, Fab', F(ab') ₂ , single-chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, intracellular antibody, IgGΔCH2, microantibody, F(ab') ₃ , tetravalent antibody, trivalent antibody, bivalent antibody, single-domain antibody, Fcab, ^mAb2 , (scFv) ₂ , or scFv-Fc. 29. The use as claimed in claim 2, wherein the fucosylation of the anti-B7-H4 antibody or its antigen-binding fragment is undetectable in the composition. 30. The use as described in claim 1 or 2, wherein the anti-PD-1 antibody or antigen-binding fragment comprises VH with an amino acid sequence as shown in SEQ ID NO:32 and VL with an amino acid sequence as shown in SEQ ID NO:33. 31. The use as described in claim 1 or 2, wherein the anti-PD-1 antibody is pembrolizumab. 32. The use as described in claim 31, wherein 200 mg of pembrolizumab is administered. 33. The use as described in claim 1 or 2, wherein (i) the anti-B7-H4 antibody or its antigen-binding fragment specifically binds to human B7-H4 and comprises the amino acid sequence VH as shown in SEQ ID NO:11 and the amino acid sequence VL as shown in SEQ ID NO:12, the anti-B7-H4 antibody or antigen-binding fragment is undetectably fucosylated, and 3, 10, or 20 mg/kg of the anti-B7-H4 antibody or its antigen-binding fragment is administered; and (ii) 200 mg of pembrolizumab is administered; (i) and (ii) were administered intravenously as separate formulations on the same day. 34. The use as claimed in claim 33, wherein the anti-B7-H4 antibody comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:21 and a light chain with an amino acid sequence as shown in SEQ ID NO:22. 35. The use as claimed in claim 1 or 2, wherein the solid tumor expresses B7-H4. 36. The use as described in claim 1 or 2, wherein the solid tumor is unresectable, locally advanced, or metastatic. 37. The use as described in claim 1 or 2, wherein 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. 38. The use as described in claim 37, wherein the solid tumor is breast cancer or ovarian cancer. 39. The use as described in claim 38, wherein the solid tumor is breast cancer. 40. The use as claimed in claim 39, wherein the breast cancer is advanced breast cancer. 41. The use as claimed in claim 38, wherein the breast cancer is triple-negative breast cancer. 42. The use as claimed in claim 38, wherein the breast cancer is hormone receptor (HR) positive breast cancer. 43. The use as described in claim 37, wherein the non-small cell lung cancer is squamous cell carcinoma. 44. The use as described in claim 1 or 2, wherein the subject has not received prior therapy with a PD-1/PD-L1 antagonist. 45. The use as described in claim 1 or 2, wherein the number of immune cells in the tumor is monitored. 46. The use as described in claim 1 or 2, wherein the number of natural killer (NK) cells, CD4+ cells and/or CD8+ cells in the tumor is monitored. 47. The use as described in claim 1 or 2, wherein cytokine levels in the subject are monitored. 48. The use as described in claim 1 or 2, wherein the levels of IL-2, IL-6, IL-10, TNF and/or interferon-γ (IFNγ) in the subject are monitored. 49. (i) the use of an anti-B7-H4 antibody that specifically binds to human B7-H4 and (ii) an anti-PD-1 antibody in the manufacture of a medicament for treating a solid tumor in a human subject, wherein (i) Administer approximately 20 mg/kg of an anti-B7-H4 antibody, said anti-B7-H4 antibody specifically binding to human B7-H4 and comprising the amino acid sequence VH as shown in SEQ ID NO:11 and the amino acid sequence VL as shown in SEQ ID NO:12; and (ii) Administer approximately 200 mg of an anti-PD-1 antibody, said anti-PD-1 antibody comprising the amino acid sequence VH as shown in SEQ ID NO:32 and the amino acid sequence VL as shown in SEQ ID NO:33. The anti-B7-H4 antibody and the anti-PD-1 antibody are administered intravenously approximately every three weeks. 50. (a) the use in manufacturing a pharmaceutical composition comprising an anti-B7-H4 antibody that specifically binds to human B7-H4 and a pharmaceutically acceptable excipient, and (b) the use in manufacturing a pharmaceutical composition comprising an anti-PD-1 antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable excipient, said pharmaceutical composition for treating a solid tumor in a human subject, wherein (a) The anti-B7-H4 antibody that specifically binds to human B7-H4 comprises the amino acid sequence VH as shown in SEQ ID NO:11 and the amino acid sequence VL as shown in SEQ ID NO:12. At least 95% of the anti-B7-H4 antibody in the composition is unfucosylated, and Administer approximately 20 mg/kg of the antibody; and (b) The anti-PD-1 antibody or its antigen-binding fragment comprises the amino acid sequence VH as shown in SEQ ID NO:32 and the amino acid sequence VL as shown in SEQ ID NO:33, and approximately 200 mg of the anti-PD-1 antibody or antigen-binding fragment is administered. (a) and (b) are administered intravenously approximately every three weeks. 51. The use as described in claim 49 or 50, wherein the anti-B7-H4 antibody comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:21 and a light chain with an amino acid sequence as shown in SEQ ID NO:22. 52. The use as described in claim 49 or 50, wherein the anti-PD-1 antibody comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:30 and a light chain with an amino acid sequence as shown in SEQ ID NO:31. 53. The use as described in claim 49 or 50, wherein the solid tumor is breast cancer or ovarian cancer. 54. The use as described in claim 53, wherein the breast cancer is triple-negative breast cancer.