EA045594B1 - ANTIBODIES TO B7-H4 AND METHODS OF THEIR APPLICATION - Google Patents

Description

Field of technology

The present invention relates to antibodies that specifically bind to human B7-H4, and to methods for producing and using these antibodies, for example, to detect B7-H4.

State of the art

B7-H4 (also known as B7x, B7-S1 and VTCN1) is an immunoregulatory molecule that shares homology with other members of the B7 family, including PD-L1. It is a type I transmembrane protein consisting of both IgV and IgC ectodomains. While B7-H4 expression in normal tissues is relatively limited at the protein level, B7-H4 is expressed in several solid tumors, such as gynecologic carcinomas of the breast, ovary, and endometrium. B7-H4 expression in tumors tends to correlate with poor prognosis. The B7-H4 receptor is unknown but is thought to be expressed on the surface of T cells. B7-H4 is thought to directly inhibit T cell activity.

Given the expression and function of B7-H4, there is a need for antibodies that specifically bind to B7-H4 and the use of these antibodies to detect B7-H4, including, for example, the use of immunohistochemistry (IHC) to detect B7-H4 in samples cancer.

The essence of the invention

The present invention provides antibodies that specifically bind to B7-H4 and the use of these antibodies to detect B7-H4, including, for example, the use of immunohistochemistry (IHC) to detect B7-H4 in cancer samples.

In certain embodiments, the present invention provides an isolated antibody or antigen binding fragment thereof that specifically binds to human B7-H4 comprising a heavy chain variable region (VH) complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 22, CDR2 VH region containing the amino acid sequence of SEQ ID NO: 23, CDR3 of the VH region containing the amino acid sequence of SEQ ID NO: 24, CDR1 of the variable light chain (VL) region containing the amino acid sequence of SEQ ID NO: 25, CDR2 of the VL region containing the amino acid sequence SEQ ID NO: 26, 27 or 28, and the CDR3 sequence of the VL region containing the amino acid sequence of SEQ ID NO: 29.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO: 6, 7, or 8. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VL region containing the amino acid sequence of SEQ ID NO: 9, 10, or eleven.

In certain embodiments, the present invention provides an isolated antibody or antigen binding fragment thereof that specifically binds to human B7-H4, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 6, 7 or 8.

In certain embodiments, the present invention provides an isolated antibody or antigen binding fragment thereof that specifically binds to human B7-H4, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9 , 10 or 11. In certain embodiments, the present invention provides an isolated antibody or antigen binding fragment thereof that specifically binds to human B7-H4, comprising a heavy chain variable region and a light chain variable region, comprising the amino acid sequences of (a) SEQ ID NO: 6 and 9 respectively;

(b) SEQ ID NO: 6 and 10 respectively;

(c) SEQ ID NO: 6 and 11, respectively;

(d) SEQ ID NO: 7 and 9 respectively;

(f) SEQ ID NO: 7 and 10, respectively;

(f) SEQ ID NO: 7 and 11 respectively;

(g) SEQ ID NO: 8 and 9 respectively;

(h) SEQ ID NO: 8 and 10 respectively;

(i) SEQ ID NO: 8 and 11 respectively;

(j) SEQ ID NO: 89 and 19, respectively;

(k) SEQ ID NO: 90 and 19, respectively; or (l) SEQ ID NO: 91 and 19, respectively.

In certain embodiments, the antibody or antigen binding fragment thereof further comprises a heavy chain constant region. In certain embodiments, the heavy chain constant region is a mouse IgG1 or IgG _2a heavy chain constant region. In certain embodiments, the antibody or antigen binding fragment further comprises a light chain constant region. In certain embodiments, the light chain constant region is a mouse IgGK light chain constant region.

In certain embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 16, 17, 18, 89, 90 or 91. In certain embodiments, the antibody or antigen binding fragment thereof comprises a light chain comprising the amino acid sequence SEQ ID NO: 16, 17, 18, 89, 90 or 91. NO: 19, 30, or 31. In certain embodiments, the antibody or antigen binding fragment further comprises a heavy chain and a light chain comprising the amino acid sequences of (a) SEQ ID NO: 16 and 19, respectively;

(b) SEQ ID NO and 30 respectively;

(c) SEQ ID NO and 31, respectively;

and 19 respectively;

(d) SEQ ID NO (e) SEQ ID NO: 17 and 30 respectively;

(f) SEQ ID NO: 17 and 31 respectively;

(g) SEQ ID NO: 18 and 19 respectively;

(h) SEQ ID NO: 18 and 30 respectively; or (i) SEQ ID NO: 18 and 31, respectively.

In certain embodiments, the present invention provides an isolated antibody or antigen binding fragment thereof that specifically binds to human B7-H4, wherein the antibody or antigen binding fragment thereof comprises a VH region CDR1, a VH region CDR2, a VH region CDR3, a VL region CDR1, a VL region CDR2 and a CDR3 region of the VL antibody selected from the group consisting of J512, J513, J514, J515, J516, J517, J518, J519, J520, J521 and J522. In certain embodiments, the CDRs are Kabat-defined CDRs, Chotia-defined CDRs, or AbM-defined CDRs. In certain embodiments, the present invention provides an isolated antibody or antigen binding fragment thereof that specifically binds to the same human B7-H4 epitope as the antibody or antigen binding fragment thereof provided by the present invention. In certain embodiments, the present invention provides an isolated antibody or antigen binding fragment thereof that competitively inhibits the binding of an antibody or antigen binding fragment thereof of another antibody or antigen binding fragment thereof of another antibody or antigen binding fragment thereof of the present invention to human B7-H4.

In certain embodiments, the antibody or antigen binding fragment thereof is a murine antibody or antigen binding fragment thereof.

In certain embodiments, the antibody or antigen-binding fragment thereof is a full-length antibody. In certain embodiments, the antibody or antigen binding fragment thereof is an antigen binding fragment. In certain embodiments, the antigen binding fragment comprises a Fab, Fab', F(ab')2, single chain Fv fragment (scFv), disulfide-linked Fv fragment, minibody, F(ab') ₃ , diabody, (scFv)2, or scFv- Fc.

In certain embodiments, the antibody or antigen-binding fragment thereof further comprises a detectable label. In certain embodiments, the present invention provides an isolated polynucleotide comprising a nucleic acid molecule encoding a heavy chain variable region, or an antibody heavy chain, or an antigen binding fragment thereof provided by the present invention. In certain embodiments, the nucleic acid molecule encodes the VH region of SEQ ID NO: 6, 7, or 8 or the heavy chain of SEQ ID NO: 16, 17, or 18.

In certain embodiments, the nucleic acid molecule contains the sequence SEQ ID NO: 12, 13, 14, 93, 94, or 95.

In certain embodiments, the present invention provides an isolated polynucleotide comprising a nucleic acid molecule encoding a light chain variable region or an antibody light chain or an antigen binding fragment thereof provided by the present invention. In certain embodiments, the nucleic acid molecule encodes the VL region of SEQ ID NO: 9, 10, or 11 or the light chain of SEQ ID NO: 19, 30, or 31. In certain embodiments, the nucleic acid molecule contains the sequence of SEQ ID NO: 15, 96 or 97.

In certain embodiments, the present invention provides an isolated polynucleotide comprising a nucleic acid molecule encoding a heavy chain variable region or heavy chain of an antibody or an antigen binding fragment thereof provided by the present invention, and a light chain variable region or light chain of an antibody or antigen binding fragment thereof provided by the present invention. in the present invention.

In certain embodiments, the present invention provides an isolated vector containing a polynucleotide provided by the present invention.

In certain embodiments, the present invention provides a host cell

- 2 045594 (for example, an isolated host cell) containing a polynucleotide proposed in the present invention, a vector proposed in the present invention, or a first vector containing a polynucleotide encoding a light chain variable region or a light chain proposed in the present invention, and a second a vector containing a polynucleotide encoding a heavy chain variable region or heavy chain as provided in the present invention. In certain embodiments, the host cell is a CHO or HEK cell.

In certain embodiments, the present invention provides a method for producing an antibody or antigen binding fragment thereof that specifically binds to human B7-H4, comprising culturing a host cell of the present invention such that the nucleic acid molecule is expressed and the antibody or its antigen-binding fragment.

In certain embodiments, the present invention provides an isolated antibody or antigen binding fragment thereof that specifically binds to human B7-H4 and is encoded by a polynucleotide of the present invention.

In certain embodiments, the present invention provides a method for detecting B7-H4 in a sample, comprising contacting the sample with an antibody or antigen binding fragment thereof provided by the present invention. In certain embodiments, the present invention provides a method for detecting B7-H4 in a sample, comprising contacting the sample with an anti-B7-H4 antibody or antigen binding fragment thereof provided by the present invention and detecting binding of the antibody or antigen binding fragment thereof to B7-H4. H4. In certain embodiments, the sample is obtained from a cancer in a subject. In certain embodiments, the method further includes administering a therapeutic anti-B7-H4 antibody or an antigen-binding fragment thereof to the subject upon detection of B7-H4. In certain embodiments, the present invention provides a method of treating a cancer expressing B7-H4 in a subject, the method comprising administering to the subject a therapeutic antibody to B7-H4 or an antigen-binding fragment thereof, wherein B7-H4 was detected in a sample obtained from the cancer. diseases using the antibody or antigen-binding fragment thereof proposed in the present invention.

In certain embodiments, the method further includes detecting B7-H4 in a sample obtained from a cancer.

In certain embodiments, the detected B7-H4 is cell membrane B7-H4. In certain embodiments, the detected B7-H4 is cytoplasmic B7-H4. In certain embodiments, the detected B7-H4 is whole cell B7-H4.

In certain embodiments, B7-H4 is found in circulating tumor cells.

In certain embodiments, the sample is solid tissue, biopsy, ascites, aspirates, fluid extract, blood plasma, serum, cerebrospinal fluid, lymph fluid, external section of skin, respiratory tract, intestinal or genitourinary tract, tears, saliva, milk, tumor, or organ obtained from the subject.

In certain embodiments, the cancer is selected from the group consisting of breast cancer, ductal carcinoma, endometrial cancer, ovarian cancer, non-small cell lung cancer, pancreatic cancer, thyroid cancer, kidney cancer, and bladder cancer. In certain embodiments, the breast cancer is triple negative breast cancer and the non-small cell lung cancer is squamous cell cancer.

In certain embodiments, the detection method uses enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), or immunohistochemistry (IHC). In certain embodiments, the detection method uses IHC and the concentration of the antibody or antigen binding fragment thereof provided herein is from about 1 μg/ml to about 50 μg/ml. In certain embodiments, the concentration of an antibody or antigen binding fragment thereof provided herein is from about 1 μg/ml to about 20 μg/ml. In certain embodiments, the concentration of the antibody or antigen binding fragment thereof provided by the present invention is about 10 μg/ml.

In certain embodiments, the subject is a human.

In certain embodiments, the present invention provides a kit comprising an antibody or antigen binding fragment thereof provided by the present invention, and a) a detection reagent, b) a B7-H4 antigen, c) a therapeutic anti-B7-H4 antibody, or d) a combination thereof.

In certain embodiments of a method or kit provided herein, the therapeutic antibody or antigen binding fragment comprises a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3 of 20502 or 22213. In certain embodiments, CDRs are CDRs defined by Ka

- 3,045,594 baht, CDR determined by Chotia or CDR determined by AbM. In certain embodiments, the VH region CDR1, VH region CDR2, VH region CDR3, VL region CDR1, VL region CDR2, and VL region CDR3 comprise the amino acid sequences of SEQ ID NOs: 32-37, respectively, or the amino acid sequences of SEQ ID NOs: 58-63, respectively . In certain embodiments, the therapeutic antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 64. In certain embodiments, the therapeutic antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 55 or SEQ ID NO: 65. In certain embodiments, a therapeutic antibody or antigen binding fragment comprises (i) a heavy chain variable region comprising an amino acid the sequence SEQ ID NO: 54, and a light chain variable region containing the amino acid sequence SEQ ID NO: 55; or (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 64 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 65.

In certain embodiments, the therapeutic antibody or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 74. In certain embodiments, the therapeutic antibody or antigen binding fragment thereof comprises a light chain comprising the amino acid sequence SEQ ID NO: 57 or SEQ ID NO: 75. In certain embodiments, the therapeutic antibody or antigen binding fragment thereof comprises (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 56 and a light chain comprising the amino acid sequence of SEQ ID NO: 57; or (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 74 and a light chain containing the amino acid sequence of SEQ ID NO: 75.

Brief description of graphic materials

In fig. 1 shows IHC staining images obtained using antibodies to B7-H4 A57.1, AET_AB_J516 and AET_AB_J512 (see example 4).

In fig. Figure 2 shows computational image analysis (Definiens) of IHC staining (see Example 4).

In fig. Figure 3 shows the segmentation of positive cells with different intensity levels (see example 4).

In fig. Figure 4 shows the number and ratio of cells associated with each level of staining intensity and obtained using antibodies to B7-H4 A57.1, AET_AB_J516 and AET_AB_J512 (see example 4).

In fig. 5A-5C show a comparison of B7-H4 expression (DAB intensity) in whole cells, membranes and cytoplasm of B7-H4 positive cells. In fig. 5A shows B7-H4 expression (DAB intensity) using antibody A57.1. In fig. 5B shows B7-H4 expression (DAB intensity) using antibody J512. In fig. 5C shows B7-H4 expression (DAB intensity) using J516 antibody (see Example 4).

Detailed description of the invention

The present invention provides antibodies (eg, monoclonal antibodies) and antigen-binding fragments thereof that specifically bind to B7-H4 (eg, human B7-H4). Antibodies to B7-H4 and their antigen-binding fragments can be used to detect B7-H4, for example, by immunohistochemistry in a cancer sample.

Also provided are isolated nucleic acids (polynucleotides), such as complementary DNA (cDNA), encoding such antibodies and their antigen-binding fragments. Additionally provided are vectors (eg, expression vectors) and cells (eg, host cells) containing nucleic acids (polynucleotides) encoding such antibodies and antigen-binding fragments thereof. Methods for producing such antibodies and their antigen-binding fragments are also proposed. In other aspects, the present invention provides methods for detecting B7-H4, for example, in a cancer sample. Related compositions (eg, detection compositions) and kits are also provided.

Terminology.

The term B7-H4 as used herein refers to mammalian B7-H4 polypeptides, including, but not limited to, native B7-H4 polypeptides and isoforms of B7-H4 polypeptides. B7-H4 covers full-length unprocessed B7-H4 polypeptides, as well as forms of B7-H4 polypeptides that arise as a result of processing within the cell. The term human B7-H4, as used herein, refers to a polypeptide comprising the amino acid sequence SEQ ID NO: 1. A B7-H4 polynucleotide, B7-H4 nucleotide or B7-H4 nucleic acid refers to a polynucleotide encoding B7-H4.

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The term antibody means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations thereof, through at least one antigen recognition site in the variable region of the immunoglobulin molecule. The term antibody as used herein includes intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, antibody-containing fusion proteins, and any other modified immunoglobulin molecule, provided that the antibodies exhibit the desired biological activity. The antibody may be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their severe constant domains. chains called alpha, delta, epsilon, gamma and mu respectively. The different classes of immunoglobulins have distinct and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

The term antibody fragment refers to a portion of an intact antibody.

An antigen-binding fragment, antigen-binding domain, or antigen-binding region refers to the portion of an intact antibody that specifically binds an antigen. The antigen binding fragment may comprise an antigen recognition site for an intact antibody (eg, complementarity determining regions (CDRs) sufficient for specific antigen binding). Examples of antigen binding antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies and single chain antibodies. The antigen-binding antibody fragment can be obtained from any animal species, such as rodents (eg, mouse, rat or hamster) and humans, or can be produced artificially.

The terms anti-B7-H4 antibody and antibody that binds to B7-H4 refer to an antibody that is capable of specifically binding to B7-H4 with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent to target B7-H4 . The terms specifically binding, immunospecifically binding, immunospecifically recognizing and specifically recognizing in the context of the present invention are similar terms in the context of antibodies or antigen-binding fragments thereof. These terms indicate that an antibody or antigen-binding fragment thereof binds to an epitope through its antigen-binding domain and that binding entails some complementarity between the antigen-binding domain and the epitope. Accordingly, an antibody that specifically binds to human B7-H4 (SEQ ID NO: 1) may also bind to B7-H4 from other species (e.g., cynomolgus, mouse, and/or rat B7-H4) and/or B7-H4 proteins produced from other human alleles, but the extent of binding of the anti-B7-H4 antibody to unbound non-B7-H4 protein (e.g., other members of the B7 protein family, such as PD-L1) is less than about 10% of the binding of the antibody to B7-H4, as measured, for example, by radioimmunoassay (RIA).

A monoclonal antibody or antigen-binding fragment thereof refers to a population of homogeneous antibody or antigen-binding fragment involved in highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants. The term monoclonal antibody or antigen-binding fragment thereof covers both intact and full-length monoclonal antibodies, as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins containing part antibodies, and any other modified immunoglobulin molecule containing a recognition site. In addition, a monoclonal antibody or antigen-binding fragment thereof refers to such antibodies and antigen-binding fragments thereof produced by any number of methods, including, but not limited to, hybridomas, phage selection, recombinant expression and transgenic animals.

The terms variable region or variable domain in the context of the present invention are used interchangeably and are common in the art. A variable region generally refers to a portion of an antibody, typically a portion of the light or heavy chain, typically the amino terminus 110 to 120 amino acids or 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which vary by sequence among antibodies and are used in the binding and specificity of a particular antibody to its particular antigen. Variation in the sequence is concentrated in those regions called complementarity determining regions (CDRs), while the more highly conserved regions in the variable domain are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the light and heavy chain CDRs are primarily responsible for antibody-antigen interaction and specificity. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region is a rodent or mouse variable region.

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

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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 known in the art and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody, or antigen binding fragment thereof. In certain aspects, CDRs may be defined in accordance with the Kabat numbering system (see, for example, Kabat E. A. & Wu T. T. (1971), Ann. NY Acad. Sci., 190:382-391; and Kabat E. A. et al. ( 1991), Sequences of Proteins of Immunological Interest, Fifth Edition, U.S., Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs in an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which may optionally include one or two additional amino acids following 35 (referred to in the Kabat numbering scheme as 35A and 35B) (CDR1) , amino acid positions 50 to 65 (CDR2) and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs in an antibody light chain molecule are typically present at amino acid positions 24-34 (CDR1), amino acid positions 50-56 (CDR2), and amino acid positions 89-97 (CDR3). In a specific embodiment, the CDRs of the antibodies described in the present invention were determined in accordance with the Kabat numbering scheme.

Chothia instead refers to the arrangement of structural loops (Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)). The end of the Chotia CDR-H1 loop when numbered according to the Kabat numbering system varies between H32 and H34 depending on the length of the loop (this is due to the fact that in the Kabat numbering scheme inserts are placed at H35A and H35B; if neither 35A nor 35B are present, the loop ends on 32; if only 35A is present, the loop ends on 33; if both 35A and 35B are present, the loop ends on 34). AbM hypervariable regions represent a compromise between Kabat's CDRs and Chotia's structural loops and are used by Oxford Molecular software to model AbM antibodies.

A loop According to Kabat ABM By Chotia L1 L24-L34 L24-L34 L24-L34 L2 L50-L56 L50-L56 L50-L56 L3 L89-L97 L89-L97 L89-L97 H1 Н31-Н35В Н26-Н35В N26-N32..34 (Numbering Kabat) H1 H31-H35 H26-H35 H26-H32 (Numbering Chotia) H2 H50-H65 H50-H58 H52-H56 NZ N95-N102 N95-N102 N95-N102

The terms constant region and constant domain are used interchangeably in the context of the present invention and have common meanings in the art. A constant region is a portion of an antibody, such as the carboxyl-terminal portion of the light and/or heavy chain, that is not directly involved in the binding of the antibody to an antigen, but which may exhibit various effector functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule usually has a more conserved amino acid sequence relative to the variable domain of the immunoglobulin. In certain aspects, the antibody or antigen-binding fragment thereof comprises a constant region or portion thereof sufficient to cause antibody-dependent cell-mediated cytotoxicity (ADCC). The term heavy chain in the context of the present invention, when used in relation to an antibody, can refer to any single type, for example alpha (a), delta (δ), epsilon (ε), gamma (γ) and mu (μ), based on the amino acid sequence of the constant domain, which leads to the antibody classes IgA, IgD, IgE, IgG, and IgM, respectively, including IgG subclasses, such as IgG1, IgG2, IgG ₃ , and IgG4. The amino acid sequences of the heavy chains are well known in the art. In specific embodiments, the heavy chain is a human heavy chain. In specific embodiments, the heavy chain is a rodent or mouse heavy chain.

The term light chain as used herein when used in relation to an antibody may refer to any other type, such as kappa (k) or lambda (λ), based on the amino acid sequence of the constant domains. The amino acid sequences of the light chains are well known in the art. In specific embodiments, the light chain is a human light chain. In specific embodiments, the light chain is a rodent or mouse light chain.

The term chimeric antibodies or antigen-binding fragments thereof refers to antibodies or antigen-binding fragments thereof, the amino acid sequence being derived from two

- 6 045594 or more species. Typically, the variable region of both the light and heavy chains corresponds to the variable region of antibodies or antigen-binding fragments thereof obtained from a single mammalian species (eg, mouse, rat, rabbit, etc.) with the desired specificity, affinity and potency, while while the constant regions are homologous to sequences of antibodies or antigen-binding fragments thereof obtained from another (usually a human) to avoid eliciting an immune response in that species. The term humanized antibody or antigen-binding fragment thereof refers to forms of non-human (eg, murine) antibodies or antigen-binding fragments thereof that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (eg, murine) sequences. Typically, humanized antibodies or antigen-binding fragments thereof are human immunoglobulins in which residues from the complementarity determining region (CDR) are replaced by residues from the CDR of a non-human species (eg, mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and characteristics (with grafted CDR) (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 residues of the human immunoglobulin Fv framework region (FR) are replaced by corresponding residues in an antibody or fragment of a non-human species that has the desired specificity, affinity and potency. The humanized antibody or antigen binding fragment thereof may be further modified by substitution of additional residues in either the Fv framework region and/or non-human CDR residues to refine and optimize the antibody, antigen binding fragment specificity, affinity and/or potency. In general, a humanized antibody or antigen binding fragment thereof will contain variable domains comprising all or substantially all of the CDR regions that correspond to a non-human immunoglobulin, while all or substantially all of the FR regions are those of the human immunoglobulin consensus sequence. The humanized antibody or antigen binding fragment thereof may also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically human immunoglobulin. Examples of methods used to create humanized antibodies are described in US Pat. No. 5,225,539; Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994); and Roguska et al., Protein Eng., 9(10):895-904 (1996). In some embodiments, the humanized antibody is a surface-edited antibody.

The term human antibody or antigen-binding fragment thereof means an antibody or antigen-binding fragment thereof having an amino acid sequence derived from the human immunoglobulin gene locus, wherein such antibody or antigen-binding fragment is produced using any method known in the art. This definition of a human antibody or antigen-binding fragment thereof includes intact or full-length antibodies and fragments thereof.

Binding affinity generally refers to the strength of the total sum of non-covalent interactions between one binding site of a molecule (eg, an antibody or an antigen-binding fragment thereof) and its binding partner (eg, an antigen). Unless otherwise specified, in the context of the present invention, binding affinity refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, an antibody or antigen-binding fragment thereof and an antigen). The affinity of a molecule X for its partner Y can usually be represented by a dissociation constant ( _KD ). Affinity can be measured and/or expressed in several ways known in the art, including, but not limited to, the equilibrium dissociation constant (KD) and the equilibrium association constant (K _A ). K _D is calculated from the ratio k _off /k _on , while K _A is calculated from the ratio k _on /k _off . k _on refers to the rate constant of association of, for example, an antibody or an antigen-binding fragment thereof with an antigen, and ko _ff refers to the dissociation of, for example, an antibody or an antigen-binding fragment thereof with an antigen, k _on and k _off can be determined by methods known to one skilled in the art techniques such as BIAcore® or KinExA.

The term epitope, as used herein, is a term in the art and refers to a localized region of an antigen to which an antibody or antigen-binding fragment thereof can specifically bind. An epitope may be, for example, contiguous amino acids of a polypeptide (linear or continuous epitope) or an epitope, for example, may be combined from two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous or non-contiguous epitopes). In certain embodiments, the epitope to which an antibody or antigen-binding fragment thereof specifically binds can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid liquid chromatography with electrospray mass spectrometry), oligopeptide scanning assays and/or mutagenesis mapping (eg, site-directed mutagenesis mapping). In the case of X-ray crystallography, crystallization can be carried out using light

- 7 045594 other methods known in the art (for example, Giege R. et al. (1994), Acta Crystallogr. D Biol. Crystallogr., 50(Pt 4):339-350; McPherson A. (1990), Eur J Biochem 189:1-23 Chayen N E (1997) Structure 5:1269-1274 McPherson A (1976) J Biol Chem 251:6300-6303 Crystals of the antibody/antigen binding fragment: antigen can be studied using well known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, for example, Meth Enzymol (1985), vols 114 & 115, eds Wyckoff H.W. 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, ed Carter C.W.; Roversi P. et al (2000), Acta Crystallogr. D Biol. Crystallogr., 56(Pt 10):1316-1323). Mutagenesis mapping studies can be carried out using any method known to one skilled in the art. See, for example, Champe M. et al. (1995), J. Biol. Chem., 270:1388-1394; and Cunningham V.S. & Wells J.A. (1989), Science, 244:1081-1085 for a description of mutagenesis methods, including alanine scanning mutagenesis methods.

An anti-B7-H4 antibody that binds to the same epitope as the reference anti-B7-H4 antibody refers to an antibody that binds to the same B7-H4 amino acid residues as the reference anti-B7-H4 antibody. The ability of an anti-B7-H4 antibody to bind to the same epitope as a reference anti-B7-H4 antibody is determined using a hydrogen/deuterium exchange assay (see Coales et al., Rapid Commun. Mass Spectrom., 2009, 23:639- 647).

An antibody is said to competitively inhibit the binding of a reference antibody to a given epitope if it binds to the same epitope or an overlapping epitope of the reference antibody such that it to some extent blocks the binding of the reference antibody to the epitope. Competitive inhibition can be determined by any method known in the art, for example, competitive ELISA. The antibody can be said to competitively inhibit the reference antibody from binding to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%. The polypeptide, antibody, polynucleotide, vector, cell or composition that is isolated is a polypeptide, antibody, polynucleotide, vector, cell or composition that is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells or compositions include those that have been purified to such an extent that they are no longer present in the form in which they occur naturally. In some embodiments, the isolated antibody, polynucleotide, vector, cell, or composition is substantially pure. The term substantially pure in the context of the present invention refers to a 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 purity of at least 99%.

The terms polypeptide, peptide and protein are used interchangeably in the present invention to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be separated by non-amino acids. In addition, these terms include an amino acid polymer that has been modified naturally or by intervention; for example, by the formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification such as conjugation with a labeled moiety. The term also includes, for example, polypeptides containing one or more amino acid analogues (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that since the polypeptides of the present invention are antibody based, in some embodiments the polypeptides may occur as single chains or linked chains.

Percent identity refers to the degree of identity between two sequences (eg, amino acid sequences or nucleic acid sequences). The percentage identity can be determined by aligning the two sequences, introducing gaps to maximize the identity of the two sequences. The alignment can be created using programs known in the art. For purposes of the present invention, nucleotide sequence alignments can be performed using the default blastn program, and amino acid sequence alignments can be performed using the default blastn program (see National Center for Biotechnology Information (NCBI) on the Internet website www.ncbi.nlm.nih.gov).

The term host cell in the context of the present invention can be any type of cell, for example, a primary cell, a cell in culture, or a cell from a cell line. In specific embodiments, the term host cell refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, for example, due to mutations or environmental influences that may occur in subsequent generations or integration of the nucleic acid molecule into the genome of the host cell.

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The term overexpression of B7-H4 in a particular sample of a tumor, tissue or cells refers to B7-H4 (a B7-H4 polypeptide or a nucleic acid encoding such a polypeptide) that is present at a level higher than that which is present in unaffected tissue or cells of the same of the same type or origin near a tumor or cancer. Such overexpression may be caused by, for example, mutation, gene amplification, increased transcription, or increased translation.

The term primary antibody or antigen-binding fragment thereof as used herein refers to an antibody or fragment that specifically binds to a target antigen in a sample. The primary antibody is typically the first antibody used in an immunohistochemistry (IHC) procedure. In one embodiment, the primary antibody is the only antibody that is used in the IHC procedure. The term secondary antibody or antigen-binding fragment thereof as used herein refers to an antibody or fragment that specifically binds to a primary antibody, thereby forming a bridge between the primary antibody and a subsequent reagent, if present. A secondary antibody is typically a second antibody that is used in an immunohistochemical procedure. The term tertiary antibody as used herein refers to an antibody that specifically binds to a secondary antibody, thereby forming a bridge between the secondary antibody and a downstream reagent, if present.

The sample according to the present invention is of biological origin. In preferred embodiments, the sample is a human sample, however, animal samples may also be used in the practice of the present invention. Non-limiting sample sources for use in the present invention include, for example, solid tissue, biopsy, ascites, aspirates, fluid extracts, blood (including circulating tumor cells), plasma, serum, cerebrospinal fluid, lymph fluid, external sections of the skin, respiratory tract , intestines and genitourinary tract, tears, saliva, milk, tumors, organs, cell cultures and/or components of cell cultures. A cancer sample is a sample that contains a cancer cell.

For purposes of the present invention, a tissue sample section refers to one part or piece of a tissue sample, for example, a thin section of tissue or cell cut from a tissue sample. It should be understood that multiple sections of tissue samples can be obtained and analyzed in accordance with the present invention. In some cases, the selected portion or section of tissue contains a homogeneous population of cells. In other cases, the selected portion comprises an area of tissue, such as a lumen, as a non-limiting example. The selected portion may be as small as one cell or two cells, or may represent several thousand cells, for example. In most cases, collection of cells is important, and although the present invention has been described for use to detect cellular components, the method can also be used to detect non-cellular components of the body (eg, by way of non-limiting example, soluble components in the blood). The word label, as used herein, refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody to produce a labeled antibody. The label may be detectable in itself (eg, radioisotope labels or fluorescent labels), or, in the case of an enzymatic label, it may catalyze a chemical change in the substrate compound or composition that is detectable. The term pharmaceutical formulation refers to a preparation that is in such a form as to provide the biological activity of the active ingredient to be effective and that does not contain any additional components that are unacceptably toxic to the subject to whom the formulation will be administered. The composition may be sterile.

The terms administer, administered, administration, and the like as used in the present invention refer to methods that can be used to provide delivery of a drug, such as an anti-B7-H4 antibody or an antigen-binding fragment thereof, to a desired site of biological action (e.g., intravenous introduction). Methods of administration that can be used with the agents and methods described in the present invention 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.

In the context of the present invention, the terms subject and patient are used interchangeably. The subject may be an animal. In some embodiments, the subject is a mammal, such as a non-human animal (eg, a cow, pig, horse, cat, dog, rat, mouse, monkey or other primate, etc.). In some embodiments, the subject is a human.

The term "therapeutically effective amount" refers to the amount of a drug, such as an anti-B7-H4 antibody or an antigen-binding fragment thereof, effective to treat a disease or disorder in a subject. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce the size or burden of the tumor

- 9 045594 li; inhibit to some extent the infiltration of cancer cells into peripheral organs; inhibit tumor metastasis to some extent; inhibit tumor growth to some extent; relieve to some extent one or more symptoms associated with cancer; and/or result in a favorable response, such as increased progression-free survival (PFS), disease-free survival (DFS), overall survival (OS), complete response (CR), partial response (PR), or in some cases stable disease ( SD), reduction in progressive disease (PD), reduction in time to progression (TTP), or any combination thereof. To the extent that a drug can prevent the growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. Terms such as cure, cure, treat, alleviate and alleviate refer to therapeutic measures that treat, slow, reduce symptoms and/or stop the progression of a pathological condition or disorder. Thus, those who need treatment include those who are already diagnosed or suspected of having the disease. In certain embodiments, a subject is successfully treated for cancer in accordance with the methods of the present invention if the patient demonstrates one or more of the following: a decrease in the number or complete absence of cancer cells; reduction in tumor size; inhibiting or preventing the penetration of cancer cells into peripheral organs, including, for example, the spread of cancer into soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; relief of one or more symptoms associated with a specific cancer; reduction in morbidity and mortality; improving quality of life; reducing tumorigenicity, oncogenic frequency or tumorigenic capacity of a tumor; reducing the number or frequency of cancer stem cells in the tumor; differentiation of oncogenic cells into a non-tumor state; increased progression-free survival (PFS), disease-free survival (DFS), overall survival (OS), complete response (CR), partial response (PR), stable disease (SD), decreased progressive disease (PD), decreased time to progression (TTP) or any combination thereof.

The terms cancer and cancerous refer to or describe a physiological condition in mammals in which a population of cells is characterized by unregulated cell growth. Examples of cancer include, but are not limited to, gynecologic cancers (eg, breast cancer (including triple negative breast cancer, ductal carcinoma, ovarian cancer, and endometrial cancer), non-small cell lung cancer, pancreatic cancer, thyroid cancer, kidney cancer (eg , renal cell carcinoma) and bladder cancer (eg, urothelial cell carcinoma). The cancer may be a cancer that expresses B7-H4 or a cancer that expresses B7-H4. Such terms refer to cancers containing cells that express B7-H4 The cancer may be a solid tumor that expresses B7-H4. The cancer may be a primary tumor or may be an advanced or metastatic cancer.

Refractory cancer is cancer that progresses even if anticancer treatment such as chemotherapy is administered to the cancer patient. Recurrent cancer is cancer that grows again, either at the original site or at a distant site, after responding to initial therapy.

As used herein and in the present disclosure and claims, the singular forms include the plural forms unless the context clearly dictates otherwise.

It should be understood that, in cases where embodiments are described in the present invention with the formulation containing, similar embodiments described in terms of consisting of and/or consisting essentially of may also be provided. In the present disclosure, contains, containing, including and having, etc. may have the meaning ascribed to them in US patent law and may mean includes, includes, etc.; consisting essentially of or consisting essentially in a like manner has the meaning given to it under US patent law, and the term is open-ended, allowing for more to be present than what is stated as long as the essential or novel characteristics of what is said are not changed by the presence of more , as stated, but excluding prior art embodiments.

Unless specifically stated or obvious from the context, the term or is understood to be inclusive as used herein. The term and/or used in a phrase such as A and/or B in the present invention is intended to include A and B, A or B, A and B. Likewise, the term and/or used in a phrase 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 (separately); B (separately); and C (separately).

The terms about and approximately in the context of the present invention, when used to change a numerical value or numerical range, indicate that deviations of 5 to 10% above and 5 to 10% below the value or range remain within the intended value of the given value or range.

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

Antibodies.

In a specific aspect, the present invention provides antibodies (eg, monoclonal antibodies) and antigen-binding fragments thereof that specifically bind to B7-H4 (eg, human B7-H4). The amino acid sequences for human B7-H4 are known in the art and are also provided herein as SEQ ID NO: 1. Human B7-H4:

MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSD

IVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTD

AGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWA SQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVT ESEIKRRSHLQLLNSKASLCVSSFFAISWALLPLSPYLMLK (SEQ Ш NO:1)

In certain embodiments, the anti-B7-H4 antibody or antigen-binding fragment thereof is an antibody described in table. 1, or is an antibody or an antigen-binding fragment thereof containing complementarity determining regions (CDRs), a heavy chain variable region, and/or a light chain variable region, and/or a heavy chain, and/or a light target of the antibody described in Table 1. 1.

Table 1

Typical antibodies to B7-H4

Clone ID VH/VL hybridomas Antibody type SEQ ID NO CDR VH VL H L J511 MB mlgGl 22-26, 29 6 9 89 19 J512 MB m!gG2a 22-26, 29 6 9 16 19 J517 M6- De-N-gly (1) mIgG2a 22-25, 27, 29 6 10 16 thirty J518 MB De-N-gly (2) mIgG2a 22-25, 28, 29 6 eleven 16 31 J513 MI mlgGl 22-26, 29 7 9 90 19 J514 MI m!gG2a 22-26, 29 7 9 17 19 J519 MI - De-N-gly (1) mIgG2a 22-25, 27, 29 7 10 17 thirty J520 MI De-N-gly (2) mIgG2a 22-25, 28, 29 7 eleven 17 31 J515 M15 mlgGl 22-26, 29 8 9 91 19 J516 M15 m!gG2a 22-26, 29 8 9 18 19 J521 M15- De-N-gly (1) mIgG2a 22-25, 27, 29 8 10 18 thirty J522 M15 De-N-gly (2) m!gG2a 22-25, 28, 29 8 eleven 18 31

In certain embodiments, the antibody or antigen binding fragment thereof described herein specifically binds to human B7-H4 and contains the six CDRs listed in Table 1. 2, i.e. six CDRs from SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, a sequence selected from the group consisting of SEQ ID NO: 26-28 and SEQ ID NO: 29.

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table 2

Amino acid sequences CDR

Description of CDR Amino acid sequence CDR (SEQ ID NO) VH-CDR1 GFSLSTYG (SEQ Ш NO:22) VH-CDR2 WWNDD (SEQ ID NO:23) VH-CDR3 VDGYYWYFDV (SEQ ID NO:24) VL-CDR1 RSSQSIVHSNRNTYLE (SEQ ID NO:25) VL-CDR2 NVSNRFS (SEQ ID NO:26) VL-CDR2, De-N- glycosylated (1) NVANRFS (SEQ ID NO:27) VL-CDR2, De-N- glycosylated (2) QVSNRFS (SEQ ID NO:28) VL-CDR3 FQGSHVPLT (SEQ ID NO:29)

In certain embodiments, an antibody or antigen binding fragment thereof described herein specifically binds to human B7-H4 and contains the VH region shown in Table 1. 3, for example in combination with area VL.

Table 3

Amino acid sequences of the variable heavy chain (VH) region

Description VH Amino acid sequence of VH (SEQ ID NO) M6 VH (J511, J512, J517, J518) QVTLKESGPGILQPSQTLSLTCSFSGFSTYGLGVGWI RQPSGKGLDWLANIWWNDDKYYNSALKSRLTISKDTS NNQVFLKISSVDTADTGTYYCAQVDGYYWYFDVWGA GTTVTVSS (SEQ ID NO:6) Mil VH (J513, J514, J519, J520) QVTLKESGPGILQPSQTLSLTCSLSGFSTYGLGVGWI RQPSGKGLGWLANIWWNDDKYYNSALKSRLTISKDTS NNQVFLKISSVDTADTGTYYCAQVDGYYWYFDVWGA GTTVTVSS (SEQ ID NO:7) M15 VH (J515, J516, J521, J522) QVTLKESGPGILQSSQTLSLTCSFSGFSTYGLGVGWI RQPSGKGLDWLANIWWNDDKYYNSALKSRLTISKDTS NNQVFLKISSVDTADTGTYYCAQVDGYYWYFDVWGA GTTVTVSS (SEQ ID NO:8)

In certain embodiments, the antibody or antigen-binding fragment thereof described in the present invention specifically binds to human B7-H4 and contains the VL region shown in table. 4, for example in combination with area VH.

Table 4

Amino acid sequences of the variable light chain (VL) region

Description VL Amino acid sequence of VL (SEQ ID NO) M6,M11,M15 VL (J511-J516) DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNRNTYLE WYLQKPGQSPKLLIYNVSNRF SGVPDRF SGSSGSGTDFT

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LKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELK (SEQ ID NO :9) De-N-glycosylated VL(1) (J517, J519, J521) DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNRNTYLE WYLQKPGQSPKLLIYNVANRF SGVPDRF SSGSGSGTDFT LKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELK (SEQ ID NO: 10) De-N-glycosylated VL (2) (J518, J520, J522) DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNRNTYLE WYLQKPGQSPKLLIYQVSNRF SGVPDRF SSGSGSGTDFT LKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELK (SEQ ID NO: 11)

In certain embodiments, an antibody or antigen binding fragment thereof described herein specifically binds to human B7-H4 and contains the VH region shown in Table 1. 3, and the region VL given in table. 4. In certain embodiments, the antibody or antigen-binding fragment thereof described in the present invention specifically binds to human B7-H4 and contains a VH region containing or consisting of the sequence shown in table. 3, and the VL region containing or consisting of the sequence shown in table. 4.

In certain embodiments, an antibody or antigen binding fragment thereof described herein specifically binds to human B7-H4 and comprises a VH region containing a sequence at least 80% identical to the VH sequence of Table 1. 3, and a VL region containing a sequence at least 80% identical to the VL sequence from table. 4. In certain embodiments, the antibody or antigen binding fragment thereof described in the present invention specifically binds to human B7-H4 and contains a VH region containing a sequence at least 85% identical to the VH sequence from table. 3, and a VL region containing a sequence at least 85% identical to the VL sequence from table. 4.

In certain embodiments, an antibody or antigen binding fragment thereof described herein specifically binds to human B7-H4 and contains a VH region consisting of a sequence that is at least 80% identical to the VH sequence of Table 1. 3, and a VL region consisting of a sequence at least 80% identical to the VL sequence from table. 4. In certain embodiments, the antibody or antigen binding fragment thereof described in the present invention specifically binds to human B7-H4 and contains a VH region consisting of a sequence that is at least 85% identical to the VH sequence of Table. 3, and a VL region consisting of a sequence at least 85% identical to the VL sequence from table. 4.

In certain embodiments, an antibody or antigen binding fragment thereof described herein specifically binds to human B7-H4 and comprises a VH region containing a sequence at least 90% identical to the VH sequence of Table 1. 3, and a VL region containing a sequence at least 90% identical to the VL sequence from table. 4. In certain embodiments, the antibody or antigen binding fragment thereof described in the present invention specifically binds to human B7-H4 and contains a VH region containing a sequence at least 95% identical to the VH sequence from table. 3, and a VL region containing a sequence at least 95% identical to the VL sequence from table. 4.

In certain embodiments, an antibody or antigen binding fragment thereof described herein specifically binds to human B7-H4 and comprises a VH region containing a sequence at least 96% identical to the VH sequence of Table 1. 3, and a VL region containing a sequence at least 96% identical to the VL sequence from table. 4. In certain embodiments, the antibody or antigen binding fragment thereof described in the present invention specifically binds to human B7-H4 and contains a VH region containing a sequence at least 97% identical to the VH sequence from table. 3, and a VL region containing a sequence at least 97% identical to the VL sequence from table. 4. In certain embodiments, the antibody or antigen-binding fragment thereof described in the present invention specifically binds to human B7-H4 and contains a VH region containing a sequence at least 98% identical to the VH sequence from table. 3, and a VL region containing a sequence at least 98% identical to the VL sequence from table. 4. In certain embodiments, an antibody or antigen-binding fragment thereof described in the present invention specifically

- 13 045594 bind to human B7-H4 and contain a VH region containing a sequence at least 99% identical to the VH sequence from table. 3, and a VL region containing a sequence at least 99% identical to the VL sequence from table. 4.

In certain embodiments, an antibody or antigen binding fragment thereof described herein specifically binds to human B7-H4 and contains a VH region consisting of a sequence that is at least 96% identical to the VH sequence of Table 1. 3, and a VL region consisting of a sequence at least 96% identical to the VL sequence from table. 4. In certain embodiments, the antibody or antigen-binding fragment thereof described in the present invention specifically binds to human B7-H4 and contains a VH region consisting of a sequence that is at least 97% identical to the VH sequence from table. 3, and a VL region consisting of a sequence at least 97% identical to the VL sequence from table. 4. In certain embodiments, the antibody or antigen binding fragment thereof described in the present invention specifically binds to human B7-H4 and contains a VH region consisting of a sequence that is at least 98% identical to the VH sequence from table. 3, and a VL region consisting of a sequence at least 98% identical to the VL sequence from table. 4. In certain embodiments, the antibody or antigen binding fragment thereof described in the present invention specifically binds to human B7-H4 and contains a VH region consisting of a sequence that is at least 99% identical to the VH sequence of Table. 3, and a VL region consisting of a sequence at least 99% identical to the VL sequence from table. 4.

In certain aspects, an antibody or antigen binding fragment thereof described in the present invention is described by its VL domain only, its VH domain only, its VL 3 CDR region only, or its VH 3 CDR region only. See, for example, Rader S. et al. (1998), PNAS, 95:8910-8915, which is incorporated herein by reference in its entirety and describes the humanization of a murine anti-αΎβ3 antibody by identifying a complementary light chain or heavy chain, respectively, from a human light chain or a heavy chain library leading to to variants of a humanized antibody having an affinity equal to or greater than that of the parent antibody. See also Clackson T. et al. (1991), Nature, 352:624-628, which is incorporated herein by reference in its entirety and describes methods for producing antibodies that specifically bind to a specific antigen using a specific VL domain (or VH domain) and screening the library for complementary VH domain (or VL domain). The screen resulted in 14 new VH domain-specific partners and 13 new VL domain-specific partners that bound stronger, as determined by ELISA. See also Kim S.J. & Hong H.J. (2007), J. Microbiol., 45:572-577, which is incorporated herein by reference in its entirety and describes methods for producing antibodies that specifically bind a specific antigen using a specific VH domain, and screening a library (e.g., a library human VL) into complementary VL domains; the selected VL domains can in turn be used to target the selection of additional complementary VH domains (eg, human).

In certain aspects, the CDR of an antibody or antigen-binding fragment thereof may be defined in accordance with the Chothia numbering scheme, which refers to the location of the structural loops of the immunoglobulin (see, for example, Chothia C. & Lesk A. M. (1987), J. Mol. Biol., 196:901-917; AlLazikani B. et al. (1997), J. Mol. Biol., 273:927-948; Chothia S. et al. (1992), J. Mol. Biol., 227:799- 817; Tramontano A. et al. (1990), J. Mol. Biol., 215(1):175-82; and US patent No. 7709226). Typically, when using the Kabat numbering system, the Chotia loop CDR-H1 is represented by heavy chain amino acids 26-32, 33, or 34, the Chotia loop CDR-H2 is represented by heavy chain amino acids 52-56, and the Chotia loop CDR-H3 is represented by amino acids 52-56 heavy chain 95-102, while the Chotia loop CDR-L1 is light chain amino acids 24-34, the Chotia loop CDR-L2 is light chain amino acids 50-56 and the Chotia loop CDR-L3 is light chain amino acids 8997. The end of the Chotia CDR-H1 loop when numbered according to the Kabat numbering system varies between H32 and H34 depending on the length of the loop (this is due to the fact that in the Kabat numbering scheme inserts are placed at H35A and H35B; if neither 35A nor 35B are present, the loop ends on 32; if only 35A is present, the loop ends on 33; if both 35A and 35B are present, the loop ends on 34).

In certain aspects, the present invention provides antibodies and antigen-binding fragments thereof that specifically bind to B7-H4 (eg, human B7-H4) and contain the CDRs of the VH and VL regions of the Chotia antibody. In certain embodiments, the antibodies or antigen-binding fragments thereof specifically bind to B7-H4 (eg, human B7-H4) and contain one or more CDRs in which the Kabat or Chotia CDRs have the same amino acid sequence. In certain embodiments, the present invention provides antibodies and antigen-binding fragments thereof that specifically bind to B7-H4 (eg, human B7-H4) and consist of combinations of Kabat CDRs and Chotia CDRs.

In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof may be identified in accordance with the IMGT numbering system as described in Lefranc M-P (1999), The Immunologist, 7:132-136; and Lefranc M-R et al. (1999), Nucleic. Acids. Res., 27:209–212. According to the IMGT numbering system, VH-CDR1 is in positions 26-35, VH-CDR2 is in positions 51-57, VH-CDR3 is in positions 93-102, VL-CDR1 is in positions 27-32, VL-CDR2 is in positions 50-52, and VL-CDR3 is in positions 89-97. In a specific embodiment, the present invention provides antibodies and antigen-binding fragments thereof that specifically bind to B7-H4 (eg, human B7-H4) and contain the CDR regions of the VH and VL of the IMGT antibodies listed in Table 1. 3 and 4, for example, as described in Lefranc M-P (1999) above and Lefranc M-P et al. (1999), supra). In certain aspects, the CDR of an antibody or antigen binding fragment thereof may be determined in accordance with MacCallum R.M. 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 Dubel, eds., chapter 31, p. 422-439, Springer-Verlag, Berlin (2001). In a specific embodiment, the present invention provides antibodies or antigen-binding fragments thereof that specifically bind to B7-H4 (eg, human B7-H4) and can be detected by the method described in MacCallum R.M. et al.

In certain aspects, the CDRs of an antibody or an antigen binding fragment thereof may be defined according to an AbM numbering scheme that refers to the hypervariable regions of the AbM, and which represents a compromise between the Kabat CDRs and the Chotia structural loops, and is used in the AbM Oxford antibody modeling software Molecular (Oxford Molecular Group, Inc.). In a specific embodiment, the present invention provides antibodies or antigen-binding fragments thereof that specifically bind to B7-H4 (eg, human B7-H4) and can be identified according to the AbM numbering scheme.

In certain embodiments, the antibody or antigen binding fragment thereof described herein specifically binds to human B7-H4 and contains the heavy chain set forth in Table 1. 5, for example, in combination with a light chain.

Table 5

Heavy chain amino acid sequences

Heavy chain description Heavy chain amino acid sequence (SEQ ID NO) M6N (J511) QVTLKESGPGILQPSQTLSLTCSFSGFSLSTYGLGVGWI RQPSGKGLDWLANIWWNDDKYYNSALKSRLTISKDTS NNQ VFLKIS S VDT ADTGT YYC AQ VDGYYW YFD VWGA GTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVK GYFPEP VT VTWNSGSLS SGVHTFP AVLESDLYTLSS SVT VPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCI CTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDP EVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIM HQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQ VYTIPPPKEQMAKDKVSLTCMITDFFPEDIT VEWQWNG QPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGN TFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:89) M6N (J512, J517, J518) QVTLKESGPGILQPSQTLSLTCSFSGFSTYGLGVGWI RQPSGKGLDWLANIWWNDDKYYNSALKSRLTISKDTS NNQ VFLKIS S VDT ADTGT YYC AQ VDGYYW YFD VWGA GTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVK GYFPEP VT VTWNSGSLS SGVHTFP AVLESDLYTLS S SVT VPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCI CTVPEVSSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDP DVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPI QHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRA PQVYVLPPPEEEMTKKQVTLTLTCMVTDFMPEDI YVEWT NNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWV ERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 16)

- 15 045594

MIN (J513) QVTLKESGPGILQPSQTLSLTCSLSGFSTYGLGVGWI RQPSGKGLGWLANIWWNDDKYYNSALKSRLTISKDTS NNQ VFLKIS S VDT ADTGT YYC AQ VDGYYW YFD VWGA GTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVK GYFPEP VT VTWNSGSLS SGVHTFP AVLESDLYTLS S S VT VPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCI CTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDP EVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIM HQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQ VYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNG QPAENYKNTQPIMNTNGSYFVYSKLNVQ KSNWEAGN TFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:90) MIN (J514, J519, J520) QVTLKESGPGILQPSQTLSLTCSLSGFSTYGLGVGWI RQPSGKGLGWLANIWWNDDKYYNSALKSRLTISKDTS NNQ VFLKIS S VDT ADTGT YYC AQ VDGYYW YFD VWGA GTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVK GYFPEP VT VTWNSGSLS SGVHTFP AVLESDLYTLS S SVT VPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCI CTVPEVSSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDP DVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPI QHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRA PQVYVLPPPEEEMTKKQVTLTLTCMVTDFMPEDI YVEWT NNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWV ERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ TO NO: 17) М15Н (J515) QVTLKESGPGILQSSQTLSLTCSFSGFSTYGLGVGWI RQPSGKGLDWLANIWWNDDKYYNSALKSRLTISKDTS NNQ VFLKIS S VDT ADTGT YYC AQ VDGYYW YFD VWGA GTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVK GYFPEP VT VTWNSGSLS SGVHTFP AVLESDLYTLS S SVT VPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCI CTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDP EVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIM HQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQ VYTIPPPKEQMAKDKVSLTCMITDFFPED ITVEWQWNG QPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGN TFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:91) M15N (J516, J521, J522) QVTLKESGPGILQSSQTLSLTCSFSGFSLSTYGLGVGWI RQPSGKGLDWLANIWWNDDKYYNSALKSRLTISKDTS NNQ VFLKIS S VDT ADTGT YYC AQ VDGYYW YFD VWGA GTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVK

- 16 045594

GYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSSVT VP S SPRP SET VTCNVAHP AS STK VDKKIVPRDCGCKPCI CTVPEVSSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDP DVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPI QHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGG RA PQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWT NNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWV ERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 18)

In certain embodiments, the antibody or antigen-binding fragment thereof described in the present invention specifically binds to human B7-H4 and contains the light chain shown in Table 1. 6, for example, in combination with a heavy chain.

Table 6

Light chain amino acid sequences

Description of light chain Light chain amino acid sequence (SEQ ID NO) M6, M11,M15L (J511-J516) DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNRNTYLE WYLQKPGQSPKLLIYNVSNRF SGVPDRF SSGSGSGTDFTL KISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELKRADA APTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKID GSERQNGVLNSWTDQDSKDSTYSMSSTLTLTK DEYERH NSYTCEATHKTSSTSPIVKSFNRNEC (SEQ ID NO: 19) De-N-glycosylated L(l) (J517, J519, J521) DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNRNTYLE WYLQKPGQSPKLLIYNVANRFSGVPDRFSGSGSGTDFTL KISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELKRADA APTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKID GSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEY ERH NSYTCEATHKTSSTSPIVKSFNRNEC (SEQ ID NO:30) De-N-glycosylated L(2) (J518, J520, J522) DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNRNTYLE WYLQKPGQSPKLLIYQVSNRF SGVPDRF SSGSGSGTDFTL KISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELKRADA APTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKID GSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERH NSYTCEATHKTSSTSPIVKSFNRNEC (SEQ ID NO:31)

In certain embodiments, the antibody or antigen binding fragment thereof described herein specifically binds to human B7-H4 and contains the heavy chain set forth in Table 1. 5, and the light chain given in table. 6.

In specific aspects, the present invention provides antibodies that contain a heavy chain and a light chain. With respect to the heavy chain, in a particular embodiment, the heavy chain of the antibody described in the present invention may be an alpha (α), delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In a specific embodiment, an antibody of the present invention that immunospecifically binds to B7-H4 (eg, human B7-H4) comprises a heavy chain wherein the amino acid sequence of the VH domain comprises the amino acid sequence set forth in Table 1. 3, and wherein the heavy chain constant region comprises the amino acid sequence of a mouse IgG2a heavy chain constant region.

In a specific embodiment, an antibody of the present invention that immunospecifically binds to B7-H4 (eg, human B7-H4) comprises a heavy chain wherein the amino acid sequence of the VH domain comprises the amino acid sequence set forth in Table 1. 3, and wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 87:

- 17 045594

AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLES

DL YTLS S S VT VP S SPRP SET VTCNVAHP AS STKVDKKIVPRDCGCKPCICT VPEVS S VFIFP

PKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVV SALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQ VTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVE RNSYSCSVVHEGL HNHHTTKSFSRTPGK (SEQ ID NO:87)

In a specific embodiment, an antibody of the present invention that immunospecifically binds to B7-H4 (eg, human B7-H4) comprises a heavy chain wherein the amino acid sequence of the VH domain comprises the amino acid sequence set forth in Table 1. 3, and wherein the heavy chain constant region comprises the amino acid sequence of a mouse IgG1 heavy chain constant region.

In a specific embodiment, an antibody of the present invention that immunospecifically binds to B7-H4 (eg, human B7-H4) comprises a heavy chain wherein the amino acid sequence of the VH domain comprises the amino acid sequence set forth in Table 1. 3, and wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 92:

AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLES

DL YTLS S S VT VP S SPRP SET VTCNVAHP AS STKVDKKIVPRDCGCKPCICT VPEVS S VFIFP

PKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVS ELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKV SLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGN TFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:92)

With respect to the light chain, in a particular embodiment, the light chain of the antibody described in the present invention is a kappa light chain or a lambda light chain. In a specific embodiment, an antibody described in the present invention that immunospecifically binds to B7-H4 (eg, human B7-H4) comprises a light chain wherein the amino acid sequence of the VL domain comprises the amino acid sequence set forth in Table 1. 4, and wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 88:

RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQ

DSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSSTSPIVKSFNRNEC (SEQ ID NO:88).

In another specific embodiment, the antibody described in the present invention, which immunospecifically binds to B7-H4 (for example, human B7-H4), contains (i) a heavy chain, and the amino acid sequence of the VH domain contains the amino acid sequence specified in table. 3, and wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 87; and (ii) a light chain, wherein the amino acid sequence of the VL domain comprises the amino acid sequence shown in table. 4, and wherein the heavy chain constant region contains the amino acid sequence of SEQ ID NO: 88.

In another specific embodiment, the antibody described in the present invention, which immunospecifically binds to B7-H4 (for example, human B7-H4), contains (i) a heavy chain, and the amino acid sequence of the VH domain contains the amino acid sequence specified in table. 3, and wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 92; and (ii) a light chain, wherein the amino acid sequence of the VL domain comprises the amino acid sequence shown in table. 4, and wherein the heavy chain constant region contains the amino acid sequence of SEQ ID NO: 88.

In a specific embodiment, an antibody described in the present invention that immunospecifically binds to B7-H4 (e.g., human B7-H4) contains a VH domain and a VL domain containing any amino acid sequence described in the present invention, and wherein the constant regions contain amino acids sequences of the constant regions of an immunoglobulin molecule IgG, IgE, IgM, IgD, IgA or IgY or a mouse immunoglobulin molecule IgG, IgE, IgM, IgD or IgA. In another specific embodiment, an antibody described in the present invention that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a VH domain and a VL domain containing any amino acid sequence described in the present invention, and wherein the constant regions comprise amino acid sequences of the constant regions of the immunoglobulin molecule IgG, IgE, IgM, IgD, IgA or IgY, of any class (for example, IgG ₁ , IgG2, IgG ₃ , IgG ₄ , IgA ₁ and IgA2) or any subclass (for example, IgG _2a and IgG _2b ) immunoglobulin molecules. In a specific embodiment, the constant regions comprise the amino acid sequences of the constant regions of a murine immunoglobulin molecule IgG, IgE, IgM, IgD, or

- 18 045594

IgA, any class (for example, IgG1, IgG2 and IgG ₃ ,) or any subclass (for example, IgG2 _a and IgG2b) of the immunoglobulin molecule. In a specific embodiment, an antibody described in the present invention that immunospecifically binds to B7-H4 (eg, human B7-H4) contains a VH domain and a VL domain containing any amino acid sequence described in the present invention and murine constant domains. In a specific embodiment, an antibody described in the present invention that immunospecifically binds to B7-H4 (eg, human B7H4) contains a VH domain and a VL domain containing any amino acid sequence described in the present invention and rabbit constant domains. In specific embodiments, the antibody or antigen binding fragment thereof comprises VH and VL sequences from the amino acid sequences of SEQ ID NO: 6 and 9, respectively. In specific embodiments, the antibody or antigen-binding fragment thereof, in the engineered embodiment of the antibody or antigen-binding fragment thereof, comprises VH and VL sequences from the amino acid sequences of SEQ ID NOs: 6 and 9, wherein one or more glycosylation sites are removed during the construction, for example, in one or more CDRs (eg VL-CDR2). In specific embodiments, the antibody or antigen binding fragment thereof comprises VH and VL sequences from amino acid sequences SEQ ID NO: 6 and 10, respectively. In specific embodiments, the antibody or antigen binding fragment thereof comprises VH and VL sequences from amino acid sequences SEQ ID NO: 6 and 11, respectively. In specific embodiments, the antibody or antigen binding fragment thereof (e.g., an antibody or antigen binding fragment thereof comprising VH of SEQ ID NO: 6 and VL of SEQ ID NO: 9, 10, or 11) further comprises a mouse IgG1 heavy chain constant domain (e.g., J511). In specific embodiments, the antibody or antigen binding fragment thereof (e.g., the antibody or antigen binding fragment thereof comprising VH of SEQ ID NO: 6 and VL of SEQ ID NO: 9, 10, or 11) further comprises a mouse IgG _2a heavy chain constant domain (e.g. , J512). In specific embodiments, the antibody or antigen binding fragment thereof comprises VH and VL sequences from the amino acid sequences of SEQ ID NO: 7 and 9, respectively. In specific embodiments, the antibody or antigen-binding fragment thereof, in an engineered embodiment of the antibody or antigen-binding fragment thereof, comprises VH and VL sequences from the amino acid sequences of SEQ ID NOs: 7 and 9, wherein the construction removes one or more glycosylation sites, for example, in one or more CDRs (eg VL-CDR2). In specific embodiments, the antibody or antigen binding fragment thereof comprises VH and VL sequences from amino acid sequences SEQ ID NO: 7 and 10, respectively. In specific embodiments, the antibody or antigen binding fragment thereof comprises VH and VL sequences from amino acid sequences SEQ ID NO: 7 and 11, respectively. In specific embodiments, the antibody or antigen binding fragment thereof (e.g., an antibody or antigen binding fragment thereof comprising VH of SEQ ID NO: 7 and VL of SEQ ID NO: 9, 10, or 11) further comprises a mouse IgG1 heavy chain constant domain (e.g., J513). In specific embodiments, the antibody or antigen binding fragment thereof (e.g., an antibody or antigen binding fragment thereof comprising VH of SEQ ID NO: 7 and VL of SEQ ID NO: 9, 10, or 11) further comprises a mouse IgG _2a heavy chain constant domain (e.g. , J514).

In specific embodiments, the antibody or antigen binding fragment thereof comprises VH and VL sequences from the amino acid sequences of SEQ ID NO: 8 and 9, respectively. In specific embodiments, the antibody or antigen-binding fragment thereof, in an engineered embodiment of the antibody or antigen-binding fragment thereof, comprises VH and VL sequences from the amino acid sequences of SEQ ID NOs: 8 and 9, wherein one or more glycosylation sites are removed during the construction, for example, in one or more CDRs (eg VL-CDR2). In specific embodiments, the antibody or antigen binding fragment thereof comprises VH and VL sequences from amino acid sequences SEQ ID NO: 8 and 10, respectively. In specific embodiments, the antibody or antigen binding fragment thereof comprises VH and VL sequences from the amino acid sequences of SEQ ID NO: 8 and 11, respectively. In specific embodiments, the antibody or antigen-binding fragment thereof (e.g., an antibody or antigen-binding fragment thereof comprising VH of SEQ ID NO: 8 and VL of SEQ ID NO: 9, 10, or 11) further comprises a mouse IgG heavy chain constant domain ₁ (e.g. , J515). In specific embodiments, the antibody or antigen binding fragment thereof (e.g., an antibody or antigen binding fragment thereof comprising VH of SEQ ID NO: 8 and VL of SEQ ID NO: 9, 10, or 11) further comprises a mouse IgG2a heavy chain constant domain (e.g., J516). In specific embodiments, the antibody or antigen binding fragment thereof comprises (i) M6 CDR sequences (eg, the amino acid sequences of SEQ ID NOs: 22-26 and 29);

(ii) sequences of the M6 VH and VL regions or De-N-glycosylated variants thereof (e.g., VH containing the amino acid sequence of SEQ ID NO: 6, and VL containing the amino acid

- 19 045594 lot sequence SEQ ID NO: 9, 10 or 11);

(iii) murine M6 IgG2a heavy and light chain sequences or De-Nglycosylated variants thereof (eg, a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a light chain containing the amino acid sequence of SEQ ID NO: 19, or 31); or (iv) murine M6 IgG1 heavy and light chain sequences (eg, a heavy chain comprising the amino acid sequence of SEQ ID NO: 89 and a light chain containing the amino acid sequence of SEQ ID NO: 19).

In specific embodiments, the antibody or antigen binding fragment thereof comprises (i) M11 CDR sequences (eg, the amino acid sequences of SEQ ID NOs: 22-25, 27 and 29);

(ii) sequences of the VH and VL regions of M11 or De-N-glycosylated variants thereof (eg, VH containing the amino acid sequence of SEQ ID NO: 7, and VL containing the amino acid sequence of SEQ ID NO: 9, 10 or 11);

(iii) murine M11 IgG2a heavy and light chain sequences or De-Nglycosylated variants thereof (eg, a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 and a light chain containing the amino acid sequence of SEQ ID NO: 19, 30 or 31); or (iv) murine M11 IgG1 heavy and light chain sequences (eg, a heavy chain comprising the amino acid sequence of SEQ ID NO: 90 and a light chain containing the amino acid sequence of SEQ ID NO: 19).

In specific embodiments, the antibody or antigen binding fragment thereof comprises (i) M15 CDR sequences (eg, the amino acid sequences of SEQ ID NOs: 22-25, 28 and 29);

(ii) sequences of the VH and VL regions of M15 or De-N-glycosylated variants thereof (eg, VH containing the amino acid sequence of SEQ ID NO: 8, and VL containing the amino acid sequence of SEQ ID NO: 9, 10 or 11);

(iii) murine M15 IgG2a heavy and light chain sequences or De-Nglycosylated variants thereof (eg, a heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain containing the amino acid sequence of SEQ ID NO: 19, 30 or 31); or (iv) murine M15 IgG1 heavy and light chain sequences (eg, a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain containing the amino acid sequence of SEQ ID NO: 19).

In another specific embodiment, an antibody or antigen binding fragment thereof described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises a VH domain containing amino acid sequences of the CDR1 region VH, CDR2 region VH and CDR3 region VH of the antibody indicated in table. 1 (eg SEQ ID NO: 22-24);

(ii) the light chain contains a VL domain containing the amino acid sequences of the VL region CDR1, VL region CDR2 and VL region CDR3 of the same antibody shown in table. 1 (for example, SEQ ID NO: 25, 26 and 29, 25, 27 and 29 or 25, 28 and 29);

(iii) the heavy chain further comprises a mouse IgG1 constant domain (eg, containing the amino acid sequence of SEQ ID NO: 92) or a mouse IgG2a constant domain (eg, containing the amino acid sequence of SEQ ID NO: 87); and (iv) the light chain further comprises a murine kappa constant domain (eg, comprising the amino acid sequence of SEQ ID NO: 88).

In another specific embodiment, an antibody or antigen binding fragment thereof described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises a VH domain containing amino acid sequence of the antibody indicated in table. 1 (for example, SEQ ID NO: 6-8);

(ii) the light chain contains a VL domain containing the amino acid sequence of the same antibody shown in Table. 1 (for example, SEQ ID NO: 9-11);

(iii) the heavy chain further comprises a mouse IgG1 constant domain (eg, containing the amino acid sequence of SEQ ID NO: 92) or a mouse IgG2a constant domain (eg, containing the amino acid sequence of SEQ ID NO: 87); and (iv) the light chain further comprises a murine kappa constant domain (eg, comprising the amino acid sequence of SEQ ID NO: 88).

In certain embodiments, an antibody or antigen-binding fragment thereof described herein that specifically binds B7-H4 (e.g., human B7-H4) comprises one, two, or more VH framework regions (FRs) having amino acid sequences the sequences described in the present invention for the antibodies shown in table. 7 (eg SEQ ID NO: 38-49). In some embodiments, an antibody or antigen binding fragment thereof described herein that specifically binds to B7-H4 (e.g., human B7-H4) comprises one, two, or more VL framework regions (FRs) having the amino acid sequences described in the present invention for the antibodies listed in table. 7 (eg SEQ ID NO: 50-53). In specific embodiments, an antibody or antigen binding fragment thereof described herein that specifically binds to B7-H4 (e.g., human B7-H4) comprises one, two, or more VH framework regions having the amino acid sequences described herein for antibodies listed in table. 7 above, and one, two or more VL framework regions having the amino acid sequences described in the present invention for the same antibody, listed in table. 7 (e.g., SEQ ID NOs: 38-41 and 50-53; SEQ ID NOs: 42-45 and 50-53; or SEQ ID NOs: 46-49 and 50-53).

Table 7

Framework amino acid sequences

VH/VL hybridomas FR1 (SEQ ID NO) FR2 (SEQ ID NO) FR3 (SEQ ID NO) FR4 (SEQ ID NO) MB VH QVTLKESGP GILQPSQTLS LTCSFS (SEQ IDNO:38) GVGWIRQPSG KGLDWLANI (SEQ ID NO:39) KYYNSALKSRLTISKD TSNNQVFLKISSVDTA DTGTYYCAQ (SEQ ID NO:40) WGAGTTVT VSS (SEQ ID NO:41) MI VH QVTLKESGP GILQPSQTLS LTCSLS (SEQ ID NO:42) GVGWIRQPSG KGLGWLANI (SEQIDNO:43) KYYNSALKSRLTISKD TSNNQVFLKISSVDTA DTGTYYCAQ (SEQ ID NO:44) WGAGTTVT VSS (SEQ ID NO:45) M15 VH QVTLKESGP GILQSSQTLS LTCSFS (SEQ ID NO:46) GVGWIRQPSG KGLDWLANI (SEQIDNO:47) KYYNSALKSRLTISKD TSNNQVFLKISSVDTA DTGTYYCAQ (SEQ ID NO:48) WGAGTTVT VSS (SEQ ID NO:49) Mb, mi, M15 VL DVLMTQTPL SLPVSLGDQ ASISC (SEQ IDNO:50) YLQKPGQSPK LLIY (SEQ ID NO:51) GVPDRFSGSGSGTDFT LKISRVEAEDLGVYY C (SEQ ID NO:52) FGAGTKLE LK (SEQ ID NO:53)

In certain embodiments, an antibody or antigen binding fragment thereof described herein that specifically binds to B7-H4 (e.g., human B7-H4) comprises VH framework regions (FRs) having at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the VH framework regions described in the present invention in table. 7. In certain embodiments, an antibody or antigen binding fragment thereof described herein that specifically binds to B7-H4 (e.g., human B7-H4) comprises VL framework regions (FR) having at least 70% 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the VL framework regions described in the present invention in table. 7. In some embodiments, an antibody or antigen binding fragment thereof described herein that specifically binds B7-H4 (e.g., human B7-H4) comprises VH framework regions (FR) having at least 70% 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the VH framework regions described in the present invention in table. 7 above, and frame regions VL (FR) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the VL framework regions described in the present invention in table. 7.

In another aspect, the present invention provides antibodies or antigen-binding fragments thereof that bind the same B7-H4 epitope (e.g., human B7-H4 epitope) as the antibody

- 21 045594 or an antigen binding fragment thereof described in the present invention (for example, J511-J522).

Competitive binding assays can be used to determine whether two antibodies bind to overlapping epitopes. Competitive binding can be determined using an assay in which the immunoglobulin of interest inhibits the specific binding of a reference antibody to a common antigen, such as B7-H4. Various types of competitive binding assays are known, for example solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), competitive sandwich assay (see Stahli C. et al. (1983), Methods Enzymol., 9: 242-253); direct solid-phase biotin-avidin EIA (see Kirkland T.N. et al. (1986), J. Immunol., 137:3614-9); solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (see Harlow E. & Lane D. (1988), Antibodies: A Laboratory Manual., Cold Spring Harbor Press); solid phase direct RIA using the I-125 label (see Morel G. A. et al. (1988), Mol. Immunol., 25(1):7-15); direct solid-phase biotin-avidin EIA (Cheung R.C. et al. (1990), Virology, 176:546-52) and direct labeled RIA. (Moldenhauer G. et al. (1990), Scand. J. Immunol., 32:77-82). Typically, such an assay involves the use of purified antigen (eg, B7-H4, such as human B7-H4) bound to a solid surface, or cells containing either of an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the immunoglobulin of interest. Usually the immunoglobulin being tested is present in excess. Typically, when a competing antibody is present in excess, it will inhibit the specific binding of the reference antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more. A competitive binding assay can be designed in a variety of different formats using a labeled antigen or a labeled antibody. In a general version of this assay, the antigen is immobilized on a 96-well plate. The ability of unlabeled antibodies to block the binding of labeled antibodies to antigen is then measured using radioactive or enzyme labels. For more information, see, for example, Wagener S. et al. (1983), J. Immunol., 130:2308-2315; Wagener S. et al. (1984), J. Immunol. Methods, 68:269-274; Kuroki M. et al. (1990), Cancer Res., 50:4872-4879; Kuroki M. et al. (1992), Immunol. Invest, 21:523-538; Kuroki M. et al. (1992), Hybridoma, 11:391-407 and Antibodies: A Laboratory Manual, ed Harlow E. & Lane D. editors supra, p. 386-389.

In one embodiment, the competition assay is performed using surface plasmon resonance (BIAcore), for example, using a tandem approach such as described by Abdiche Y.N. et al. (2009), Analytical. Biochem., 386:172-180, where the B7-H4 antigen is immobilized on the surface of a chip, for example a CM5 sensor chip, and anti-B7-H4 antibodies are then passed through the chip. To determine whether an antibody or antigen binding fragment thereof competes with the anti-B7H4 antibody described in the present invention, the anti-B7-H4 antibody is first passed through the surface of the chip to saturate and then a potential competing antibody is added. Binding of the competing antibody or antigen-binding fragment thereof can then be detected and quantified relative to a non-competing control.

In one embodiment, Fortebio Octet competitive binding is used to determine that an anti-B7-H4 antibody or antigen binding fragment thereof competitively inhibits the binding of another anti-B7-H4 antibody or antigen binding fragment thereof to B7-H4.

In another aspect, the present invention provides antibodies that competitively inhibit (e.g., in a dose-dependent manner) the binding of an antibody or antigen binding fragment thereof described in the present invention (e.g., J511-J522) to B7-H4 (e.g., human B7-H4), as determined by assays known to one skilled in the art or described herein (eg, competitive ELISA assays, or suspension array assays, or surface plasmon resonance assays).

In specific aspects, the present invention provides an antibody or antigen binding fragment that competitively inhibits (e.g., in a dose-dependent manner) binding to B7-H4 (e.g., human B7-H4) of a VH domain-containing antibody having the amino acid sequence set forth in SEQ ID NO: 6, and a VL domain having the amino acid sequence set forth in SEQ ID NO: 9.

In specific aspects, the present invention provides an antibody or antigen binding fragment that competitively inhibits (e.g., in a dose-dependent manner) binding to B7-H4 (e.g., human B7-H4) of a VH domain-containing antibody having the amino acid sequence set forth in SEQ ID NO: 7, and a VL domain having the amino acid sequence set forth in SEQ ID NO: 9.

In specific aspects, the present invention provides an antibody or antigen binding fragment that competitively inhibits (e.g., in a dose-dependent manner) binding to B7-H4 (e.g., human B7-H4) of a VH domain-containing antibody having the amino acid sequence set forth in SEQ ID NO: 8, and a VL domain having the amino acid sequence set forth in SEQ ID NO: 9.

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In specific aspects, the present invention provides an antibody or antigen binding fragment that competitively inhibits (e.g., in a dose-dependent manner) binding to B7-H4 (e.g., human B7-H4) of a VH domain-containing antibody having the amino acid sequence set forth in SEQ ID NO: 6, and a VL domain having the amino acid sequence set forth in SEQ ID NO: 10.

In specific aspects, the present invention provides an antibody or antigen binding fragment that competitively inhibits (e.g., in a dose-dependent manner) binding to B7-H4 (e.g., human B7-H4) of a VH domain-containing antibody having the amino acid sequence set forth in SEQ ID NO: 7, and a VL domain having the amino acid sequence set forth in SEQ ID NO: 10.

In specific aspects, the present invention provides an antibody or antigen binding fragment that competitively inhibits (e.g., in a dose-dependent manner) binding to B7-H4 (e.g., human B7-H4) of a VH domain-containing antibody having the amino acid sequence set forth in SEQ ID NO: 8, and a VL domain having the amino acid sequence set forth in SEQ ID NO: 10.

In specific aspects, the present invention provides an antibody or antigen binding fragment that competitively inhibits (e.g., in a dose-dependent manner) binding to B7-H4 (e.g., human B7-H4) of a VH domain-containing antibody having the amino acid sequence set forth in SEQ ID NO: 6, and a VL domain having the amino acid sequence set forth in SEQ ID NO: 11.

In specific aspects, the present invention provides an antibody or antigen binding fragment that competitively inhibits (e.g., in a dose-dependent manner) binding to B7-H4 (e.g., human B7-H4) of a VH domain-containing antibody having the amino acid sequence set forth in SEQ ID NO: 7, and a VL domain having the amino acid sequence set forth in SEQ ID NO: 11.

In specific aspects, the present invention provides an antibody or antigen binding fragment that competitively inhibits (e.g., in a dose-dependent manner) binding to B7-H4 (e.g., human B7-H4) of a VH domain-containing antibody having the amino acid sequence set forth in SEQ ID NO: 8, and a VL domain having the amino acid sequence set forth in SEQ ID NO: 11.

In specific aspects, the present invention provides an antibody or antigen binding fragment thereof that immunospecifically binds to the same B7-H4 epitope (eg, human B7-H4) as any of J511-J522. In a specific aspect, an antigen binding fragment as described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) is selected from the group consisting of Fab, Fab', F(ab')2, and ScFv, wherein Fab, Fab', F(ab') ₂ or ScFv contains the heavy chain variable region and light chain variable region sequence of an anti-B7-H4 antibody or an antigen binding fragment thereof as described in the present invention. Fab, Fab', F(ab') ₂ or scFv can be obtained using any method known to one skilled in the art, including without limitation those described in section 5.3 below. The anti-B7-H4 antibody or antigen-binding fragment thereof may be fused or conjugated (eg, covalently or non-covalently linked) to a detectable label or substance. Examples of detectable tags or substances include enzyme tags such as glucose oxidase; radioisotopes such as iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹²¹ In) and technetium ( ⁹⁹ Tc); luminescent tags such as luminol; and fluorescent labels such as fluorescein, rhodamine and biotin. Such labeled antibodies or antigen-binding fragments thereof can be used to detect B7-H4 protein (eg, human B7-H4). See for example section 5.4.1 below.

Antibody production.

Antibodies and antigen-binding fragments thereof that immunospecifically bind to B7H4 (eg, human B7-H4) can be produced by any method known in the art for the synthesis of antibodies and antigen-binding fragments thereof, for example, chemical synthesis or recombinant expression methods. The methods described in the present invention use, unless otherwise indicated, conventional methods of molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization and related fields within the art . These techniques are described, for example, in the references cited in the present invention, and are fully explained in the literature. See, for example, Sambrook J. et al. (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel F.M. et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984), Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991), Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al. (eds.) (1999), Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

In a certain aspect, the present invention provides a method for producing an antibody or anti-gene binding fragment that immunospecifically binds to B7-H4 (eg, human B7-H4), comprising culturing a cell or host cell described in the present invention. In a certain aspect, the present invention provides a method for producing an antibody or antigen binding fragment thereof that immunospecifically binds to B7-H4 (e.g., human B7-H4), comprising expressing (e.g., recombinant expression) the antibody or antigen binding fragment thereof using a cell or a host cell described in the present invention (eg, a cell or host cell containing polynucleotides encoding an antibody or antigen binding fragment thereof described in the present invention). In a specific embodiment, the cell is an isolated cell. In a specific embodiment, exogenous polynucleotides have been introduced into a cell. In a specific embodiment, the method further includes the step of purifying an antibody or antigen binding fragment obtained from a cell or host cell. Methods for producing polyclonal antibodies are known in the art (see, for example, Chapter 11 in Short Protocols in Molecular Biology (2002), 5th ed., Ausubel F.M. et al., eds., John Wiley and Sons, New York).

Monoclonal antibodies or antigen binding fragments thereof can be produced using a wide range of methods known in the art, including the use of hybridoma, recombinant and phage display technologies, yeast-based presentation technologies, or combinations thereof. For example, monoclonal antibodies or antigen binding fragments thereof can be produced using hybridoma techniques, including those known in the art and described, for example, in Harlow E. & Lane D., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed ., 1988); Hammerling G.J. et al., Monoclonal Antibodies and T-Cell Hybridomas, 563681 (Elsevier, N.Y., 1981), or as described in Kohler G. & Milstein S. (1975), Nature, 256:495. Examples of yeast-based presentation methods that can be used to select and produce antibodies described in the present invention include methods described, for example, in WO 2009/036379 A2; WO 2010/105256; and WO 2012/009568, each of which is incorporated herein by reference in its entirety.

In specific embodiments, a monoclonal antibody or antigen-binding fragment thereof can be produced using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), as mentioned above. In the hybridoma method, a mouse or other suitable host animal is immunized as described above to induce the production of lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization, for example, extracellular domain (ECD) protein B7-H4 ( SEQ ID NO: 20). The lymphocytes are then fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to produce a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986). In certain embodiments, a monoclonal antibody or its antigen-binding fragment is an antibody or antigen-binding fragment derived from a clonal cell (e.g., a hybridoma or a host cell producing a recombinant antibody or antigen-binding fragment), wherein the antibody or antigen-binding fragment immunospecifically binds to B7-H4 (e.g., human B7-H4) , as determined, for example, by ELISA or other antigen binding assays known in the art or described in the examples provided herein. In specific embodiments, the monoclonal antibody or antigen binding fragment thereof may be a rodent or mouse antibody or antigen binding fragment thereof. fragment. In specific embodiments, the monoclonal antibody or antigen binding fragment thereof may be a chimeric or humanized antibody or antigen binding fragment thereof. In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof may be a Fab fragment or an F(ab')2 fragment. Monoclonal antibodies or antigen binding fragments thereof described in the present invention can, for example, be produced using the hybridoma method as described in Kohler G. & Milstein C. (1975), Nature, 256:495, or can, for example, be isolated from phage libraries, using, for example, methods as described in the present invention. Other methods for producing clonal cell lines and the monoclonal antibodies they express and their antigen binding fragments are well known in the art (see, for example, Chapter 11 in Short Protocols in Molecular Biology (2002), 5th ed., Ausubel F.M. et al., supra ).

Antigen binding fragments of the antibodies described in the present invention can be obtained using any method known to one skilled in the art. For example, the Fab and F(ab')2 fragments described in the present invention can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') )2-fragments). The Fab fragment corresponds to one of the two identical arms of the tetrameric antibody molecule and contains the complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment contains two antigen-binding arms of a tetrameric antibody molecule linked by disulfide bonds at the hinge region.

Moreover, the antibodies or antigen-binding fragments thereof described in the present invention

- 24 045594 can also be produced using various yeast-based phage display and/or presentation methods known in the art. In phage display methods, proteins are displayed on the surface of phage particles that carry polynucleotide sequences encoding them. In particular, DNA sequences encoding the VH and VL domains are amplified from animal cDNA libraries (eg, human or murine disease tissue cDNA libraries). DNA encoding the VH and VL domains is recombined with the scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated into E. coli and E. coli is infected with helper phage. The phage used in these methods is typically a filamentous phage, including fd and M13, and the VH and VL domains are typically recombinantly fused to phage gene III or gene VIII. A phage expressing an antibody or antigen-binding fragment thereof that specifically binds to a particular antigen can be selected or identified by the antigen, for example, using a labeled antigen or an antigen bound or captured on a solid surface or bead. Examples of phage display methods that can be used to produce antibodies or fragments described in the present invention include those described by Brinkman U. et al. (1995), J. Immunol. Methods, 182:41-50; Ames R.S. et al. (1995), J. Immunol. Methods, 184:177-186; Kettleborough C.A. et al. (1994), Eur. J Immunol 24:952-958; Persic L. et al. (1997), Gene, 187:9-18; Burton D.R. & Barbas C.F. (1994), Advan. Immunol., 57:191-280; in PCT application No. PCT/GB91/001134; international publications No. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401 and WO 97/13844; and also in US patents No. 5698426, 5223409, 5403484, 5580717, 5427908, 5750753, 5821047, 5571698, 5427908, 5516637, 5780225, 5658727, 5733743 and 5969108.

The antibody or antigen binding fragment thereof may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and from any isotype, including IgG1, _IgG2 , _IgG3 and IgG4.

Polynucleotides.

In certain aspects, the present invention provides polynucleotides comprising a nucleotide sequence encoding an antibody or antigen binding fragment thereof described herein, or a domain thereof (e.g., a light chain variable region and/or a heavy chain variable region) that immunospecifically binds to the B7 antigen. -H4 (eg, human B7-H4), as well as vectors, eg, vectors containing such polynucleotides for recombinant expression in host cells (eg, E. coli and mammalian cells).

In specific aspects, the present invention provides polynucleotides comprising nucleotide sequences encoding antibodies or antigen binding fragments thereof that immunospecifically bind to a B7-H4 polypeptide (e.g., human B7-H4) and contain an amino acid sequence as described herein, as well as antibodies or antigen binding fragments that compete with such antibodies or antigen binding fragments for binding to the B7-H4 polypeptide (eg, in a dose-dependent manner) or that bind to the same epitope as such antibodies or antigen binding fragments.

The present invention also provides a polynucleotide containing a nucleotide sequence encoding a polypeptide containing a sequence selected from the group consisting of SEQ ID NO: 6-8. In some embodiments, the antibody or antigen binding fragment thereof comprising the polypeptide immunospecifically binds to B7-H4.

The present invention also provides a polynucleotide containing a nucleotide sequence encoding a polypeptide containing a sequence selected from the group consisting of SEQ ID NOs: 9-11. In some embodiments, the antibody or antigen binding fragment thereof comprising the polypeptide immunospecifically binds to B7-H4.

The present invention also provides a polynucleotide comprising a nucleotide sequence encoding a polypeptide containing a sequence selected from the group consisting of SEQ ID NOs: 22-24, or containing all of the amino acids from SEQ ID NOs: 22-24. In some embodiments, the antibody or antigen binding fragment thereof comprising the polypeptide immunospecifically binds to B7-H4. The present invention also provides a polynucleotide containing a nucleotide sequence encoding a polypeptide containing a sequence selected from the group consisting of SEQ ID NOs: 25-29, or containing all of SEQ ID NOs: 25, 26 and 29, all of SEQ ID NOs : 25, 27 and 29 or all of SEQ ID NOs: 25, 28 and 29. In some embodiments, the antibody or antigen binding fragment thereof comprising the polypeptide immunospecifically binds to B7-H4.

The present invention also provides a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 16-18, 89, 90 and 91. In some embodiments, an antibody or antigen binding fragment thereof comprising a polypeptide immunospecifically bind to B7-H4. The present invention also provides a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 19, 30, and 31. In some embodiments, the antibody or antigen binding fragment thereof comprising the polypeptide immunospecifically binds to B7 -H4.

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The present invention also provides polynucleotides containing a nucleotide sequence encoding a heavy chain and/or a light chain and shown in table. 8, for example, wherein an antibody or antigen binding fragment thereof comprising an encoded heavy chain and/or light chain specifically binds to B7-H4.

Table 8 Polynucleotide sequences encoding the heavy chain and light chain

Antibody Polynucleotide sequence encoding heavy/light chain (SEQ Ш NO) J511 NS ATGGGCAGGCTTACTTCTTCATTCCTGCTACTGATTGTCCCTGCATAT GTCCTGTCCCAGGTCACTCTGAAAGAGTCTGGCCCTGGGATATTGCA GCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTTCACT GAGCACTTATGGTCTGGGTGTAGGTTGGATTCGTCAGCCTTCAGGGA AGGGTCTGGACTGGCTGGCCAACATTTGGTGGAATGATGATAAATAC TATAACT CAGCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTC CAACAACCAGGTATTCCTCAAGATCTCCAGTGTGGACACTGCAGATA CTGGCACATACTACTGTGCTCAAGTTGATGGTTACTACTGGTACTTCG ATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGCCAAAACG ACACCACCAAGTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAAC TAACTCCATGGTGACCC TGGGATGCCTGGTCAAGGGCTATTTCCCTG AGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTG CACACCTTCCCAGCTGTCCTGGAGTCTGACCTCTACACTCTGAGCAG CTCAGTGACTGTCCCCTCCAGCCCTCGGCCCAGCGAGACCGTCACCT GCAACGTTGCCCACCCGGCCAGCAGCACCAAAGTGGACAAGAAAAT TGTGCCCAGGATTGTGGTTGTA AGCCTTGCATATGTACAGTCCCAG AAGTATCATCTGTCTTCATCTTCCCCCAAAGCCCAAGGATGTGCTCA CCATTACTCTGACTCcTAAGGTCACGTGTGTTGTGGTAGACATCAGCA AGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAG GTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCA CTTTCCGCTCAGTCAGTGAACT TCCCATCATGCACCAGGACTGGCTC AATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGC

- 26 045594

CCCCATCGAGAAAACCATCTCCCAAAACCAAAGGCAGACCGAAGGCT CCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGG ATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGAC ATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACA AGAACACTCAGCCCATCATGAACACGAATGGCTCTTACTTCGTCTAC AGCAAGCTCAATG TGCAGAAGAGCAACTGGGAGGCAGGAAATACTT TCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAG AAGAGCCTCTCCCACTCTCCTGGTAAATGA (SEQ ID NO:93) J512, J517, J518HC ATGGGCAGGCTTACTTCTTCATTCCTGCTACTGATTGTCCCTGCATAT GTCCTGTCCCAGGTCACTCTGAAAGAGTCTGGCCCTGGGATATTGCA GCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTTCACT GAGCACTTATGGTCTGGGTGTAGGTTGGATTCGTCAGCCTTCAGGGA AGGGTCTGGACTGGCTGGCCAACATTTGGTGGAATGATGATAAATAC TATAACT CAGCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTC CAACAACCAGGTATTCCTCAAGATCTCCAGTGTGGACACTGCAGATA CTGGCACATACTACTGTGCTCAAGTTGATGGTTACTACTGGTACTTCG ATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGCCAAAACG ACACCACCAAGTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAAC TAACTCCATGGTGACCC TGGGATGCCTGGTCAAGGGCTATTTCCCTG AGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTG CACACCTTCCCAGCTGTCCTGGAGTCTGACCTCTACACTCTGAGCAG CTCAGTGACTGTCCCCTCCAGCCCTCGGCCCAGCGAGACCGTCACCT GCAACGTTGCCCACCCGGCCAGCAGCACCAAAGTGGACAAGAAAAT TGTGCCCAGGATTGTGGTTGTA AGCCTTGCATATGTACAGTCCCAG AAGTATCATCTGTCTTCATCTTCCCCCAAAGATCAAGGATGTACTCA TGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGC GAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGA AGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGT ACTCTCCGGGTGGTCAGTGCCCTC CCCATCCAGCACCAGGACTGGAT GAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCA GCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAG CTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAG AAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGA CATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCT AAACTAC AAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTA

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CAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAG CTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGA CTAAGAGCTTCTCCCGGACTCCGGGTAAATAGTAA (SEQ ID NO: 12) J513HC ATGGGCAGGCTTACTTCTTCATTCCTGCTACTGATTGTCCCTGCATAT GTCCTGTCCCAGGTCACTCTGAAAGAGTCTGGCCCTGGGATATTGCA GCCCTCCCAGACCCTCAGTCTGACTTGTTCTCTCTCTGGGTTTTTCACT GAGCACTTATGGTCTGGGTGTAGGTTGGATTCGTCAGCCTTCAGGGA AGGGTCTGGGCTGGCTGGCCAACATTTGGTGGAATGATGATAAATAC TATAACT CAGCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTC CAACAACCAGGTATTCCTCAAGATCTCCAGTGTGGACACTGCAGATA CTGGCACATACTACTGTGCTCAAGTTGATGGTTACTACTGGTACTTCG ATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGCCAAAACG ACACCACCAAGTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAAC TAACTCCATGGTGACCC TGGGATGCCTGGTCAAGGGCTATTTCCCTG AGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTG CACACCTTCCCAGCTGTCCTGGAGTCTGACCTCTACACTCTGAGCAG CTCAGTGACTGTCCCCTCCAGCCCTCGGCCCAGCGAGACCGTCACCT GCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAAT TGTGCCCAGGATTGTGGTTGTA AGCCTTGCATATGTACAGTCCCAG AAGTATCATCTGTCTTCATCTTCCCCCAAAGCCCAAGGATGTGCTCA CCATTACTCTGACTCcTAAGGTCACGTGTGTTGTGGTAGACATCAGCA AGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAG GTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCA CTTTCCGCTCAGTCAGTGAACT TCCCATCATGCACCAGGACTGGCTC AATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGC CCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCT CCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGG ATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGAC ATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAG CGGAGAACTACA AGAACACTCAGCCCATCATGAACACGAATGGCTCTTACTTCGTCTAC AGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTT TCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAG AAGAGCCTCTCCCACTCTCCTGGTAAATGA (SEQ ID NO:94) J514, J519, J520 NS ATGGGCAGGCTTACTTCTTCATTCCTGCTACTGATTGTCCCTGCATAT GTCCTGTCCCAGGTCACTCTGAAAGAGTCTGGCCCTGGGATATTGCA

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GCCCTCCCAGACCCTCAGTCTGACTTGTTCTCTCTTGGGTTTTCACT GAGCACTTATGGTCTGGGTGTAGGTTGGATTCGTCAGCCTTCAGGGA AGGGTCTGGGCTGGCTGGCCAACATTTGGTGGAATGATGATAAATAC TATAACTCAGCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTC CAACAACCAGGTATTCCTCAAGATCTCCAGTGTGGACACTGCAGATA CTGG CACATACTACTGTGCTCAAGTTGATGGTTACTACTGGTACTTCG ATGTCTGGGGCGCCAGGGACCACGGTCACCGTCTCCTCAGCCAAAACG ACACCACCAAGTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAAC TAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTG AGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTG CACACCTTCCCAGC TGTCCTGGAGTCTGACCTCTACACTCTGAGCAG CTCAGTGACTGTCCCCTCCAGCCCTCGGCCCAGCGAGACCGTCACCT GCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAAT TGTGCCCAGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAG AAGTATCATCTGTCTTCATCTTCCCCCCCAAAGATCAAGGATGTACTCA TGATCTCCCTGAGCCCCAT AGTCACATGTGTGGTGGTGGATGTGAGC GAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGA AGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGT ACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGAT GAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCA GCGCCCATCGAGAGAACCATCTCA AAACCCAAAAGGGTCAGTAAGAG CTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAG AAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGA CATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTAC AAGAACACTGAACCAGTCCTGGACTCTGATCGTTCTTACTTCATGTA CAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAA GAAATAG CTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGA CTAAGAGCTTCTCCCGGACTCCGGGTAAATAGTAA (SEQ ID NO: 13) J515HC ATGGGCAGGCTTACTTCTTCATTCCTGCTACTGATTGTCCCTGCATAT GTCCTGTCCCAGGTCACTCTGAAAGAGTCTGGCCCTGGGATATTGCA GTCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTTCACT GAGCACTTATGGTCTGGGTGTAGGTTGGATTCGTCAGCCTTCAGGGA AGGGTCTGGACTGGCTGGCCAACATTTGGTGGAATGATGATAAATAC TATAACTCA GCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTC CAACAACCAGGTATTCCTCAAGATCTCCAGTGTGGACACTGCAGATA

- 29 045594

CTGGCACATACTACTGTGCTCAAGTTGATGGTTACTACTGGTACTTCG ATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGCCAAAACG ACACCACCAAGTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAAC TAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTG AGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTG CACACC TTCCCAGCTGTCCTGGAGTCTGACCTCTACACTCTGAGCAG CTCAGTGACTGTCCCCTCCAGCCCTCGGCCCAGCGAGACCGTCACCT GCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAAT TGTGCCCAGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAG AAGTATCATCTGTCTTCATCTTCCCCCAAAGCCCAAGGATGTGCTCA CCATTACTCTG ACTCcTAAGGTCACGTGTGTTGTGGTAGACATCAGCA AGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAG GTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCA CTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTC AATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGC CCCCATCGAG AAAACCATCTCCAAACCAAAGGCAGACCGAAGGCT CCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGG ATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGAC ATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACA AGAACACTCAGCCCATCATGAACACGAATGGCTCTTACTTCGTCTAC AGCAAGCTCAATGTGCAGAAGAGCA ACTGGGAGGCAGGAAATACTT TCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAG AAGACCTCTCCCACTCTCCTGGTAAATGA (SEQ ID NO:95) J516, J521, ATGGGCAGGCTTACTTCTTCATTCCTGCTACTGATTGTCCCTGCATAT J522 NS GTCCTGTCCCAGGTCACTCTGAAAGAGTCTGGCCCTGGGATATTGCA GTCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACT GAGCACTTATGGTCTGGGTGTAGGTTGGATTCGTCAGCCTTCAGGGA AGGGTCTGGACTGGCTGGCCAACATTTGGTGGAATGATGATAAATAC TATAACTCAGCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTC CAACAAC CAGGTATTCCTCAAGATCTCCAGTGTGGACACTGCAGATA CTGGCACATACTACTGTGCTCAAGTTGATGGTTACTACTGGTACTTCG ATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGCCAAAACG ACACCACCAAGTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAAC TAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTG AGCCAGTGACAGTGAC CTGGAACTCTGGATCCCTGTCCAGCGGTGTG

- 30 045594

CACACCTTCCCAGCTGTCCTGGAGTCTGACCTCTACACTCTGAGCAG CTCAGTGACTGTCCCCTCCAGCCCTCGGCCCAGCGAGACCGTCACCT GCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAAT TGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAG AAGTATCATCTGTCTTCATCTTCCCCCAAAGATCAAGGATGTACTCA TG ATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGC GAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGA AGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGT ACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGAT GAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCA GCGCCCAT CGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAG CTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAG AAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGA CATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTAC AAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTA CAGCAAGCTGAGAGTGGAAAAGA AGAACTGGGTGGAAAGAAATAG CTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGA CTAAGAGCTTCTCCCGGACTCCGGGTAAATAGTAA (SEQ ID NO: 14) J511-J516 ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCT L.C. TCCGGCAGTGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTC AGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCAT TGTACATAGTAATAGAAACACCTATTTAGAATGGTACCTGCAGAAAC CAGGCCAGTCTCCAAAGCTCCTGATCTACAACGTTTCCAACCGATTTT CTGGGGTCCCAGACAGTTCAGTGGCAGTGGATCAGGACAGATTTC ACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTA CTGCTTTCAAGGTTCACATGTTCCGCTCACGTTCGGTGCTGGGACCAA GCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCC CACCATCCAGTGAGCAGTTGACATCTGGAGGTGCCTCAGTCGTGTGC TTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGAT TGA TGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATC AGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTT GACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCC ACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAA TGAGTGTTAG (SEQ ID NO: 15)

- 31 045594

J517, J519 and J521 LC ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCT TCCGGCAGTGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTC AGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCAT TGTACATAGTAATAGAAACACCTATTTAGAATGGTACCTGCAGAAAC CAGGCCAGTCTCCAAAGCTCCTGATCTACAACGTTGCCAACCGATTT TCT GGGGTCCCAGACAGTTCAGTGGCAGTGGATCAGGGACAGATTT CACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATT ACTGCTTTCAAGGTTCACATGTTCCGCTCACGTTCGGTGCTGGGACCA AGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTC CCACCATCCAGTGAGCAGTTGACATCTGGAGGTGCCTCAGTCGTGTG CTTCTTGAA CAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGA TTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGAT CAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGT TGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGC CACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGA ATGAGTGTTAG (SEQ Ш NO:9 6) J518, J520 and J522 LC ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCT TCCGGCAGTGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTC AGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCAT TGTACATAGTAATAGAAACACCTATTTAGAATGGTACCTGCAGAAAC CAGGCCAGTCTCCAAAGCTCCTGATCTACCAGGTTTCCAACCCGATTTT CTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTC ACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTA CTGCTTTCAAGGTTCACATGTTCCGCTCACGTTCGGTGCTGGGACCAA GCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCC CACCATCCAGTGAGCAGTTGACATCTGGAGGTGCCTCAGTCGTGTGC TTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGAT TGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATC AGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTT GACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCC ACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAA TGAGTGTTAG (SEQ ID NO:97)

The present invention also provides a polynucleotide containing a nucleic acid encoding a heavy chain variable domain, wherein the nucleic acid encoding a heavy chain variable domain contains the three CDR encoding sequence SEQ ID NO: 12. The present invention also provides a polynucleotide containing a nucleic acid encoding a heavy chain variable domain, wherein the nucleic acid encoding the heavy chain variable domain comprises the three CDR encoding sequence of SEQ ID NO: 13. The present invention also provides a polynucleotide comprising a nucleic acid encoding a heavy chain variable domain, wherein the nucleic acid encoding the variable domain heavy chain, contains the three-CDR encoding sequence SEQ ID NO: 14. The present invention also provides a polynucleotide containing a nucleic acid encoding a heavy chain variable domain, wherein the nucleic acid encoding a heavy chain variable domain contains the three-CDR encoding sequence SEQ ID NO: 93. The present invention also provides a polynucleotide comprising a nucleic acid encoding a heavy chain variable domain, wherein the nucleic acid encoding a heavy chain variable domain comprises a tri-CDR encoding sequence SEQ ID NO: 94. The present invention also provides a polynucleotide comprising a nucleic acid , encoding a heavy chain variable domain, wherein the nucleic acid encoding the heavy chain variable domain contains the three CDR encoding sequence SEQ ID NO: 95.

The present invention also provides a polynucleotide containing a nucleic acid encoding a light chain variable domain, wherein the nucleic acid encoding the variable

- 32 045594 light chain domain, contains the three CDR encoding sequence SEQ ID NO: 15. The present invention also provides a polynucleotide containing a nucleic acid encoding a light chain variable domain, wherein the nucleic acid encoding a light chain variable domain contains an encoding three CDRs sequence SEQ ID NO: 96. The present invention also provides a polynucleotide comprising a nucleic acid encoding a light chain variable domain, wherein the nucleic acid encoding a light chain variable domain comprises the three CDR encoding sequence SEQ ID NO: 97.

The present invention also provides a composition comprising (i) a polynucleotide containing a nucleic acid encoding a heavy chain variable domain, wherein the nucleic acid encoding a heavy chain variable domain comprises a three CDR encoding sequence of SEQ ID NO: 12 or 93; and (ii) a polynucleotide comprising a nucleic acid encoding a light chain variable domain, wherein the nucleic acid encoding the light chain variable domain comprises the three CDR encoding sequence of SEQ ID NO: 15, 96 or 97.

The nucleic acid encoding the heavy chain variable domain and the nucleic acid encoding the light chain variable domain may be present in the same polynucleotide or in different polynucleotides. The nucleic acid encoding the heavy chain variable domain and the nucleic acid encoding the light chain variable domain may be in the same vector or in different vectors. The present invention also provides a composition comprising (i) a polynucleotide comprising a nucleic acid encoding a heavy chain variable domain, wherein the nucleic acid encoding a heavy chain variable domain comprises a three CDR encoding sequence of SEQ ID NO: 13 or 94; and (ii) a polynucleotide comprising a nucleic acid encoding a light chain variable domain, wherein the nucleic acid encoding the light chain variable domain comprises the three CDR encoding sequence of SEQ ID NO: 15, 96 or 97.

The nucleic acid encoding the heavy chain variable domain and the nucleic acid encoding the light chain variable domain may be present in the same polynucleotide or in different polynucleotides. The nucleic acid encoding the heavy chain variable domain and the nucleic acid encoding the light chain variable domain may be in the same vector or in different vectors. The present invention also provides a composition comprising (i) a polynucleotide containing a nucleic acid encoding a heavy chain variable domain, wherein the nucleic acid encoding a heavy chain variable domain comprises a three CDR encoding sequence of SEQ ID NO: 14 or 95; and (ii) a polynucleotide comprising a nucleic acid encoding a light chain variable domain, wherein the nucleic acid encoding the light chain variable domain comprises the three CDR encoding sequence of SEQ ID NO: 15, 96 or 97.

The nucleic acid encoding the heavy chain variable domain and the nucleic acid encoding the light chain variable domain may be present in the same polynucleotide or in different polynucleotides. The nucleic acid encoding the heavy chain variable domain and the nucleic acid encoding the light chain variable domain may be in the same vector or in different vectors. The present invention also provides a polynucleotide containing a nucleic acid encoding a heavy chain variable domain, wherein the nucleic acid encoding a heavy chain variable domain contains the variable domain encoding sequence SEQ ID NO: 12. The present invention also provides a polynucleotide containing a nucleic acid encoding a heavy chain variable domain, wherein the nucleic acid encoding the heavy chain variable domain comprises the variable domain encoding sequence SEQ ID NO: 13. The present invention also provides a polynucleotide comprising a nucleic acid encoding a heavy chain variable domain, wherein the nucleic acid encoding the variable domain heavy chain, contains the variable domain encoding sequence SEQ ID NO: 14. The present invention also provides a polynucleotide containing a nucleic acid encoding a heavy chain variable domain, wherein the nucleic acid encoding a heavy chain variable domain contains the variable domain encoding sequence SEQ ID NO: 93. The present invention also provides a polynucleotide comprising a nucleic acid encoding a heavy chain variable domain, wherein the nucleic acid encoding a heavy chain variable domain comprises the variable domain encoding sequence SEQ ID NO: 94. The present invention also provides a polynucleotide comprising a nucleic acid , encoding a heavy chain variable domain, wherein the nucleic acid encoding the heavy chain variable domain comprises the variable domain encoding sequence SEQ ID NO: 95.

The present invention also provides a polynucleotide containing a nucleic acid encoding a light chain variable domain, wherein the nucleic acid encoding a light chain variable domain contains the variable domain encoding sequence SEQ ID NO: 15. The present invention also provides a polynucleotide containing a nucleic acid encoding

- 33 045594 a light chain variable domain, wherein the nucleic acid encoding the light chain variable domain comprises the variable domain encoding sequence SEQ ID NO: 96. The present invention also provides a polynucleotide comprising a nucleic acid encoding a light chain variable domain, wherein the nucleic acid , encoding a light chain variable domain, contains the variable domain encoding sequence SEQ ID NO: 97. Also provided by the present invention is a composition comprising (i) a polynucleotide containing a nucleic acid encoding a heavy chain variable domain, wherein the nucleic acid encoding a heavy chain variable domain , contains the variable domain coding sequence SEQ ID NO: 12 or 93; and (ii) a polynucleotide comprising a nucleic acid encoding a light chain variable domain, wherein the nucleic acid encoding a light chain variable domain comprises a variable domain encoding sequence of SEQ ID NO: 15, 96, or 97.

The nucleic acid encoding the heavy chain variable domain and the nucleic acid encoding the light chain variable domain may be present in the same polynucleotide or in different polynucleotides. The nucleic acid encoding the heavy chain variable domain and the nucleic acid encoding the light chain variable domain may be in the same vector or in different vectors. The present invention also provides a composition comprising (i) a polynucleotide comprising a nucleic acid encoding a heavy chain variable domain, wherein the nucleic acid encoding a heavy chain variable domain comprises a variable domain encoding sequence of SEQ ID NO: 13 or 94; and (ii) a polynucleotide comprising a nucleic acid encoding a light chain variable domain, wherein the nucleic acid encoding a light chain variable domain comprises a variable domain encoding sequence of SEQ ID NO: 15, 96, or 97.

The nucleic acid encoding the heavy chain variable domain and the nucleic acid encoding the light chain variable domain may be present in the same polynucleotide or in different polynucleotides. The nucleic acid encoding the heavy chain variable domain and the nucleic acid encoding the light chain variable domain may be in the same vector or in different vectors.

The present invention also provides a composition comprising (i) a polynucleotide comprising a nucleic acid encoding a heavy chain variable domain, wherein the nucleic acid encoding a heavy chain variable domain comprises a variable domain encoding sequence of SEQ ID NO: 14 or 95; and (ii) a polynucleotide comprising a nucleic acid encoding a light chain variable domain, wherein the nucleic acid encoding a light chain variable domain comprises a variable domain encoding sequence of SEQ ID NO: 15, 96, or 97.

The nucleic acid encoding the heavy chain variable domain and the nucleic acid encoding the light chain variable domain may be present in the same polynucleotide or in different polynucleotides. The nucleic acid encoding the heavy chain variable domain and the nucleic acid encoding the light chain variable domain may be in the same vector or in different vectors.

The present invention also provides polynucleotides containing a nucleotide sequence that encodes SEQ ID NO: 6, 7, 8, 9, 10, 11, 22, 23, 24, 25, 26, 27, 28 or 29, respectively.

The present invention also provides polynucleotides that are at least about 80, 85, or 90% identical to the nucleotide sequence encoding SEQ ID NO: 6, 7, 8, 9, 10, 11, 22, 23, 24, 25, 26 , 27, 28 or 29 respectively.

The present invention also provides polynucleotides containing a nucleotide sequence that is at least about 95% identical to the nucleotide sequence that encodes SEQ ID NO: 6, 7, 8, 9, 10, 11, 22, 23, 24, 25, 26 , 27, 28 or 29 respectively. The present invention also provides polynucleotides containing a nucleotide sequence that is at least about 96% identical to the nucleotide sequence that encodes SEQ ID NO: 6, 7, 8, 9, 10, 11, 22, 23, 24, 25, 26 , 27, 28 or 29 respectively. The present invention also provides polynucleotides containing a nucleotide sequence that is at least about 97% identical to the nucleotide sequence that encodes SEQ ID NO: 6, 7, 8, 9, 10, 11, 22, 23, 24, 25, 26 , 27, 28 or 29 respectively. The present invention also provides polynucleotides containing a nucleotide sequence that is at least about 98% identical to the nucleotide sequence that encodes SEQ ID NO: 6, 7, 8, 9, 10, 11, 22, 23, 24, 25, 26 , 27, 28 or 29 respectively. The present invention also provides polynucleotides containing a nucleotide sequence that is at least about 99% identical to the nucleotide sequence that encodes SEQ ID NO: 6, 7, 8, 9, 10, 11, 22, 23, 24, 25, 26 , 27, 28 or 29 respectively.

In a specific embodiment, a polynucleotide, or combination of polynucleotides, is provided

- 34 045594 used in the present invention contain a nucleotide sequence or combination of nucleotide sequences encoding an antibody or antigen binding fragment thereof that immunospecifically binds to B7-H4 (for example, human B7-H4), wherein the antibody or antigen binding fragment thereof comprises a heavy chain, wherein the heavy chain contains a heavy chain variable domain containing the amino acid sequence shown in table. 3 (eg, SEQ ID NO: 6-8), and a constant region containing the amino acid sequence of a mouse gamma (γ) heavy chain constant region (eg, IgG1 or IgG2a).

In a specific embodiment, a polynucleotide or combination of polynucleotides provided by the present invention comprises a nucleotide sequence or combination of nucleotide sequences encoding an antibody or antigen-binding fragment thereof that immunospecifically binds B7-H4 (e.g., human B7-H4), wherein the antibody or antigen-binding fragment thereof the fragment contains a light chain, wherein the light chain contains a light chain variable domain containing the amino acid sequence shown in table. 4 (eg, SEQ ID NO: 9-11), and a constant region containing the amino acid sequence of the mouse kappa light chain constant region. In a specific embodiment, the present invention provides polynucleotides comprising a nucleotide sequence encoding an anti-B7-H4 antibody or an antigen binding fragment thereof, or a domain thereof, as defined herein; see, for example, table. 1.

The present invention also provides polynucleotides encoding an anti-B7-H4 antibody or an antigen-binding fragment thereof described in the present invention, or a domain thereof, that are optimized, for example, by codon/RNA optimization, replacement with heterologous signal sequences, and by eliminating unstable elements mRNA. Methods for generating optimized nucleic acids encoding an anti-B7-H4 antibody or an antigen-binding fragment or domain (e.g., heavy chain, light chain, VH domain, or VL domain) for recombinant expression by making changes to the codons (e.g., changing the codon encoding the same amino acid due to the degeneracy of the genetic code) and/or elimination of inhibitory regions in the mRNA, can be accomplished by adapting optimization methods described, for example, in US patents No. 5965726, 6174666, 6291664, 6414132 and 6794498, respectively. A polynucleotide encoding an antibody or antigen binding fragment thereof described in the present invention, or a domain thereof, can be obtained from a nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods ). For example, PCR amplification using synthetic primers that are capable of hybridizing to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to produce nucleic acids containing a sequence encoding the light chain and/or heavy chain of an antibody or antigen-binding fragment thereof. Such PCR amplification methods can be used to produce nucleic acids containing a sequence encoding a light chain variable region and/or a heavy chain variable region of an antibody or an antigen binding fragment thereof.

The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example, to produce chimeric or humanized antibodies or antigen binding fragments thereof. The polynucleotides provided by the present invention may be, for example, in the form of RNA or in the form of DNA. DNA includes genomic DNA and synthetic DNA, and DNA can be double-stranded or single-stranded. Being single-stranded, DNA can be a coding strand or a non-coding (antisense) strand. In certain embodiments, the polynucleotide is cDNA or DNA that lacks one or more endogenous introns. In certain embodiments, the polynucleotide is a non-naturally occurring polynucleotide. In certain embodiments, the polynucleotide is produced recombinantly. In certain embodiments, the polynucleotides are isolated. In certain embodiments, the polynucleotides are substantially pure. In certain embodiments, the polynucleotide is purified from natural components.

Cells and vectors.

In certain aspects, the present invention provides vectors (eg, expression vectors) containing polynucleotides containing nucleotide sequences encoding anti-B7-H4 antibodies and antigen binding fragments or a domain thereof, for recombinant expression in host cells, preferably mammalian cells. The present invention also provides cells, for example host cells, containing such vectors for the recombinant expression of anti-B7-H4 antibodies or antigen-binding fragments thereof described in the present invention. In a specific aspect, the present invention provides methods for producing an antibody or antigen binding fragment thereof described herein, comprising expressing such

- 35 045594 antibody or antigen-binding fragment thereof in the host cell.

In certain embodiments, the recombinant expression of an antibody, or an antigen binding fragment, or a domain thereof described in the present invention (e.g., a heavy or light chain described in the present invention) that specifically binds B7-H4 (e.g., human B7-H4) involves constructing an expression vector containing a polynucleotide that encodes an antibody, or an antigen-binding fragment, or a domain thereof. After obtaining a polynucleotide encoding an antibody, or an antigen-binding fragment, or a domain thereof (e.g., a heavy or light chain variable domain) described in the present invention, a vector for generating the antibody or antigen-binding fragment thereof can be produced by recombinant DNA technology using methods , well known in the art. Thus, the present invention describes methods for producing a protein by expressing a polynucleotide comprising an antibody, or an antigen binding fragment thereof, or a domain (eg, light chain or heavy chain) encoding a nucleotide sequence thereof. Methods that are well known to those skilled in the art can be used to construct expression vectors containing an antibody, or an antigen binding fragment thereof, or a domain (e.g., light chain or heavy chain), coding sequences, and corresponding transcriptional and translational control signals . These methods include, for example, in vitro recombinant DNA techniques, in vivo synthesis techniques, and genetic recombination. Also provided are replicable vectors containing a nucleotide sequence encoding an antibody or antigen-binding fragment thereof described in the present invention, a heavy or light chain, a heavy or light chain variable domain, or a heavy or light chain CDR operably linked to a promoter. Such vectors, for example, may contain a nucleotide sequence encoding the constant region of an antibody or an antigen-binding fragment thereof (see, for example, International Publications No. WO 86/05807 and WO 89/01036; and US Pat. No. 5,122,464), and the variable domains of the antibody or its antigen binding fragment can be cloned into such a vector to express the entire heavy chain, the entire light chain, or both the entire heavy and all light chains.

The expression vector can be transferred into a cell (eg, a host cell) using conventional techniques, and the resulting cells can then be cultured using conventional techniques to produce the antibody or antigen binding fragment thereof described in the present invention (eg, the antibody or antigen binding fragment thereof containing six CDRs from SEQ ID NOs: 22-25, 26, 27 or 28, and 29, a VH region from SEQ ID NOs: 6-8, a VL region from SEQ ID NOs: 9-11, a VH region from SEQ ID NOs: : 6-8 and the VL region of SEQ ID NO: 9-11, the heavy chain of SEQ ID NO: 16-18, 89, 90 or 91, the light chain of SEQ ID NO: 19, 30, 31, or the heavy chain of SEQ ID NO: 16-18, 89, 90 or 91 and the light chain from SEQ ID NO: 19, 30 or 31). Thus, the present invention provides host cells containing a polynucleotide encoding an antibody or antigen binding fragment thereof described in the present invention, for example, an antibody or antigen binding fragment thereof containing six CDRs from SeQ ID NO: 22-25, 26, 27 or 28, and 29, VH region of SEQ ID NO: 6-8, VL region of SEQ ID NO: 9-11, VH region of SEQ ID NO: 6-8, and VL region of SEQ ID NO: 9-11, heavy chain from SEQ ID NO: 16-18, 89, 90 or 91, light chain from SEQ ID NO: 19, 30, 31, or heavy chain from SEQ ID NO: 16-18, 89, 90 or 91 and light chain from SEQ ID NO: 19, 30 or 31, operably linked to a promoter for expression of such sequences in a host cell. In certain embodiments, for the expression of double-chain antibodies or antigen-binding fragments thereof, vectors encoding both the heavy and light chains can be co-expressed individually in a host cell to express the entire immunoglobulin, as described below. In certain embodiments, the host cell contains a vector containing a polynucleotide encoding the heavy chain and light chain of an antibody of the present invention, or a domain thereof. In specific embodiments, the host cell contains two different vectors, wherein the first vector contains a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody or antigen binding fragment thereof described in the present invention, and the second vector contains a polynucleotide encoding the light chain or light chain variable region. chains of an antibody described in the present invention (for example, an antibody containing the six CDRs of SEQ ID NOs: 22-25, 26, 27 or 28, and 29), or a domain thereof. In other embodiments, the first host cell contains a first vector containing a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody or antigen binding fragment thereof described in the present invention, and the second host cell contains a second vector containing a polynucleotide encoding the light chain or a light chain variable region of an antibody or antigen binding fragment thereof described in the present invention (eg, an antibody or antigen binding fragment thereof comprising the six CDRs of SEQ ID NOs: 22-25, 26, 27, or 28 and 29). In specific embodiments, the heavy chain/heavy chain variable region expressed by the first cell is linked to the light chain/light chain variable region of the second cell to form an anti-B7-H4 antibody or antigen-binding fragment thereof described herein (e.g., an antibody or antigen-binding fragment thereof) fragment containing six CDRs from SEQ ID NO: 22-25, 26, 27 or 28 and 29). In certain embodiments, the present invention provides a population of host cells comprising such a first host cell and such a second host cell.

In a specific embodiment, the present invention provides a population of vectors comprising a first vector containing a polynucleotide encoding the light chain/light chain variable region of an anti-B7-H4 antibody or an antigen binding fragment thereof described in the present invention, and a second vector containing a polynucleotide encoding a heavy chain. the chain/heavy chain variable region of an anti-B7-H4 antibody or antigen-binding fragment thereof described herein (eg, an antibody or antigen-binding fragment thereof comprising the CDRs of SEQ ID NOs: 22-25, 26, 27, or 28 and 29). Alternatively, a single vector may be used that encodes and is capable of expressing heavy and light chain polypeptides.

A variety of host expression vector systems can be used to express the antibodies and antigen binding fragments thereof described herein (e.g., an antibody or antigen binding fragment thereof comprising the CDRs of SEQ ID NOs: 22-25, 26, 27, or 28 and 29). ., for example, US patent No. 5807715). Such host expression systems provide vehicles by which coding sequences of interest can be produced and then purified, and they provide cells that, when transformed or transfected with the appropriate nucleotide coding sequences, can express the antibody or antigen binding fragment thereof described in the present invention, in situ. These include, but are not limited to, microorganisms such as bacteria (eg, E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (eg, Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (eg, baculovirus) containing antibody coding sequences; plant cell systems (eg green algae such as Chlamydomonas reinhardtii) infected with recombinant viral expression vectors (eg cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (eg Ti plasmid), containing sequences encoding antibodies; or mammalian cell systems (eg, COS cells (eg, COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa and NIH 3T3, HEK-293T, HepG2, SP210 , R1.1, B-W, L-M, BSCl, BSC40, YB/20 and BMT10) carrying recombinant expression constructs containing promoters derived from the genome of mammalian cells (for example, the metallothionein promoter) or from mammalian viruses (for example, the adenovirus late promoter; vaccinia virus promoter 7.5K). In a specific embodiment, the cells for expressing the antibodies or antigen binding fragments thereof described in the present invention (e.g., antibodies or antigen binding fragments thereof comprising the CDRs of SEQ ID NOs: 22-25, 26, 27, or 28 and 29) are CHO cells, for example, CHO cells from the CHO GS System™ (Lonza). In a specific embodiment, the cells for expressing the antibodies described in the present invention are human cells, for example, human cell lines. In a specific embodiment, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In a specific embodiment, bacterial cells, such as Escherichia coli, or eukaryotic cells (eg, mammalian cells), especially for expressing a whole recombinant antibody molecule, are used to express the recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells in combination with a vector such as the core intermediate early gene promoter element from human cytomegalovirus are an effective expression system for antibodies (Foecking M.K. & Hofstetter H. (1986), Gene, 45:101-105; and Cockett M. I. et al (1990), Biotechnology, 8:662-667). In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention are produced by CHO cells or NS0 cells.

In some embodiments, the signal peptide is used in the construction of a vector containing the VH or VL region of an antibody or antigen binding fragment thereof provided by the present invention. In one specific embodiment, the nucleotide sequence and amino acid sequence of the signal peptide used to construct an expression vector for the VH sequence are provided in SEQ ID NO: 2 and 4, respectively. In another specific embodiment, the nucleotide sequence and amino acid sequence of the signal peptide used to construct an expression vector for the VL sequence are provided in SEQ ID NO: 3 and 5, respectively.

In one particular embodiment, the nucleotide sequence of the VH signal peptide is ATGGGC AGGCTT ACTTCT TCATTC CTGCTA CTGATT GTCCCT GCATAT GTCCTG TCC (SEQ ID NO: 2). In another specific embodiment, the amino acid sequence of the VH signal peptide is MGRLTSSFLLLIVPAYVLS (SEQ ID NO: 4). In one specific embodiment, the nucleotide sequence of the VL signal peptide

- 37 045594 is presented as ATGAAG TTGCCT GTTAGG CTGTTG GTGCTG ATGTTC TGGATT CCTGCT TCCGGC AGT (SEQ ID NO: 3). In another specific embodiment, the amino acid sequence of the VL signal peptide is MKLPVRLLLVLMFWIPASGS (SEQ ID NO: 5). In addition, a host cell strain may be selected that modulates the expression of the inserted sequences or modifies and processes the gene product in a particular desired manner. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products can contribute to protein function. For this purpose, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D cells, NSO (a murine myeloma cell line that does not endogenously produce no immunoglobulin chains), CRL7O3O, COS (eg COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSCl, BSC40, YB/20, BMT10 and HsS78Bst. In certain embodiments, the anti-B7-H4 antibodies described herein (e.g., an antibody or antigen binding fragment thereof comprising CDR SEQ ID NOs: 22-25, 26, 27, or 28 and 29) are produced in mammalian cells, such as SNO.

In certain embodiments, the anti-B7-H4 antibodies described herein (e.g., an antibody or antigen binding fragment thereof comprising the CDRs of SEQ ID NOs: 22-25, 26, 27, or 28 and 29) are produced in Potelligent® CHOK1SV cells.

Once an antibody or antigen binding fragment thereof described in the present invention has been produced by recombinant expression, it can be purified by any method known in the art for purifying an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, in particular, by affinity for a specific antigen after protein A, and column chromatography with size distribution); centrifugation, differential solubility, or any other standard protein purification technique. Moreover, the antibodies or antigen binding fragments thereof described in the present invention can be fused to heterologous polypeptide sequences described in the present invention or known in the art to facilitate purification.

In specific embodiments, an antibody or antigen binding fragment thereof described in the present invention is isolated or purified. Typically, an isolated antibody or antigen binding fragment thereof is one that is substantially free of other antibodies or antigen binding fragments thereof with an antigenic specificity different from the isolated antibody or antigen binding fragment thereof. For example, in a particular embodiment, the antibody composition or antigen binding fragment thereof described in the present invention is substantially free of cellular material and/or chemical precursors.

Applications and methods.

Detection and diagnostic applications.

The anti-B7-H4 antibody or antigen-binding fragment thereof described in the present invention (see, for example, section 5.2) can be used to quantify the levels of B7-H4 protein in a biological sample using methods known to those skilled in the art, including immunoassays , such as enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), immunohistochemistry (IHC), immunoprecipitation, and Western blotting.

Suitable labels for antibody assays are known in the art and include enzyme labels such as horseradish peroxidase (HRP) and glucose oxidase; radioisotopes such as iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹²¹ In) and technetium ( ⁹⁹ Tc); haptens, fluorescent tags, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles and/or ligands such as biotin. In some embodiments, the enzyme (enzyme tag) will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, hydrogen (horseradish) peroxidase and/or glucose oxidase.

Such labels can be used to label an antibody or antigen binding fragment thereof described in the present invention. Alternatively, a second antibody or antigen binding fragment thereof that recognizes an anti-B7-H4 antibody or an antigen binding fragment thereof described herein can be labeled and used in combination with an anti-B7-H4 antibody or an antigen binding fragment thereof to detect levels of the B7-H4 protein. In some embodiments, such a secondary antibody or antigen binding fragment thereof, such as a mouse or rodent antibody, is labeled with an enzyme (eg, horseradish peroxidase) and detected with an enzyme substrate (eg, 3,3'-diaminobenzidine (DAB)).

Several methods are known in the art for attaching or conjugating an antibody to its conjugate fragment. Some coupling methods include the use of a metal chelate complex using, for example, an organic chelating agent such as diethylenetriaminepentaacetic acid anhydride (DTPA); ethylene triaminetetraacetic acid;

- 38 045594

N-chloro-p-toluenesulfonamide and/or tetrachloro-3a-6a-diphenylglycouryl-3 attached to an antibody (US Pat. No. 4,472,509 and 4,938,948, each of which is incorporated herein by reference). Monoclonal antibodies can also react with the enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of such coupling agents or by reaction with isothiocyanate. In US Pat. No. 4,938,948, imaging of breast tumors, for example, is achieved using monoclonal antibodies, and the detectable imaging moieties are linked to the antibody using linkers such as methyl p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate .

In other embodiments, derivatization of immunoglobulins by selectively introducing sulfhydryl groups into the Fc region of the immunoglobulin is discussed using reaction conditions that do not alter the binding site of the antibody. Antibody conjugates produced according to this methodology have been described to exhibit improved durability, specificity, and sensitivity (US Pat. No. 5,196,066, incorporated herein by reference). Site-specific coupling of effector or reporter molecules, where the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region, has also been described in the literature (O'Shannessy et al., Biotechnol. Appl. Biochem., 1987 Dec, 9(6):488 -96). A B7-H4 protein expression assay is intended to involve a qualitative or quantitative measurement or assessment of the B7-H4 protein level in a first biological sample, either directly (e.g., by determining or estimating the absolute protein level) or indirectly (e.g., by comparison with the B7 protein level -H4 in the second biological sample or reference sample). The expression level of the B7-H4 polypeptide in a first biological sample may be measured or assessed and compared to a reference level of B7H4 protein, wherein the reference is determined from a second unaffected biological sample or determined by averaging levels in a population of unaffected samples. As will be appreciated by those skilled in the art, once the reference level of the B7-H4 polypeptide is known, it can be systematically used as a reference standard. The term biological sample, as used herein, refers to any biological sample obtained from a subject, cell line, tissue, or other source of cells potentially expressing B7H4. Non-limiting sources of biological sample for use in the present invention include, for example, solid tissue, biopsy, ascites, aspirates, fluid extracts, blood (including circulating tumor cells), plasma, serum, cerebrospinal fluid, lymph fluid, external sections of the skin, respiratory tract, intestinal and genitourinary tract, tears, saliva, milk, tumors, organs, cell cultures and/or cell culture components. Methods for obtaining biopsy samples of tissues and body fluids from animals (eg, humans) are well known in the art. Examples of methods for obtaining circulating tumor cells (CTCs) are described in Ferreira et al., Molecular Oncology, 10:374-394, which is incorporated herein by reference in its entirety. For example, CTCs can be enriched from a blood sample using immunoaffinity or biophysics-based strategies. CTCs can also be obtained using direct imaging techniques. Examples of methods for obtaining and processing biopsy samples that can be used in immunohistochemical (IHC) analyzes are presented in the present invention in Example 4.

The anti-B7-H4 antibody described in the present invention can be used in diagnostic applications, including in vitro applications, which are well known and standard to those skilled in the art and are also based on the present disclosure. In vitro B7-H4 diagnostic assays and kits can be used to study patient samples, including samples known to have cancer or suspected cancer. This type of prognostic and diagnostic monitoring and evaluation is used in practice, for example, using antibodies to the HER2 protein in breast cancer (HercepTest™, Dako), where the test is used to evaluate a patient for antibody therapy using Herceptin®.

Antibodies to B7-H4 and their antigen binding fragments described in the present invention may carry a detectable or functional label. When fluorescent tags are used, currently available microscopy and fluorescence activated cell sorting (FACS), or a combination of the two methods known in the art, can be used to identify and quantify specific binding members. Antibodies to B7-H4 or antigen binding fragments thereof described in the present invention may carry a fluorescent label. Examples of fluorescent labels include, for example, reactive and conjugated probes such as aminocoumarin, fluorescein and Texas red, Alexa Fluor dyes, Cy dyes and DyLight dyes. The antibody to B7-H4 can carry a radioactive label, such as the isotopes ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁶⁷ Cu, ⁹⁰ Y, ⁹⁹ Tc, ¹ⁿ In, ¹¹⁷ Lu, ¹²¹ I, ¹²⁴ 1, ¹²⁵ 1, ¹³¹ I, ¹⁹⁸ Au, ²¹¹ At, ²¹³ Bi, ²²⁵ Ac and ¹⁸⁶ Re. When radioactive labels are used, currently available counting methods known in the art can be used to identify and quantify the specific binding of an antibody to B7-H4 or an antigen binding fragment to B7-H4

- 39 045594 (for example, B7-H4 human). In the case where the label is an enzyme, detection can be performed using any method currently used in the art: colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric method. This can be achieved by contacting the sample or control with an anti-B7-H4 antibody or antigen-binding fragment thereof under conditions that allow the formation of a complex between the antibody or antigen-binding fragment thereof and B7-H4. All complexes formed between the antibody or its antigen-binding fragment and B7-H4 are detected and compared in the sample and control sample. In light of the specific binding of the antibodies or antigen binding fragments thereof described in the present invention to B7-H4, the antibodies or antigen binding fragments thereof can be used to specifically detect B7-H4 expression, for example, in whole cells, on cell membranes or in the cytoplasm. Antibodies or antigen binding fragments thereof described in the present invention can also be used to purify B7-H4 by immunoaffinity purification.

Also included in the present invention is an assay system that can be provided as an assay kit to quantify the presence of, for example, B7-H4. The assay system or kit may contain a labeled component, such as a labeled antibody or antigen binding fragment, as well as one or more additional immunochemical reagents. For more information on kits, see, for example, section 5.4.3 below.

In some aspects, methods for detecting B7-H4 in a sample in vitro include contacting the sample with an antibody or antigen-binding fragment thereof, as described in the present invention. In some aspects, the present invention provides the use of an antibody or antigen binding fragment thereof, as described herein, to detect B7-H4 in a sample in vitro. In one aspect, the present invention provides an antibody or antigen binding fragment thereof described herein for use in detecting B7-H4 in a subject or in a sample obtained from a subject. In one aspect, the present invention provides an antibody or antigen binding fragment thereof described herein for use in diagnostic purposes. In one embodiment, the antibody contains a detectable label. In one preferred embodiment, B7-H4 is human B7-H4. In one preferred embodiment, the subject is a human. In a further embodiment, the subject, for example, a human subject, has cancer. In some aspects, the present invention provides immunoassay methods for binding and detecting B7-H4. Antibodies prepared in accordance with the present invention can be used to detect B7-H4. Some immunoassay methods include, but are not limited to, immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, immunofluorescence assay, chemiluminescent assay, bioluminescent assay, and Western blotting. The steps of various immunoassay methods have been described in the scientific literature, such as Doolittle M.H., Ben-Zeev O., Methods Mol. Biol., 1999, 109:215-37; Gulbis V, Galand P, Hum Pathol., 1993 Dec, 24(12):1271-85; and De Jager R. et al., Semin. Nucl. Med., 1993 Apr, 23(2):165-79, each of which is incorporated herein by reference.

In general, immunobinding methods involve obtaining a sample, such as a sample suspected of containing B7-H4, and contacting the sample with a first anti-B7-H4 antibody of the present invention under conditions effective to form immune complexes.

Contacting a selected biological sample with an antibody under effective conditions and for a period of time sufficient to form immune complexes (primary immune complexes) typically involves adding an antibody composition to the sample and incubating the mixture for a period of time sufficient to form antibodies of immune complexes, i.e. for specific binding to any B7-H4 present. Thereafter, the sample antibody composition, such as a tissue section, ELISA plate, dot blot, or Western blot, is typically washed to remove any nonspecifically bound antibodies, allowing detection of only those antibodies that are specifically bound to the primary immune complexes.

In general, detection of immune complex formation is well known in the art and can be achieved using a variety of approaches. Such methods are typically based on detection of a label or marker, such as any of a radioactive, fluorescent, biological or enzymatic label. US patents covering the use of such marks include US Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, each of which is incorporated herein by reference. In some embodiments, a secondary binding agent, such as a secondary antibody and/or a biotin/avidin ligand binding system, may be used in accordance with methodologies known in the art.

In some embodiments, a first antibody that binds within the primary immune kits can be detected by a second binding agent having binding affinity for the antibody. In such cases, the second coupling agent may be bound

- 40 045594 with detectable tag. The second binding agent is often itself an antibody, which may thus be referred to as a secondary antibody. Primary immune complexes are contacted with a labeled secondary binding agent, or antibody, under effective conditions and for a period of time sufficient to form secondary immune complexes. The secondary immune complexes are then typically washed to remove any nonspecifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

Additional methods include detection of primary immune complexes using a two-step approach. A second binding agent, such as an antibody, which has binding affinity for the antibody, is used to form secondary immune complexes as described above. After washing, the secondary immune complexes are brought into contact with a third binding agent or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to form immune complexes (tertiary immune complexes). A third ligand or antibody is bound to the detectable label, allowing detection of tertiary immune complexes formed in this manner. Such a system can provide signal amplification if desired.

In another embodiment, a biotinylated monoclonal or polyclonal antibody is used to detect target antigens, and the second step antibody is then used to detect biotin attached to the antibody complex. In this method, the sample to be analyzed is first incubated in a solution containing the first-stage antibody. When a target antigen is present, some antibodies specifically bind to the antigen to form a biotinylated antibody-antigen complex. The antibody-antigen complex is then amplified by incubation in subsequent solutions of streptavidin (or avidin) and biotinylated DNA and/or complementary biotinylated DNA, with additional biotin sites added to the antibody-antigen complex at each step. The amplification steps are repeated until a suitable level of amplification is achieved, after which the sample is incubated in a solution containing the second-step anti-biotin antibody. Such a second step antibody is labeled with, for example, an enzyme that can be used to detect the presence of an antibody-antigen complex by histoenzymology using a chromogenic substrate. With suitable amplification, a conjugate can be obtained that is macroscopically visible.

In one embodiment, immunohistochemistry (IHC) is used for immunological detection. Using IHC, detection of B7-H4 in a sample is achieved by targeting the sample with a binding agent, such as an anti-B7-H4 antibody or antigen-binding fragment thereof. The binding agent may be linked, directly or indirectly, to the detectable label, or may be detected by another binding agent that is directly or indirectly linked to the detectable label. In one embodiment, the IHC assay uses 3,3'-diaminobenzidine (DAB) to detect the primary antibody associated with B7-H4. In one embodiment, the concentration of the anti-B7-H4 antibody or antigen binding fragment thereof in the IHC assay is from about 1 μg/ml to about 50 μg/ml. In one embodiment, the concentration of the anti-B7-H4 antibody or antigen binding fragment thereof in the IHC assay is from about 1 μg/ml to about 20 μg/ml. In one embodiment, the concentration of the anti-B7-H4 antibody or antigen-binding fragment thereof in the IHC assay is about 10 μg/ml. IHC can be performed on cells, cell pellets, tissues, blood products, plasma, serum or lymph fluid, etc. In some embodiments, the samples are fixed patterns. In some embodiments, the samples are paraffin-embedded samples. In some embodiments, the samples are formalin-fixed, paraffin-embedded samples.

In one embodiment, flow cytometry is used for immunological detection. Thus, for example, the number of bound antibodies per cell ABC can be assessed using flow cytometry.

Therapeutic uses and methods.

In one aspect, the present invention provides methods for treating a cancer expressing B7-H4 in a subject, comprising administering to the subject a therapeutic antibody to B7-H4 or an antigen-binding fragment thereof, wherein B7-H4 expression has been detected in a sample obtained from the subject, using an antibody or antigen-binding fragment thereof as provided in the present invention. In some embodiments, the method further includes detecting B7-H4 in a sample obtained from the subject.

In certain embodiments, the cancer provided herein is selected from the group consisting of: breast cancer (e.g., triple negative breast cancer, hormone receptor (HR) positive breast cancer, ductal cancer), endometrial cancer, ovarian cancer, non-small cell lung cancer (eg, squamous cell carcinoma), pancreatic cancer, thyroid cancer, kidney cancer (eg, renal cell carcinoma), and bladder cancer (eg, urothelial cell carcinoma).

- 41 045594

Therapeutic antibodies to B7-H4 or antigen binding fragments thereof include, for example, 20502 and 22213, as well as antigen binding fragments thereof. The amino acid and nucleotide sequences of antibodies 20502 and 22213 are presented in table. 9 and 10.

Table 9

Amino acid and nucleotide sequences of antibody 20502

Antibody domain (AA/N) Sequence (SEQ ID NO) VHCDR1 (AA) GSIKSGSYYWG (SEQ Ш NO:58) VH CDR2 (AA) NIYYSGSTYYNPSLRS (SEQ ID NO:59) VH CDR3 (AA) AREGSYPNQFDP (SEQ ID NO:60) VL CDR1 (AA) RASQSVSSNLA (SEQ ID NO:61) VL CDR2 (AA) GASTRAT (SEQ ID NO:62) VL CDR3 (AA) QQYHSFPFT (SEQ ID NO:63) VH(AA) QLQLQESGPGLVKPSETLSLTCTVSGGSIKSGSYYWGWI RQPPGKGLEWIGNIYYSGSTYYNPSLRSRVTISVDTSKN QF SLKLS S VT AADT AVYYC AREGS YPNQFDPWGQGTL V TVSS (SEQ ID NO:64) VL (AA) EIVMTQSPATLSVSPGERATLLSCRASQSVSSNLAWYQQK PGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQS EDFAVYYCQQYHSFPFTFGGGTKVEIK (SEQ ID NO:65) VH FR1 (AA) QLQLQESGPGLVKPSETLSLTCTVSG (SEQ ID NO:66) VH FR2 (AA) WIRQPPGKGLEWIG (SEQ ID NO:67) VH FR3 (AA) RVTISVDTSKNQFSLKLSSVTAADTAVYYC (SEQ ID NO :68) VH FR4 (AA) WGQGTLVTVSS (SEQ ID NO:69) VL FR1 (AA) EIVMTQSPATLSVSPGERATLSC (SEQ ID NO:70) VL FR2 (AA) WYQQKPGQAPRLLIY (SEQ ID NO:71) VL FR3 (AA) GIPARFSGSGSGTEFTLTISSLQSEDF AVYYC (SEQ ID NO :72) VL FR4 (AA) FGGGTKVEIK (SEQ ID NO:73) Heavy chain (AA) QLQLQESGPGLVKPSETLSLTCTVSGGSIKSGSYYWGWI RQPPGKGLEWIGNIYYSGSTYYNPSLRSRVTISVDTSKN QF SLKLS SVT AADT AVYYC AREGS YPNQFDPWGQGTL V TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVL

- 42 045594

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ Ш NO:74) Light chain (AA) EIVMTQSPATLSVSPGERATLLSCRASQSVSSNLAWYQQK PGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQS EDFAVYYCQQYHSFPFTFGGGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACE VTHQGLSSPVTKSFNRGEC (SEQ ID NO:75) VH(N) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTG AAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCT CTGGTGGCTCCATCAAAAGTGGTAGTTACTACTGGGG CTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTG GATTGGGAACATCTATTATAGTGGGAGCACCTACTAC AACCCGTCCCCTCAGAAGTCGAGTCACCATATCCGTAG ACACGTCCAAGAACC AGTTCTCCCTGAAGCTGAGTTC TGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCC AGAGAAGGATCTTACCCCAATCAGTTTTGATCCATGGG GACAGGGTACATTGGTCACCGTCTCCTCA (SEQ ID NO :76) VL(N) GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTG TGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGC CAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAG CAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATG GTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTT CAGTGGCAGTGGGTCTGGGACAGGTTCACTCTCACC ATCAGCAGCCTGCAGTC TGAAGATTTGCAGTTTATT ACTGTCAGCAGTACCACTCCTTTCCCTTTCACTTTTGGC GGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO:77)

- 43 045594

Table 10

Amino acid and nucleotide sequences of antibody 22213

Antibody domain (AA/N) Sequence (SEQ ID NO) VH CDR1 (AA) GSIGRGSYYWG (SEQ Ш NO:32) VH CDR2 (AA) NIYYSGSTYYNPSLKS (SEQ ID NO:33) VH CDR3 (AA) AREGSYTTVLNV (SEQ ID NO:34) VL CDR1 (AA) RASQSVASSHLA (SEQ ID NO:35) VL CDR2 (AA) DAVSRAT (SEQ ID NO:36) VL CDR3 (AA) QQAASYPLT (SEQ ID NO:37) VH(AA) QLQLQESGPGLVKPSETLSLTCTVSGGSIGRGSYYWGWI RQPPGKGLEWIGNIYYSGSTYYNPSLKSRVTISVDTSKN QFSLKLSSVTAADTAVYYCAREGSYTTVLNVWGQGTM VTVSS (SEQ ID NO:54) VL (AA) EIVLTQSPGTLLSLSPGERATLSCRASQSVASSHLAWYQQ KPGQAPRLLIYDAVSRATGIPDRFSGSGGTDFTLTISRL EPEDFAVYYCQQAASYPLTFGGGTKVEIK (SEQ ID NO:55) VH FR1 (AA) QLQLQESGPGLVKPSETLSLTCTVSG (SEQ ID NO:78) VH FR2 (AA) WIRQPPGKGLEWIG (SEQ ID NO:79) VH FR3 (AA) RVTISVDTSKNQFSLKLSSVTAADTAVYYC (SEQ ID NO :80) VH FR4 (AA) WGQGTMVTVSS (SEQ ID NO:81) VL FR1 (AA) EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO:82) VL FR2 (AA) WYQQKPGQAPRLLIY (SEQ ID NO:83) VL FR3 (AA) GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO :84) VL FR4 (AA) FGGGTKVEIK (SEQ ID NO:98) Heavy chain (AA) QLQLQESGPGLVKPSETLSLTCTVSGGSIGRGSYYWGWI RQPPGKGLEWIGNIYYSGSTYYNPSLKSRVTISVDTSKN QFSLKLSSVTAADTAVYYCAREGSYTTVLNVWGQGTM VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE

- 44 045594

DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:56) Light chain (AA) C EVTHQGLSSPVTKSFNRGEC (SEQ ID NO:57) VH(N) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTG AAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCT CTGGTGGCTCCATCGGGAGGGGGAGTTACTACTGGGG CTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTG GATTGGGAACATCTATTATAGTGGGAGCACCTACTAC AACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAG ACACGTCCAAGAACCAG TTCTCCCTGAAGCTGAGTTC TGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCC AGAGAAGGATCTTACACAACCGTGTTAAACGTATGG GGTCAGGGTACAATGGTCACCGTCTCCTCA (SEQ ID NO:85) VL(N) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTT TGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGC CAGTCAGAGTGTTGCCAGCAGCCACTTAGCCTGGTAC CAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCT ATGACGCAGTCAGCAGGGCCACTGGCATCCCAGACA GGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCT CACCATCAGCAGACTGGAGCCTG AAGATTTTGCAGTG TATTACTGTCAGCAGGCCGCCAGTTACCCTCTCACTTT TGGCGGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO :86)

In some embodiments, the anti-B7-H4 therapeutic antibody or antigen binding fragment comprises the VH region CDR1, VH region CDR2, VH region CDR3, VL region CDR1, VL region CDR2, and VL region CDR3 of antibody 20502. The CDRs may be Kabat-defined CDRs, CDRs determined by Chotia or CDRs determined by AbM.

In some embodiments, the anti-B7-H4 therapeutic antibody or antigen binding fragment comprises the VH region CDR1, VH region CDR2, VH region CDR3, VL region CDR1, VL region CDR2, and VL region CDR3 of antibody 22213. The CDRs may be Kabat-defined CDRs, CDRs determined by Chotia or CDRs determined by AbM.

In some embodiments, the anti-B7-H4 therapeutic antibody or antigen binding fragment comprises a VH CDR1 region, a VH CDR2 region, a VH CDR3 region, a VL CDR1 region, a VL CDR2 region, and a VL CDR3 region comprising the amino acid sequences of SEQ ID NOs: 32-37, respectively. In some embodiments, the anti-B7-H4 therapeutic antibody or antigen binding fragment comprises a VH CDR1 region, a VH CDR2 region, a VH CDR3 region, a VL CDR1 region, a VL CDR2 region, and a VL CDR3 region comprising the amino acid sequences of SEQ ID NOs: 58-63, respectively.

In some embodiments, the anti-B7-H4 therapeutic antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the anti-B7-H4 therapeutic antibody or antigen-binding fragment comprises

- 45 045594 fragment contains a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 64.

In some embodiments, the anti-B7-H4 therapeutic antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, the anti-B7-H4 therapeutic antibody or antigen-binding fragment comprises the fragment comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 65.

In some embodiments, the anti-B7-H4 therapeutic antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 55. In some embodiments, the anti-B7-H4 therapeutic antibody comprises the amino acid sequence SEQ ID NO: 55. The B7-H4 or antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 64 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, the anti-B7-H4 therapeutic antibody or antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 56. In some embodiments, the therapeutic anti-B7-H4 antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the therapeutic anti-B7-H4 antibody or antigen-binding fragment the fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 57. In some embodiments, the anti-B7-H4 therapeutic antibody or antigen binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 75. In some embodiments, the anti-B7-H4 therapeutic antibody or the antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 56 and a light chain comprising the amino acid sequence of SEQ ID NO: 57. In some embodiments, the anti-B7-H4 therapeutic antibody or antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 74, and a light chain containing the amino acid sequence of SEQ ID NO: 75.

Sets.

The present invention provides kits containing one or more of the antibodies or antigen-binding fragments thereof described in the present invention, or conjugates thereof (eg, detection conjugates). As proposed in the present invention, the kits can be used in diagnostic methods. In one embodiment, the kit contains an antibody or antigen binding fragment thereof described in the present invention, preferably a purified antibody or antigen binding fragment thereof, in one or more containers.

In a specific embodiment, the kits described in the present invention contain essentially isolated B7-H4 antigen (eg, human B7-H4), which can be used as a control. In specific embodiments, the kit of the present invention may contain a recombinantly produced or chemically synthesized B7-H4 antigen. The B7-H4 antigen provided in the kit can also be attached to a solid support.

In another specific embodiment, the kits described in the present invention further contain a control antibody or antigen binding fragment thereof that does not react with the B7-H4 antigen. In another specific embodiment, the kits described in the present invention contain one or more elements for detecting the specific binding of an antibody or antigen binding fragment thereof to the B7-H4 antigen (for example, the antibody or antigen binding fragment thereof may be conjugated to a detectable substrate, such as a fluorescent compound , an enzyme, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody or an antigen-binding fragment thereof that recognizes the first antibody or an antigen-binding fragment thereof may be conjugated to a detectable substrate). In yet another specific embodiment, the detection elements from the kit described above include a solid support to which the B7-H4 antigen is attached. Such a kit may also include an unattached reporter-labeled mouse/rodent antibody or antigen binding fragment thereof. In this embodiment, binding of the antibody or antigen binding fragment thereof to the B7-H4 antigen can be detected by binding of said reporter-labeled antibody or antigen binding fragment thereof.

In another specific embodiment, the kits described in the present invention further contain a therapeutic anti-B7-H4 antibody or antigen-binding fragment thereof and information that the therapeutic anti-B7-H4 antibody or antigen-binding fragment thereof is to be administered when B7-H4 is detected in the sample using an anti-B7-H4 antibody or an antigen-binding fragment thereof as provided in the present invention.

The following examples are provided for purposes of illustration and not limitation.

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Examples

The examples in this section (i.e., Section 6) are offered as illustrations and not as limitations.

Example 1. Preparation of a hybridoma.

A panel of antibodies that selectively bind to B7-H4 was generated using a recombinant soluble variant of the B7-H4 extracellular domain (ECD) protein:

MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSD

IVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTD AGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWA SQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVT ESEIKRRSHLQLLNSKAS GSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK (SEQ Ш NO:20).

ECD B7-H4 protein resuspended in the Sigma Adjuvant System® (Sigma-Aldrich, Inc., St. Louis, MO) was injected into each hind footpad of a NZB/W mouse. 21 days after the first injection, serum was collected and titrated by enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), and flow cytometry to ensure a good immune response in the mouse. ELISA and FACS screenings are described in detail in Examples 2 and 3, respectively. 3 days after the last injection, popliteal cells obtained from titer-positive mice were fused with mouse myeloma cell lines (see, for example, Chuntharapai et al., 1997, Methods Enzymol., 288:15-27). About 2880 rat hybridoma clones were obtained.

The hybridoma clones thus obtained were then screened for the production of monoclonal antibodies that bind to the B7-H4 ECD protein (SEQ ID NO: 20), an Fc fusion protein of the N-terminal IgV domain of B7-H4 (amino acids l-151) -huIgG (SEQ ID NO: 21) and with HEK 293 cells stably expressing full-length human B7-H4 (SEQ ID NO: 1) on their surface.

MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSD IVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTD AGTYKCYIITSKGKGNANLEYKTGAFSGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK (SEQ Ш NO:21).

Example 2. ELISA screening.

To screen the hybridoma clones obtained in Example 1, an ELISA was performed generally as described in Baker et al., 2002, Trends Biotechnol, 20:149-156. Briefly, 96-well plates were coated with 50 μl of B7-H4 ECD protein (SEQ ID NO: 20) or B7-H4 N-terminal Fc fusion protein (SEQ ID NO: 21) at a concentration of 2 μg/ml in coating buffer (0. 05 M carbonate buffer, pH 9.6), was sealed and stored overnight at 40°C. After removing the coating solution, 200 μl of assay/blocking solution containing 0.5% bovine serum albumin (BSA) and 0.05% Tween®-20 in PBS (pH 7.4) (ELISA diluent) was added to each well of a 96-well plate ). The plates were incubated at room temperature for one hour with stirring. The wells were then washed three times with 300 μl of 0.05% Tween®-20 in PBS (wash buffer). After washing, 100 μl of hybridoma supernatant in ELISA diluent was added to each well, and the plates were incubated at room temperature for one hour with mixing. The wells were washed three times with wash buffer as described above. After washing, 100 μl of a 1:1000 dilution of sheep anti-mouse IgG combined with horseradish peroxidase in ELISA diluent was added to each well. The plates were incubated at room temperature for one hour with agitation, washed three times with wash buffer as described above, and dried. Wells were treated by adding 100 μl of tetramethylbenzidine (TMB) horseradish peroxidase microwell substrate to each well and incubating at room temperature for 5-10 minutes or until a color change occurred. Treatment was stopped by adding 100 μl of TMB stop solution to each well. The plates were analyzed at 650 nm. Pre-selection and polyserum were used as controls. Pre-selection samples contained mouse serum before immunization, and polyserum samples contained mouse antiserum after immunization. Antibodies that gave a positive ELISA signal were selected for subsequent screening using fluorescence-activated cell sorting (FACS).

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Example 3: FACS Screening.

To further screen the hybridoma clones obtained in Example 1, fluorescence-activated cell sorting (FACS) was performed using HEK 293 cells stably expressing full-length human B7-H4 (SEQ ID NO: 1). Standard HEK 293 cell lines served as negative controls. Cells were resuspended and centrifuged at 500g for 5 minutes at 40°C. The medium was aspirated and the cells were resuspended in BD FACSFlow™ Stain Buffer (BD Biosciences, San Jose, CA) with 1% fetal bovine serum (FBS) (cell staining buffer) at 40°C. Cells were centrifuged as described previously, the medium was aspirated, and the cells were resuspended in cell staining buffer at 40°C to a final concentration of 2 x 10 ⁶ cells/ml. Cells were then added to 96-well round-bottom plates at a concentration of 50 μl/well. 100 μl of supernatant from each hybridoma clone was added to each well, such that each hybridoma supernatant was incubated with one well containing the HEK 293 cell line stably expressing B7-H4 or the negative control cell line. The plates were incubated on ice for 30 minutes and centrifuged as previously described. The supernatants were then aspirated. Each well was resuspended in 200 μl of cell staining buffer at 40°C. The plates were then centrifuged as previously described, and the cell staining buffer was aspirated. After the washing step, the cells in each well were resuspended in 100 μl of a 1:1000 dilution of goat anti-mouse IgG Fc coupled with R-phycoerythrin (Jackson Immunoresearch, West Grove, PA) in cell staining buffer at 40°C. and the plates were incubated in the dark on ice for 30 minutes. The plates were centrifuged as previously described, and the supernatants were aspirated. Each well was resuspended in 200 μl of cell staining buffer at 40°C, and the plates were centrifuged as previously described. Cell staining buffer was aspirated. Cells in each well were resuspended in 200 μl cell staining buffer at 40°C and transferred to 1.2 ml microtiter tubes. FACS was performed on a FACScan™ or FACSCalibur™ instrument (BD Biosciences, San Jose, CA). Antibodies that demonstrated cellular binding in FACS analysis were selected for further screening by immunohistochemistry (IHC).

Example 4: IHC screening.

To further screen for the hybridoma clones obtained in Example 1, immunohistochemical (IHC) staining was performed on sections obtained from a subject with invasive ductal carcinoma of the breast. Formalin-fixed paraffin-embedded (FFPE) sections were cut at 5 microns, air dried on charged Superfrost Plus slides, and baked for one hour at 60°C. Sections were deparaffinized and stained on a Discovery Ultra automated staining system (Ventana Medical Systems, Tucson, AZ). After 1 hour of antigen retrieval with Ultra CC1 at 95°C, B7-H4 was detected using mouse anti-B7-H4 primary antibodies for 1 hour at room temperature followed by the Omni-Map HRP-conjugated anti-mouse antibody detection system. , 3,3'-diaminobenzidine (DAB) ChromoMap and counterstained with hematoxylin according to the manufacturer's instructions.

Seven primary antibodies to B7-H4 were used: six recombinant antibodies obtained in example 1 (at a concentration of 10 μg/ml), as well as A57.1 (see US patent No. 7619068) (at a concentration of 1.25 μg/ml), which was used as a control. Two recombinant antibodies (J516 and J512) detected B7-H4 on cell membranes and in the cytoplasm in a manner similar to A57.1, with the majority of B7-H4 positive cells observed in the tumor compartment, as shown in FIG. 1 and 2. Recombinant antibodies detected fewer B7-H4-expressing cells with signal intensity 1+ than A57.1 (35% with J512 versus 35% with J516% versus 58% with A57.1 ), as shown in Fig. 3 and 4. When B7-H4 expression was measured using computational image analysis (Definiens, Cambridge, MA), J516 and J512 showed a similar distribution of B7-H4 signal across cellular compartments as A57.1, as shown in FIG. 5A-5C.

Example 5: Determination of sequences.

The heavy chain (HC) and light chain (LC) sequences of anti-B7-H4 antibodies M6, M11 and M15, all of which showed IHC staining, were determined according to the following procedure.

RNA was collected from hybridomas using TRIzol reagent (Thermofisher Scientific, Carlsbad, CA), and cDNA was prepared using Smartscribe reverse transcriptase (Clontech, Mountain View, CA) and Advantage 2 polymerase (Clontech, Mountain View, CA). PCR was performed using Phusion Taq polymerase (NEB, Ipswich, MA) under standard PNR conditions. The PCR products were then cloned using the Zero Blunt® TOPO® cloning kit (Thermofisher Scientific, Carlsbad, CA).

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Claims (21)

Clones were screened for insertions, and multiple clones were sequenced to verify the original heavy chain and light chain gene sequences. Polynucleotide sequences encoding the heavy chain and light chain are presented in table. 8 above. The anti-B7-H4 variable domains were then subcloned into expression vectors with different Fc regions to produce antibodies with mouse IgG2a and mouse IgG1 constant regions. The anti-B7-H4 heavy chain variable domains were then cloned into expression vectors with different F regions to produce antibodies with mouse IgG2a and mouse IgG1 constant regions. The light chain variable domains of the anti-B7-H4 antibody were cloned into expression vectors with the mouse IgK constant region. After checking the sequences, the isolated antibodies were assigned ID J511-J516, as indicated in Table. 1 above. The invention should not be limited in scope to the specific embodiments described in the present invention. Indeed, various modifications of the invention in addition to those described will be apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to be within the scope of the appended claims. All references (e.g., publications, or patents, or patent applications) cited in the present invention are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication, or patent or patent application) is specifically and individually incorporated by reference in its entirety for all purposes. Other embodiments are within the scope of the following claims. CLAIM 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, comprising a heavy chain variable region (VH) complementarity determining region (CDR) 1 containing the amino acid sequence of SEQ ID NO: 22, VH region CDR2 containing the amino acid sequence SEQ ID NO: 23, VH region CDR3 containing the amino acid sequence of SEQ ID NO: 24, light chain variable region (VL) CDR1 containing the amino acid sequence of SEQ ID NO: 25, VL region CDR2 containing the amino acid sequence of SEQ ID NO: 26, 27 or 28, and the CDR3 sequence of the VL region containing the amino acid sequence of SEQ ID NO: 29. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a VH containing the amino acid sequence of SEQ ID NO: 6, 7 or 8, and/or a VL containing the amino acid sequence of SEQ ID NO: 9, 10 or 11. 3. An isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, comprising a heavy chain variable region and a light chain variable region containing the amino acid sequences of (a) SEQ ID NO: 6 and 9, respectively; (b) SEQ ID NO: 6 and 10 respectively; (c) SEQ ID NO: 6 and 11 respectively; (d) SEQ ID NO: 7 and 9 respectively; (e) SEQ ID NO: 7 and 10 respectively; (f) SEQ ID NO: 7 and 11 respectively; (g) SEQ ID NO: 8 and 9 respectively; (h) SEQ ID NO: 8 and 10 respectively; or (i) SEQ ID NO: 8 and 11, respectively. 4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment comprises a heavy chain and a light chain containing the amino acid sequences (a) SEQ ID NO: 16 and 19, respectively; (b) SEQ ID NO: 16 and 30 respectively; (c) SEQ ID NO: 16 and 31, respectively; (d) SEQ ID NO: 17 and 19 respectively; (e) SEQ ID NO: 17 and 30 respectively; (f) SEQ ID NO: 17 and 31 respectively; (g) SEQ ID NO: 18 and 19 respectively; (h) SEQ ID NO: 18 and 30 respectively; (i) SEQ ID NO: 18 and 31 respectively; (j) SEQ ID NO: 89 and 19, respectively; (k) SEQ ID NO: 90 and 19, respectively; or (l) SEQ ID NO: 91 and 19, respectively. 5. An isolated antibody or its antigen-binding fragment that specifically binds - 49 045594 with human B7-H4, wherein the antibody or antigen-binding fragment thereof contains the VH region CDR1, the VH region CDR2, the VH region CDR3, the VL region CDR1, the VL region CDR2 and the VL region CDR3 of an antibody selected from the group consisting of J512, comprising the VH amino acid sequence of SEQ ID NO: 6 and the VL amino acid sequence of SEQ ID NO: 9, J513 or J514, each of which contains the VH amino acid sequence of SEQ ID NO: 7 and the VL amino acid sequence of SEQ ID NO: 9, J515 or J516, each comprising the VH amino acid sequence of SEQ ID NO: 8 and the VL amino acid sequence of SEQ ID NO: 9, J517 comprising the VH amino acid sequence of SEQ ID NO: 6 and the VL amino acid sequence of SEQ ID NO: 10, J518, containing the amino acid sequence of VH from SEQ ID NO: 6 and the amino acid sequence of VL from SEQ ID NO: 11, J519, containing the amino acid sequence of VH from SEQ ID NO: 7 and the amino acid sequence of VL from SEQ ID NO: 10, J520, containing the amino acid the VH sequence of SEQ ID NO: 7 and the VL amino acid sequence of SEQ ID NO: 11, J521 containing the VH amino acid sequence of SEQ ID NO: 8 and the VL amino acid sequence of SEQ ID NO: 10, and J522 containing the VH amino acid sequence of SEQ ID NO: 8 and the VL amino acid sequence of SEQ ID NO: 11. 6. The antibody or antigen binding fragment thereof according to claim 5, wherein the CDRs are Kabat CDRs, Chotia CDRs, or AbM CDRs. 7. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which is an antigen-binding fragment, wherein the antigen-binding fragment comprises Fab, Fab', F(ab') ₂ , single chain Fv fragment (scFv), disulfide-linked Fv fragment , minibody, F(ab') ₃ , diabody, (scFv) ₂ or scFv-Fc. 8. Conjugate of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7 and a toxin. 9. An isolated polynucleotide containing a nucleic acid molecule encoding the variable region of the heavy chain of an antibody or its antigen-binding fragment according to claim 2 or 3. 10. An isolated polynucleotide containing a nucleic acid molecule encoding the variable region of the light chain of an antibody or its antigen-binding fragment according to claim 2 or 3. 11. An isolated polynucleotide containing a nucleic acid molecule encoding a light chain variable region or a light chain of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7. 12. The isolated polynucleotide of claim 11, wherein the nucleic acid molecule encodes the VL region of SEQ ID NO: 9, 10 or 11 or the light chain of SEQ ID NO: 19, 30 or 31. 13. An isolated polynucleotide containing a nucleic acid molecule encoding a heavy chain variable region or a heavy chain of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7. 14. An isolated polynucleotide containing a nucleic acid molecule encoding a variable region of a heavy chain or a heavy chain of an antibody, or an antigen-binding fragment thereof according to any one of claims 2-4 and a variable region of a light chain or a light chain of an antibody or an antigen-binding fragment thereof according to any one of claims. 2-4. 15. An isolated expression vector containing a polynucleotide according to any one of claims 9-14. 16. A host cell containing a polynucleotide according to any one of claims 9 to 13, a vector according to claim 15, or a first vector containing a polynucleotide encoding a heavy chain variable region according to claim 9 or 13, and a second vector encoding a light chain variable region according to any one of claims 10-12. 17. A method for producing an antibody or an antigen-binding fragment thereof that specifically binds to human B7-H4, comprising culturing a host cell containing a polynucleotide encoding a heavy chain variable region and a light chain variable region according to any one of claims 9 to 14, thus so that a polynucleotide encoding a heavy chain variable region and a polynucleotide encoding a light chain variable region are expressed and an antibody or an antigen-binding fragment thereof is produced. 18. A method for detecting B7-H4 in a sample, comprising contacting the sample with an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7 and detecting the binding of the antibody or an antigen-binding fragment thereof to B7-H4. 19. The method of claim 18, wherein the sample is obtained from a subject having cancer. 20. The method of claim 19, wherein the cancer is selected from the group consisting of breast cancer, ductal carcinoma, endometrial cancer, ovarian cancer, non-small cell lung cancer, pancreatic cancer, thyroid cancer, kidney cancer and bladder cancer. 21. Use of a therapeutic antibody to B7-H4 or an antigen binding fragment thereof for the treatment of cancer expressing B7-H4, wherein B7-H4 expression was detected in a sample obtained from a subject using the antibody or antigen binding fragment thereof according to any one of claims 1 to 7 wherein the therapeutic antibody or antigen binding fragment comprises (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54 and a light chain variable region comprising the amino acid sequence -