Anti-B7-H4 antibody and application thereof

Abstract

The present invention provides an ScFv-FC antibody of B7-H4. The ScFv-FC antibody of The invention further provides amino acid sequences of the antibodies, cloning or expression vectors, host cells and methods for expressing or isolating the antibodies, epitopes for identifying the antibodies, and methods of treating cancer and other diseases with the antibodies.

Description

Technical Field

[0001] The present invention relates to anti-B7-H4 antibodies and compositions thereof, as well as methods for expressing or isolating antibodies, and methods for treating cancer using the antibodies. Background Art

[0002] Tumors are one of the most serious diseases threatening human health due to their incurable nature, high mortality rate, and poor clinical manifestations after recovery. For this reason, researchers have tried a variety of tumor treatment methods and technologies, including surgical treatment, radiotherapy, chemotherapy, gene therapy, etc., but no effective treatment methods have been found so far. With the deepening of biotechnology and immunology research, tumor immunotherapy and anti-tumor antibodies have received attention in the industry. Since the 1980s, hundreds of antibodies have been approved for tumor treatment worldwide, and currently thousands of antibodies are submitted for clinical trials each year.

[0003] B7-H4 is highly expressed in many cancers, including ovarian cancer, breast cancer, lung cancer, endometrial cancer, gastric cancer, renal cell carcinoma, melanoma, colorectal cancer, pancreatic cancer and prostate cancer. Therefore, B7-H4 protein has been identified as a new target for tumor treatment.

[0004] Therefore, it is necessary to design antibodies against B7-H4 for tumor treatment. Summary of the invention

[0005] In view of this, the present invention provides anti-B7-H4 ScFv-FC antibodies, polynucleotides encoding the antibodies, cells containing the antibodies or polynucleotides, and methods of using the same.

[0006] According to one aspect of the present application, the present application provides an antibody or an antigen-binding fragment thereof that binds to B7-H4, wherein the antibody or the antigen-binding fragment thereof comprises:

[0007] 1) a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and

[0008] 2) a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR1 amino acid sequence, and the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR2 amino acid sequence.

[0009] The VL CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR3 amino acid sequence;

[0010] wherein the selected VH CDR 1, 2 and 3 amino acid sequences are shown by SEQ ID NOs: 3, 4, 5, respectively, and the selected VL CDR 1, 2 and 3 amino acid sequences are shown by SEQ ID NOs: 6, 7, 8, respectively;

[0011] According to certain embodiments of the present application, the VH comprises CDRs 1, 2, 3 having amino acid sequences shown in SEQ ID NOs: 3, 4, and 5, respectively, and the VL comprises CDRs 1, 2, 3 having amino acid sequences shown in SEQ ID NOs: 6, 7, and 8, respectively.

[0012] According to certain embodiments of the present application, the amino acid sequence comprised by the antibody or antigen-binding fragment has at least 80% identity with the amino acid sequence of SEQ ID NO:2.

[0013] According to certain embodiments of the present application, the antibody or antigen-binding fragment specifically binds to human B7-H4.

[0014] According to certain embodiments of the present application, the antibody or antigen-binding fragment inhibits B7-H4; the antibody or antigen-binding fragment binds to the epitope of SEQ ID NO: 11.

[0015] According to certain embodiments of the present application, the antibody or antigen-binding fragment is a humanized antibody or an antigen-binding fragment thereof.

[0016] According to certain embodiments of the present application, the antibody or antigen-binding fragment is a single-chain variable fragment (scFv).

[0017] According to one aspect of the present application, the present application provides a nucleic acid comprising a polynucleotide encoding a polypeptide, wherein the polypeptide comprises:

[0018] 1) an immunoglobulin heavy chain or a fragment thereof having a heavy chain variable region (VH), wherein the heavy chain variable region (VH) comprises a sequence represented by SEQ ID

[0019] NO:3, 4 and 5, and wherein the VH binds to B7-H4 when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:13; or

[0020] 2) an immunoglobulin light chain or a fragment thereof, wherein the VL comprises a light chain variable region (VL) comprising SEQ ID NOs: 6, 7 and 8, respectively.

[0021] CDRs 1, 2 and 3 of the amino acid sequence shown in SEQ ID NO: 12, and wherein the VL binds to B7-H4 when paired with a VH comprising the amino acid sequence shown in SEQ ID NO: 12.

[0022] According to certain embodiments of the present application, the nucleic acid comprises a polynucleotide encoding a polypeptide, which comprises an immunoglobulin heavy chain or a fragment thereof containing VH, wherein the VH comprises CDRs 1, 2 and 3 containing the amino acid sequences shown in SEQ ID NOs: 3, 4 and 5, respectively.

[0023] According to certain embodiments of the present application, the nucleic acid comprises a polynucleotide encoding a polypeptide, which comprises an immunoglobulin light chain or a fragment thereof containing VL, wherein the VL comprises CDRs 1, 2 and 3 containing the amino acid sequences shown in SEQ ID NOs: 6, 7 and 8, respectively.

[0024] According to certain embodiments of the present application, the VH specifically binds to human B7-H4 when paired with the VL, or the VL specifically binds to human B7-H4 when paired with the VH.

[0025] According to certain embodiments of the present application, the immunoglobulin heavy chain or fragment thereof is a humanized immunoglobulin heavy chain or fragment thereof, and the immunoglobulin light chain or fragment thereof is a humanized immunoglobulin light chain or fragment thereof.

[0026] According to certain embodiments of the present application, the nucleic acid encodes a single-chain variable fragment (scFv).

[0027] According to one aspect of the present application, the present application provides a vector comprising one or more of the nucleic acids.

[0028] According to one aspect of the present application, the present application provides a vector comprising two of the nucleic acids, wherein the vector encodes a VL region and a VH region, and the VL region and the VH region together bind to B7-H4.

[0029] According to one aspect of the present application, the present application provides a vector pair, wherein each vector comprises one of the nucleic acids, wherein the vector pair jointly encodes a VL region and a VH region, and the VL region and the VH region together bind to B7-H4.

[0030] According to one aspect of the present application, the present application provides a cell, comprising the vector or the vector pair.

[0031] According to one aspect of the present application, the present application provides a cell comprising one or more of the nucleic acids.

[0032] According to one aspect of the present application, the present application provides a cell comprising two of the nucleic acids.

[0033] According to certain embodiments of the present application, the two nucleic acids jointly encode a VL region and a VH region, and the VL region and the VH region together bind to B7-H4.

[0034] According to one aspect of the present application, the present application provides a method for producing an antibody or an antigen-binding fragment thereof, the method comprising

[0035] (a) culturing the cells under conditions sufficient for the cells to produce antibodies or antigen-binding fragments; and

[0036] (b) collecting the antibodies or antigen-binding fragments produced by the cells.

[0037] According to one aspect of the present application, the present application provides a transgenic animal, wherein the transgenic animal expresses the antibody.

[0038] According to one aspect of the present application, the present application provides an antibody or an antigen-binding fragment thereof that binds to B7-H4, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises an amino acid sequence that is at least 90% identical to a selected VH sequence, and the light chain variable region (VL) comprises an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 12, and the selected VL sequence is SEQ ID NO: 13.

[0039] According to one aspect of the present application, the present application provides an antibody or an antigen-binding fragment thereof, comprising VH CDR 1, 2, 3 and VL CDR 1, 2, 3 of the antibody or the antigen-binding fragment thereof.

[0040] According to one aspect of the present application, the present application provides an antibody or an antigen-binding fragment thereof that cross-competes with the antibody or an antigen-binding fragment thereof for binding to B7-H4.

[0041] According to one aspect of the present application, the present application provides a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

[0042] According to one aspect of the present application, the present application provides an antibody-drug conjugate, which comprises the antibody or antigen-binding fragment thereof covalently bound to a therapeutic agent.

[0043] According to certain embodiments of the present application, the therapeutic agent is a cytotoxic agent or a cytostatic agent.

[0044] According to one aspect of the present application, the present application provides a pharmaceutical composition, which comprises the antibody-drug conjugate and a pharmaceutically acceptable carrier.

[0045] According to one aspect of the present application, the present application provides a method for treating a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective dose of a composition comprising the antibody or antigen-binding fragment thereof, or the pharmaceutical composition or antibody-drug conjugate.

[0046] According to certain embodiments of the present application, B7-H4 is highly expressed in the cancer.

[0047] According to certain embodiments of the present application, the cancer is colorectal cancer; and the colorectal cancer is colorectal adenocarcinoma.

[0048] According to certain embodiments of the present application, the present application provides a method for treating a B7-H4-related disease, wherein the method administers the anti-B7-H4 antibody or its antigen-binding fragment or the pharmaceutical composition or antibody-drug conjugate to a subject.

[0049] According to certain embodiments of the present application, B7-H4 is highly expressed in the B7-H4-related disease.

[0050] According to certain embodiments of the present application, the B7-H4-related disease is colorectal cancer; and the colorectal cancer is colorectal adenocarcinoma.

[0051] According to one aspect of the present application, the present application provides a method for reducing tumor growth rate, the method comprising administering an effective dose of a composition comprising the antibody or its antigen-binding fragment, or the pharmaceutical composition or antibody-drug conjugate to a subject in need thereof.

[0052] According to one aspect of the present application, the present application provides a method for killing tumor cells, the method comprising administering an effective dose of a composition comprising the antibody or its antigen-binding fragment, or the pharmaceutical composition or antibody-drug conjugate to a subject in need thereof.

[0053] According to one aspect of the present application, the present application provides a method for detecting the presence or content of B7-H4 in a sample, the method comprising contacting the sample with the anti-B7-H4 antibody or its antigen-binding fragment, and determining the presence or content of B7-H4 in the sample.

[0054] According to certain embodiments of the present application, the sample is a cell expressing B7-H4; the cell expressing B7-H4 is a cancer cell; the cancer is colorectal cancer; the colorectal cancer is colorectal adenocarcinoma.

[0055] According to one aspect of the present application, the present application provides a method for diagnosing a disease or condition associated with B7-H4 in a subject, the method comprising: a) obtaining a sample from the subject; b) contacting the sample with the anti-B7-H4 antibody or its antigen-binding fragment; c) determining the presence or content of B7-H4 in the sample; d) correlating the presence or content of B7-H4 with the disease or condition of the subject.

[0056] Beneficial effects of the present invention:

[0057] Compared with the prior art, the anti-B7-H4 antibody of the present invention has the following advantages and characteristics:

[0058] 1) The present invention relates to an antibody and its application in anti-tumor.

[0059] 2) The present invention provides a novel monoclonal antibody targeting B7-H4, which can bind to the target antigen human B7-H4 with high affinity and strong specificity. The reported B7-H4 antibody kills tumor cells by activating T cells, while this antibody can directly act on colon cancer cells HT29 and LS174T and inhibit the proliferation of colon cancer cells HT29 and LS174T compared with the previously reported B7-H4 antibody.

[0060] 3) The present invention also discovered the mechanism of action of this antibody, namely, inhibiting the proliferation of colon cancer cells HT29 and LS174T by inhibiting the phosphorylation of JAK2 / STAT3 protein, and blocking the cell cycle of colon cancer cells HT29 and LS174T cells at the S phase, which is not done by other B7-H4 antibodies.

[0061] 4) The present invention also discovered the antigenic epitope of the antibody, providing a new research basis for the development of related anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The antibodies obtained after protein A purification are shown;

[0063] Figure 2 A shows the expression of antibodies after plasmid transfection into 293F cells; Figure 2 B shows Coomassie blue staining and Western assay;

[0064] Figure 3A and B show the results of Western blotting to detect B7-H4 protein expression on the cell membrane of 293T, DLD1, HT29, and LS174T cells; Figure 3 CF anti-B7-H4 antibody inhibits the proliferation of 293T, LS174T, HT29 and DLD1 cells;

[0065] Figure 4 Immunofluorescence assays were performed to detect the binding of anti-B7-H4 antibodies XF04-4, XF04-9, and XF04-12 to LS174T cell membrane surface antigens;

[0066] Figure 5 Immunofluorescence assay of anti-B7-H4 antibody XF04-4 and 293T ( Figure 5 A), LS174T( Figure 5 B), HT29( Figure 5 C), DLD1( Figure 5 D) Binding of cell surface antigens;

[0067] Figure 6 A shows the binding model of the B7-H4 antibody XF04-4 VL region and the B7-H4 molecule docking results; Figure 6 B shows the binding model of the B7-H4 antibody XF04-4 VH region and the B7-H4 molecule docking results; Figure 6 C shows the molecular docking results analysis;

[0068] Figure 7 AF shows the competition binding experiment between the predicted epitope peptide and XF04-4 antibody; wherein the abscissa is the peptide concentration used; Figure 7 A is the epitope polypeptide EGVL, Figure 7 B is the epitope polypeptide QLTDAGTY, Figure 7 C is the epitope polypeptide SEQDEMFRGRT, Figure 7 D is the epitope peptide SCTFEPDI, Figure 7 E is the epitope peptide HSITVTTVA, Figure 7 F is epitope peptide IVGN;

[0069] Figure 8 A competition blocking experiment is shown, where the abscissa is the concentration of B7-H4 protein;

[0070] Fig. 9 The results of the experiment showing the peptide antagonizing the cell inhibitory activity of XF04-4 antibody; Fig. 9 A is LS174T cells, Fig. 9 B is HT29 cells, Fig. 9 C is DLD1 cell; Fig. 9A and B show that for LS174T cells and HT29 cells, treatment with B7-H4 antibody XF04-4 caused a significant decrease in cell survival, while after incubation of polypeptide chain SEQDEMFRGRT with B7-H4 antibody XF04-4, cell survival rate was significantly increased;

[0071] Fig.10 It shows that the B7-H4 antibody XF04-4 can specifically bind to the B7-H4 antigen, but does not bind to TIM3 and PBS control;

[0072] Fig.11 The phosphorylation of JAK2 and STAT3 proteins in cells was detected by Western blot after HT29 and LS174T cells were treated with anti-B7-H4 antibody XF04-4;

[0073] Fig.12 AC shows the inhibition results of cell proliferation by different concentrations of anti-B7-H4 antibody; Fig.12 A is DLD1 cell, Fig.12 B is LS174T cells, Fig.12 C is HT29 cells;

[0074] Fig.13 The anti-B7-H4 antibody showed the effect of LS174T ( Fig.13 A), HT29( Fig.13 B), DLD1( Fig.13 C) The effect on the cell cycle of cells. DETAILED DESCRIPTION DETAILED DESCRIPTION OF THE INVENTION

[0076] sequence

[0077] According to certain embodiments of the present application, the structure of the anti-B7-H4 antibody XF04-4 is a ScFv-FC antibody, wherein the ScFv is of mouse origin and the FC is of human origin.

[0078] DNA sequence of anti-B7-H4 antibody XF04-4 (SEQ ID NO: 1)

[0079] CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATATCCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATCGCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCACTTATTACTGCCAGCAGTATCATAGTTACCCACTCACGTTCGGTGCTGGGACAAAGTTGGAAATAAAACGTACGGGTGGTGGCGGCTCGGGAGGAGGAGGATCCCAGGTCCAACTGCAACAGCCTGGGGCTAAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGACTTCTGGCTACTCCTTCACCAGCTACTTGATGAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGCATGATTCATCCTTCCGATAGTGAAACTAGGTTAAATCAGAGGTTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCCGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCCCATTACTTCGGTAGTAGTTCCTATAAAATGGATTATTGGGGTCAAGGAACCACGCTCACCGTCTCCTCA

[0080] Amino acid sequence of anti-B7-H4 antibody XF04-4 (SEQ ID NO: 2)

[0081] Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly GluLys Val Thr Ile Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Tyr Trp Tyr GlnGln Lys Pro Gly Ser Ser Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Arg Thr SerAsn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser TyrSer Leu Thr Ile Ser Met Glu Ala Glu Asp Ala Thr Tyr Tyr Cys Gln GlnTyr His Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg ThrGly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Glu Leu Gln Gln ProGly Ala Lys Leu Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Thr Ser GlyTyr Ser Phe Thr Ser Tyr Leu Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly LeuGlu Trp Ile Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Arg PheLys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln LeuSer Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser His Tyr PheGly Ser Ser Ser Tyr Lys Met Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val SerSer

[0082] VH amino acid sequence of anti-B7-H4 antibody XF04-4 (SEQ ID NO: 12)

[0083] QVQLQQPGAKLVRPGASVKLSCKTSGYSFTSYLMNWVKQRPGQGLEWIGMIHPSDSETRLNQRFKDKATLTVDKSSSTAYMQLSS PTSEDSAVYYCARSHYFGSSSYKMDYWGQGTTLTVSS

[0084] VL amino acid sequence of anti-B7-H4 antibody XF04-4 (SEQ ID NO: 13)

[0085] QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATY YCQQYHSYPLTFGAGTKLEIKRT

[0086] CDR sequences of anti-B7-H4 antibody XF04-4 (Kabat definition) (SEQ ID NO: 3-8)

[0087] SEQ ID NO:3:VH-CDR1 SYLMN

[0088] SEQ ID NO:4:VH-CDR2 MIHPSDSETRLNQR

[0089] SEQ ID NO:5:VH-CDR3 YCARSHYF

[0090] SEQ ID NO:6:VL-CDR1 SASSSVSYMY

[0091] SEQ ID NO:7:VL-CDR2 TSNLAS

[0092] SEQ ID NO:8:VL-CDR3 QYHSYPLTF

[0093] CDR sequences of anti-B7-H4 antibody XF04-4 (Chothia definition) (SEQ ID NOs: 14-19)

[0094] SEQ ID NO:14:VH-CDR1 GYSFTSYLM

[0095] SEQ ID NO:15:VH-CDR2 HPSDS

[0096] SEQ ID NO:16:VH-CDR3 YCARSHYF

[0097] SEQ ID NO:17:VL-CDR1 SASSSVSYMY

[0098] SEQ ID NO:18:VL-CDR2 TSNLAS

[0099] SEQ ID NO:19:VL-CDR3 QYHSYPLTF

[0100] IgG2 FC sequence of anti-B7-H4 antibody XF04-4 (SEQ ID NO: 9)

[0101] GAGCGGAAGTGCTGCGTGGAGTGCCCTCCCTGCCCTGCTCCTCCTGTGGCCGGACCTTCCGTGTTCCTGTTCCCTCCTAAGCCCAAGGACACCCTGATGATCTCTAGAACACCTGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTTCAACTCCACCTTCCGGGTGGTGTCCGTGCTGACCGTGGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCGCTCCTATCGAGAAGACCATCTCCAAGACCAAGGGCCAGCCTCGGGAGCCTCAGGTGTACACCCTGCCTCCTTCCAGAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACACCTCCTATGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCTGGCAAG

[0102] The complete amino acid sequence of B7-H4 (SEQ ID NO: 10)

[0103] HSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKN VQLTDAGTYKCYIITSKGKGNANLEYKTGH

[0104] B7-H4 epitope sequence recognized by anti-B7-H4 antibody XF04-4 (SEQ ID NO: 11)

[0105] SEQDEMFRGRT

[0106] DNA sequence of anti-B7-H4 antibody XF04-5 (SEQ ID NO: 21)

[0107] GATATTCTGATGACCCAGACTCCCAAATTCCTGCTTGTATCAGCAGGAGACAGGGTTACCATAACCTGCAAGGCCAGTCAGAGTGTGAGTAATGATGTAGCTTGGTACCAACAGAAGCCAGGGCAGTCTCCTAAACTGCTGATATACTATGCATCCAATCGCTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCACTGTGCAGGCTGAAGACCTGGCAGTTTATTTCTGTCAGCAGGATTATAGCTCTCCATTCACGTTCGGCTCGGGGACAAAGCTGGAAATAAAACGTACGGGTGGTGGCGGCTCGGGAGGAGGAGGATCCCAGGTCCAGCTGCAGCAGCCTGGGGCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACTCCTTCACCAGCTACTTGATGAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGCATGATTCATCCTTCCGATAGTGAAACTAGGTTAAATCAGAGGTTCAAGGACAAGGCCACATTGGCTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCCGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCCCATTACTTCGGTAGTAGCTCCTATAAAATGGACTACTGGGGTCAAGGAACCTCTGTCACCGTCTCCTCA

[0108] Amino acid sequence of anti-B7-H4 antibody XF04-5 (SEQ ID NO: 22)

[0109] DILMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPFTFGSGTKLEIKRTGGGGSGGGGSQVQLQQPGAELVRPGASVKLSCKASGYSFTSYLMNWVKQRPGQGLEWIGMIHPSDSETRLNQRFKDKATLAVDKSSSTAYMQLSSPTSEDSAVYYCARSHYFGSSSYKMDYWGQGTSVTVSS

[0110] DNA sequence of anti-B7-H4 antibody XF04-9 (SEQ ID NO: 23)

[0111] GACATCCAGCTGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGGGAATATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTGGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATCATTATGGTTCTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGCTGAAACGTACGGGTGGTGGCGGCTCGGGCGGCGGCGGGTCGGGAGGAGGAGGATCCCAGGTCCAGCTGCAACAGCCTGGGGCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACTCCTTCACCAGCTACTTGATGAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGCATGATTCATCCTTCCGATGGTGAAACTAGGTTAAATCAGAGGTTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCCGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCCCATTACTTCGGTAGTAGCTCCTATAAAATGGACTACTGGGGTCAAGGAACCACAGTCACCGTCTCCTCA

[0112] Amino acid sequence of anti-B7-H4 antibody XF04-9 (SEQ ID NO: 24)

[0113] DIQLTQSPASLSASVGETVTITCRASGNIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGSPYTFGGGTKLELKRTGGGGSGGGGSGGGGSQVQLQQPGAELVRPGASVKLSCKASGYSFTSYLMNWVKQRPGQGLEWIGMIHPSDGETRLNQRFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCARSHYFGSSSYKMDYWGQGTTVTVSS

[0114] DNA sequence of anti-B7-H4 antibody XF04-10 (SEQ ID NO: 25)

[0115] GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGCTGAAACGTACGGGTGGTGGCGGCTCGGGCGGCGGCGGGTCGGGAGGAGGAGGATCCCAGGTCCAGCTGCAACAGCCTGGGGCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGACTTCTGGCTACTCCTTCACCAGCTACTTGATGAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGCATGATTCATCCTTCCGATAGTGAAACTAGGTTAAATCAGAGGTTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCCGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCCCATTACTTCGGTAGTAGTTCCTATAAAATGGATTATTGGGGTCAAGGAACCACAGTCACAGTCTCCTCA

[0116] Amino acid sequence of anti-B7-H4 antibody XF04-10 (SEQ ID NO: 26)

[0117] DVVMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLELKRTGGGGSGGGGSGGGGSQVQLQQPGAELVRPGASVKLSCKTSGYSFTSYLMNWVKQRPGQGLEWIGMIHPSDSETRLNQRFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCARSHYFGSSSYKMDYWGQGTTVTVSS

[0118] DNA sequence of anti-B7-H4 antibody XF04-12 (SEQ ID NO: 27)

[0119] GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTATATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGTACGGGCGGCGGCGGGTCGGGAGGAGGAGGATCCGAGGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGTCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTAGAGTGGGTCGCAACCATTAGTAGTGGTGATAATTACACCTACTATCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGACCGGACGTAACTACGGCTACGTTTGTTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0120] Amino acid sequence of anti-B7-H4 antibody XF04-12 (SEQ ID NO: 27)

[0121] DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPLTFGAGTKLELKRTGGGGSGGG GSEVKLVESGGGLVKPGGSLKLSCAASGFTFSSYVMSWVRQTPEKRLEWVATISSGDNYTYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARPDVTTATFVYWGQGTLVTVSA

[0122] definition

[0123] cancer

[0124] The term "cancer" or "cancer" as used herein refers to any medical condition characterized by malignant cell growth or tumor, abnormal proliferation, infiltration or metastasis, and includes solid tumors and non-solid cancers (hematological malignancies) such as leukemia. Some examples of cancer include cells with abnormal states or conditions characterized by rapidly proliferating cell growth. "Solid tumor" used in this application refers to a solid mass of tumors and / or malignant cells. Examples of cancer or tumors include hematological malignancies, oral cancer (e.g., cancer of the lips, tongue or pharynx), digestive organ cancer (e.g., esophagus, stomach, small intestine, colon, large intestine or rectum), peritoneal cancer, liver or biliary tract cancer, pancreatic cancer, respiratory system, such as laryngeal or lung (small cell or non-small cell) cancer, bone cancer, connective tissue cancer, skin cancer (e.g., melanoma), breast cancer, reproductive organ (fallopian tube, uterus, cervix, testicle, ovary or prostate) cancer, urethra (e.g., bladder or kidney) cancer, brain and endocrine gland (e.g., thyroid) cancer. In some embodiments, the cancer is selected from ovarian cancer, breast cancer, head and neck cancer, kidney cancer, bladder cancer, hepatocellular carcinoma, and colorectal cancer. In some embodiments, the cancer is a blood cancer. In some embodiments, the hematology cancer is selected from non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), Richter syndrome, Burkitt's lymphoma or follicular lymphoma.

[0125] The "cancer" includes cancerous growths, such as tumors; carcinogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of the histopathological type or degree of infiltration. The "cancer" also includes malignancies of various organ systems, such as the respiratory, cardiovascular, renal, reproductive, hematological, nervous, hepatic, gastrointestinal, and endocrine systems; and adenocarcinomas, which include malignancies such as most colon cancers, renal cell carcinomas, prostate cancers, and / or testicular tumors, non-small cell lung cancers, and small intestinal cancers.

[0126] "Naturally occurring" cancer includes any cancer that is not experimentally induced by implanting cancer cells into a subject, and includes, for example, cancers that arise spontaneously, cancers caused by exposure of a patient to a carcinogen, cancers caused by the insertion of an oncogene or the knockout of a tumor suppressor gene, and cancers caused by infection (e.g., a viral infection).

[0127] The term "carcinoma" is art-recognized and refers to a malignancy of epithelial or endocrine tissue. The term also includes carcinosarcoma, which includes malignant tumors composed of cancerous and sarcomatous tissue.

[0128] "Adenocarcinoma" refers to a cancer that arises from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0129] The term "sarcoma" is art-recognized and refers to a malignant tumor of mesenchymal origin.

[0130] The term "hematopoietic neoplastic disorder" includes diseases involving proliferative / tumor cells of hematopoietic origin. Hematopoietic neoplastic disorders may arise from cells of the myeloid, lymphoid or erythroid lineages, or their precursors.

[0131] Colorectal cancer

[0132] Colorectal cancer is the third most common type of cancer and causes high mortality and socioeconomic impact in developed countries. Although new strategies in chemotherapy, surgery, and prevention may help improve survival, patients with advanced metastatic colorectal cancer remain at risk for fatal outcomes. Therefore, new treatment options with high efficacy and few adverse effects are urgently needed.

[0133] Colorectal cancer is a biologically diverse disease with many factors, including laterality (lesions on the left side of the colon versus those on the right side) and oncogene and tumor suppressor gene mutations that influence tumor behavior. The boundary between colon and rectal tumors is based on distance from the anal verge, but molecularly they are very similar. There are some observed differences depending on the site of origin of the primary tumor (left-sided versus right-sided colon cancer). Colorectal cancer in younger populations is most common on the left side, and left-sided tumors located in the rectum or distal part of the colon are more likely to respond to anti-epidermal growth factor receptor (EGFR) therapy.

[0134] Colorectal cancer is almost always adenocarcinoma, which originates from the intestinal mucosal lining and can arise in any part of the large intestine. Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are thought to be the initiating event in most colorectal cancers, followed by other mutations such as p53 and KRAS. RAS oncogene mutations are found in 50% of colorectal cancer patients and are associated with more severe disease and poorer survival. BRAF V600E mutations are found in approximately 9% of colorectal tumors and are associated with a significantly worse prognosis.

[0135] B7-H4 is particularly highly expressed in intestinal glandular epithelial cells of colon cancer. According to certain embodiments of the present application, the B7-H4 antibody XF04-4 can inhibit the proliferation of colon cancer cells HT29 and LS174T. HT29 and LS174T are mainly adenocarcinoma cells in colon cancer.

[0136] HT29 cell line includes positive oncogenes c-myc, K-ras, H-ras, N-ras, Myb, sis, fos. P53 gene is overexpressed and there is a G to A mutation at codon 273.

[0137] LS174T is a trypsinized variant of LS180 colon cancer cell line, which is easier to passage than the parental line and produces a large amount of carcinoembryonic antigen (CEA) like LS180. It is negative for P53 antigen expression but positive for mRNA expression; positive for keratin; and positive for the expression of oncogenes c-myc, N-myc, H-ras, Myb and fos.

[0138] B7-H4-related diseases

[0139] As used herein, "B7-H4-related disease" refers to any disease or condition caused, exacerbated, or otherwise associated with increased or decreased expression or activity of B7-H4. B7-H4 protein is expressed at low levels in normal tissues, but is highly expressed in a variety of tumor cells, including colorectal cancer.

[0140] In some embodiments, diseases associated with B7-H4 include ovarian cancer, breast cancer, lung cancer, endometrial cancer, gastric cancer, renal cell carcinoma, melanoma, colorectal cancer, pancreatic cancer, prostate cancer, rheumatoid disease, systemic lupus erythematosus, preeclampsia in pregnant women, etc.

[0141] B7-H4

[0142] B7-H4 is an important member of the B7 family of molecules. B7-H4 mRNA is widely expressed in normal tissues, but the expression of B7-H4 protein is strictly controlled. To date, the receptor of B7-H4 remains unknown. A large number of studies have confirmed that B7-H4 is highly expressed in tumors, inflammatory and autoimmune diseases, viral infections and pregnant patients. B7-H4 promotes tumorigenesis and tumor development through immune escape, and has a positive effect on type 1 diabetes and pancreatic islet cell transplantation by inhibiting autologous and allogeneic reactive T cells. In addition, B7-H4 also has biological functions such as liver protection, kidney protection, and pregnancy protection. Studies have shown that B7-H4 is closely related to classical signaling pathways, such as IL6 / JAK / STAT3, PI3K / AKT, CXCL12 / CXCR4, etc. Studies have shown that B7-H4 is regulated by cytokines, metalloproteinases, TFs (transcription factors), miRNAs, etc. Notably, B7-H4-based immunotherapeutic drugs, including monoclonal antibodies, ADCs, anti-b7-H4 / CD3 BsAbs, and CAR-T cells, have been developed and achieved acceptable efficacy. Therefore, B7-H4 is a promising therapeutic target for cancer, inflammatory and autoimmune diseases, and organ transplantation.

[0143] B7-H4 is a type I transmembrane protein containing 282 amino acids. B7-H4 contains a large hydrophobic transmembrane domain and an intracellular domain of only 2 amino acids. There are 7 potential N-glycosylation sites in its intracellular domain.

[0144] B7-H4 protein is an important member of the B7 family and plays an important role in the occurrence and development of tumors. B7-H4 protein negatively regulates the immune response of T cells by inhibiting the proliferation of T cells, the production of cytokines, and the progression of the cell cycle, thereby promoting the proliferation, invasion, and metastasis of tumor cells.

[0145] B7-H4 is highly expressed in many cancers, including ovarian cancer, breast cancer, lung cancer, endometrial cancer, gastric cancer, renal cell carcinoma, melanoma, colorectal cancer, pancreatic cancer and prostate cancer. Therefore, B7-H4 protein has been identified as a new target for tumor treatment.

[0146] There are no antibody drugs targeting B7-H4 on the market yet, but there are currently two B7-H4 antibodies undergoing clinical trials. They include:

[0147] (1) Monoclonal antibodies. This type of antibody has high sensitivity, strong specificity, few cross-reactions, and low preparation costs. It is currently the most widely used antibody drug. Currently, researchers are targeting the B7-H4 target and only one monoclonal antibody, FPA150, is undergoing clinical trials. The study includes a phase 1a FPA150 monotherapy dose increase, a phase 1a monotherapy dose exploration, a phase 1a combined safety introduction (FPA150 + pembrolizumab), a phase 1b FPA150 monotherapy dose expansion, and a phase 1b combined dose expansion.

[0148] (2) Antibody-drug conjugate (ADC) is a drug that connects a small molecule drug with biological activity to a monoclonal antibody through a chemical link. The monoclonal antibody acts as a carrier to transport the small molecule drug into the target cell. This combination drug has both the targeting of biological molecules and the strong tumor killing ability of chemical drugs. In order to reduce toxic side effects, biological molecules are often required to have high specificity. The linker connecting biological molecules and chemical drugs also needs to be precisely designed to give full play to the synergistic effect. There is an ADC drug targeting B7-H4 in clinical trials, HS-20089. HS-20089 is a new dar6 antibody-drug conjugate (ADC) targeting B7-H4. In preclinical studies, it inhibited the growth of tumor cells expressing B7-H4 in vitro and in vivo. The first human trial aims to evaluate the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of HS-20089 in patients with advanced solid tumors to evaluate the pharmacokinetics, safety and preliminary anti-tumor activity of HS-20089.

[0149] Although there are some reports on tumor immunotherapy targeting B7-H4, there are few studies on antibody drugs that directly act on the B7-H4 protein on the cell membrane surface, and the effect of B7-H4 protein on the proliferation of colon cancer cells and its mechanism of action have not been fully explored.

[0150] According to certain embodiments of the present application, the B7-H4 antibody XF04-4 described in the present application can directly inhibit the proliferation of colon cancer cells by binding to the B7-H4 protein expressed by colon cancer cells. This is different from other reports in the literature that B7-H4 antibodies inhibit the proliferation of tumor cells by promoting the proliferation of T cells, and there is no literature reporting that B7-H4 antibodies can inhibit colon cancer.

[0151] The conclusions of this application provide new ideas for B7-H4 therapeutic targets and provide a basis for the treatment of colon cancer.

[0152] B7-H4 ligands

[0153] The ligand of B7-H4 has not yet been discovered and no research has been reported (Jia-Yu Wang and Wei-Peng Wang, B7-H4, A promising target for immunotherapy, Cellular Immunology 347 (2020) 104008)

[0154] Researchers have identified some molecules that may be ligands for B7-H4. For example, ICOSL, VCAM-1, NOTCH-1, NOTCH-2, and STEAP4 have all been shown to interact with B7-H4. According to certain embodiments of the present invention, VCAM-1 is selected as the target protein for the competitive blocking experiment between the B7-H4 antibody XF04-4 and the B7-H4 ligand.

[0155] STAT3 pathway

[0156] The STAT3 pathway is a well-known "bridge" connecting inflammation and cancer, and is involved in the occurrence and development of tumors. Studies have found that in esophageal squamous cell carcinoma (ESCC), there is a positive feedback relationship between B7-H4 and the IL-6 / STAT3 pathway. B7-H4 stimulates IL-6 secretion and upregulates B7-H4 expression by activating the JAK2 / STAT3 pathway, thereby enhancing the growth and tumorigenicity of cancer cells.

[0157] Another study also confirmed that in human histiocytic lymphoma U-937 cells, B7-H4 promoted the production of IL-10 and TGF-β1 through the JAK2 / STAT3 pathway. B7-H4 mediates the interaction between glioma-initiating cells and macrophages through the IL6 / JAK / STAT3 pathway, leading to poor prognosis of glioma patients.

[0158] Studies have found that B7-H4 siRNA effectively inhibits the proliferation, invasion and migration of human colon cancer cell LoVo cells by targeting JAK2 / STAT3 signal transduction, which may be a new research direction for the treatment of colorectal cancer. In the HT29 and LS174T cells used in this application, JAK2 / STAT3 phosphorylation is increased.

[0159] According to certain embodiments of the present application, the anti-B7-H4 antibody XF04-4 inhibits the proliferation of colon cancer cells by inhibiting the JAK2-STAT3 signaling pathway in colon cancer cells.

[0160] The STAT3 pathway is a well-known "bridge" between inflammation and cancer and is involved in the occurrence and development of tumors. B7-H4 stimulates the secretion of IL-6 by activating the JAK2 / STAT3 pathway, and IL-6 in turn upregulates the expression of B7-H4, thereby enhancing the growth and tumorigenicity of cancer cells. The interaction between primary glioma cells and macrophages is mediated by B7-H4 through the IL6 / JAK / STAT3 pathway, leading to poor prognosis in glioma patients. B7-H4 siRNA effectively inhibits the proliferation, invasion and migration of LoVo cells by targeting JAK2 / STAT3 signal transduction, and may become a new target for the treatment of colorectal cancer. The anti-B7-H4 antibody XF04-4 in this application inhibits the proliferation of colorectal cancer cells HT29 and LS174T cells by inhibiting the phosphorylation of JAK2 / STAT3.

[0161] Cell cycle

[0162] The cell cycle refers to the entire process that a cell goes through from the completion of one division to the end of the next division, which is divided into two stages: interphase and division.

[0163] The interphase is divided into three phases, namely the pre-DNA synthesis phase (G1 phase), the DNA synthesis phase (S phase) and the late DNA synthesis phase (G2 phase).

[0164] 1. G1 phase (first gap) is a period from mitosis to DNA replication, also known as the presynthesis phase. This phase mainly synthesizes RNA and ribosomes. This phase is characterized by active material metabolism, rapid synthesis of RNA and protein, and significant increase in cell volume. The main significance of this phase is to prepare materials and energy for DNA replication in the next stage, the S phase.

[0165] 2. S phase (synthesis) is the DNA synthesis phase. In this phase, in addition to DNA synthesis, histones are also synthesized. The enzymes required for DNA replication are synthesized during this phase.

[0166] 3. The G2 phase (second gap) is the late stage of DNA synthesis and the preparation period for mitosis. During this period, DNA synthesis stops and a large amount of RNA and proteins, including microtubules and maturation-promoting factors, are synthesized.

[0167] The cell division phase, namely the M phase.

[0168] Mitosis is a continuous process that goes through the prophase, the middle phase, the anaphase and the telophase. It is a process in which one mother cell divides into two daughter cells. It usually takes 1 to 2 hours.

[0169] 1. In the prophase, the chromatin filaments are highly spiraled and gradually form chromosomes. The chromosomes are short, thick, and highly basophilic. The two centrosomes move in opposite directions and form two poles in the cell; then the microtubules are synthesized starting from the centriole satellites to form the spindle. As the nucleolus spirals with the chromatin, the nucleolus gradually disappears. The nuclear envelope begins to disintegrate into discrete vesicular endoplasmic reticulum.

[0170] 2. Metaphase: The cell becomes spherical, and the nucleolus and nuclear envelope have completely disappeared. The chromosomes move to the equatorial plane of the cell, and the microtubules emanating from the two poles of the spindle attach to the centromere of each chromosome. A complete set of chromosomes can be separated from metaphase cells, a total of 46, of which 44 are autosomes and 2 are sex chromosomes. The chromosome karyotype of males is 44+XY, and that of females is 44+XX. The separated chromosomes are short and thick rods or hairpins, and are composed of two chromatids connected by a narrow centromere.

[0171] 3. Anaphase: Due to the activity of the spindle microtubules, the centromere splits longitudinally, and the two chromatids of each chromosome separate and move in opposite directions, approaching their respective centrosomes, and the chromatids are divided into two groups. At the same time, the cell is elongated, and due to the activity of the circular microfilament bundles under the cell membrane at the equator, this part narrows, and the cell becomes dumbbell-shaped.

[0172] 4. In the telophase, the chromatids gradually unwind, and the chromatin filaments and nucleoli reappear; the endoplasmic reticulum vesicles combine to form the nuclear envelope; the equator of the cell narrows and deepens, and finally completely divides into two diploid daughter cells.

[0173] Link between B7-H4 and the cell cycle

[0174] The expression of B7-H4 in the nuclear membrane is significantly correlated with the expression of Ki67 (a cell proliferation marker), indicating that B7-H4-positive tumor cells have a higher proliferation activity. Human B7-H4 has a predicted nuclear localization sequence (NLS) motif and is a cytoplasmic nuclear shuttling protein. Studies have shown that B7-H4 enters the cell nucleus, upregulates the expression of cyclin E / D1, promotes the transition of the cell cycle G1 / S phase, and thus promotes the proliferation of tumor cells.

[0175] Antibody

[0176] "Antibodies" in this application include any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody or bispecific (bivalent) antibody that can bind to a specific antigen. A natural complete antibody is "Y"-shaped, containing two heavy chains and two light chains. Each heavy chain consists of a variable region (VH) and the first, second and third constant regions (constant regions); each light chain consists of a variable region (VL) and a constant region (constant region). The neck of the antibody "Y"-shaped structure is composed of the second and third constant regions of the two heavy chains, and is bound by disulfide bonds. Each arm of the "Y"-shaped structure includes the variable region and the first constant region of one of the heavy chains, combined with the variable region and constant region of one light chain. The variable regions of the light chain and the heavy chain determine the binding of the antigen. The variable region of each chain contains three hypervariable regions, called complementary determining regions (CDRs). The CDR of the light chain (L) includes LCDR1, LCDR2, and LCDR3; the CDR of the heavy chain (H) includes HCDR1, HCDR2, and HCDR3. The three CDRs are separated by a lateral continuous part called the framework region (FR), which is more highly conserved than the CDR and forms a scaffold to support the hypervariable loop. The constant regions of the heavy and light chains are not related to antigen binding, but have a variety of effector functions. The heavy chains of mammals can be divided into α, δ, ε, γ, and μ, and the light chains of mammals can be divided into λ or κ. Antibodies can be divided into several categories based on the amino acid sequence of the heavy chain constant region. Depending on whether they contain α, δ, ε, γ, and μ heavy chains, antibodies can be divided into five major classifications or isomers: IgA, IgD, IgE, IgG, and IgM. Several major antibody classifications can be further divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain), etc.

[0177] Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, the antibody may include the Fc region of a human antibody. The term antibody also includes derivatives, such as bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0178] According to certain embodiments of the present application, the antibody is a complete immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). IgG subclasses (IgG1, IgG2, IgG3 and IgG4) are highly conserved, differing in their constant regions, especially their hinge and upper CH2 domains. The sequences and differences of IgG subclasses are known in the art and are described in, for example, the following documents: Vidarsson, et al, "IgG subclasses and allotypes: from structure to effector functions." Frontiers in immunology 5 (2014); Irani, et al. "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases." Molecular immunology 67.2 (2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016. Each of these documents is incorporated herein by reference in its entirety.

[0179] Fab

[0180] The term "antigen binding fragment" in this application refers to an antibody fragment formed by an antibody portion containing one or more CDRs or any other antibody fragment that binds to an antigen but does not have a complete antibody structure. Examples of antigen binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (dsdiabody), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), bivalent single-chain antibodies (BsFv), multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. The antigen binding fragment can bind to the same antigen as the parent antibody. In certain embodiments, the antigen binding fragment can contain one or more CDRs from a particular human antibody, grafted to the framework region from one or more different human antibodies.

[0181] According to certain embodiments of the present application, the antigen-binding fragment generally includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). The antigen-binding fragment does not necessarily have to include an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). For example, an antibody fragment may consist only of a VH domain, but still retain some antigen-binding functions of a complete antibody. According to certain embodiments of the present application, the antigen-binding fragment includes an antibody light chain variable region (VL). According to certain embodiments of the present application, the antigen-binding fragment includes an antibody heavy chain variable region (VH).

[0182] The "Fab" fragment of an antibody refers to the portion of the antibody molecule composed of a light chain (including the variable region and the constant region) and the variable region and the constant region of a heavy chain bound together by disulfide bonds.

[0183] The "Fab'" fragment refers to the Fab fragment including part of the hinge region.

[0184] "F(ab')2" refers to a dimer of Fab.

[0185] The "Fc" of an antibody refers to the part of the antibody composed of the second and third constant regions of the heavy chain bound by disulfide bonds. The Fc segment of an antibody is responsible for a variety of different effector functions such as ADCC and CDC, but does not participate in antigen binding.

[0186] The "Fv" fragment of an antibody refers to the smallest antibody fragment that contains a complete antigen binding site. The Fv fragment consists of the variable region of one light chain and one heavy chain.

[0187] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody in which the light chain variable region is directly linked to the heavy chain variable region or connected through a peptide chain (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)).

[0188] "Single-chain antibody Fv-Fc" or "scFv-Fc" refers to an engineered antibody consisting of a scFv linked to the Fc portion of an antibody.

[0189] "Single-chain antibody" refers to a single polypeptide comprising at least two immunoglobulin variable domains (eg, variable domains of a mammalian immunoglobulin heavy or light chain) that is capable of specific binding to an antigen.

[0190] "Camelized single domain antibody", "heavy chain antibody" or "HCAb (Heavy-chain-only antibodies, HCAb)" all refer to antibodies containing two VH domains but no light chain (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231 (1-2): 25-38 (1999); Muyldermans S., J Biotechnol. Jun; 74 (4): 277-302 (2001); WO94 / 04678; WO94 / 25591; US ​​Patent No. 6,005,079). Heavy chain antibodies were originally derived from camelids (camels, dromedaries and llamas). Although lacking light chains, camelized antibodies have confirmed full antigen binding function (Hamers Casterman C. et al., Nature. Jun 3; 363(6428): 446-8 (1993); Nguyen VK. et al., "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation," Immunogenetics. Apr; 54(1): 39-47 (2002); Nguyen VK. et al., Immunology. May; 109(1): 93-101 (2003)). The variable region (VHH domain) of the heavy chain antibody is the smallest known antigen binding unit produced by adaptive immunity (Koch-Nolte F. et al., FASEB J. Nov; 21(13): 3490-8. Epub 2007 Jun 15 (2007)).

[0191] "Nanobody" refers to an antibody fragment that consists of a VHH domain from a heavy chain antibody and two constant regions CH2 and CH3.

[0192] "Diabodies" include small antibody fragments with two antigen binding sites, wherein the fragment contains a VH domain and a VL domain (VH-VL or VH-VL) connected on the same polypeptide chain (see, Holliger P. et al., Proc Natl Acad Sci USA. Jul 15; 90 (14): 6444-8 (1993); EP404097; WO93 / 11161). The linker between the two domains is very short, so that the two domains on the same chain cannot pair with each other, thereby forcing the two domains to pair with the complementary domains of another chain to form two antibody binding sites. The two antibody binding sites can target and bind to the same or different antigens (or antigenic epitopes).

[0193] "Domain antibodies" refer to antibody fragments that contain only one heavy chain variable region or one light chain variable region. In some cases, two or more VH domains are covalently linked by a polypeptide linker to form a bivalent domain antibody. The two VH domains of a bivalent domain antibody can target the same or different antigens.

[0194] In certain embodiments, "(dsFv)2" contains three peptide chains: two VH groups are connected by a polypeptide linker and are bound to two VL groups by disulfide bonds.

[0195] In certain embodiments, a "bispecific ds bifunctional antibody" contains VL1-VH2 (connected by a polypeptide linker) and VH1-VL2 (also connected by a polypeptide linker), and the two are linked by a disulfide bond between VH1 and VL1.

[0196] "Bispeci?c dsFv" or "dsFv-dsFv" contains three polypeptide chains: a VH1-VH2 group, wherein the heavy chains of the two are connected by a polypeptide linker (e.g., a long flexible linker) and are bound to the VL1 and VL2 groups, respectively, by disulfide bonds, and each pair of heavy and light chains paired by disulfide bonds has different antigenic specificity.

[0197] According to certain embodiments of the present application, "scFv dimer" is a bivalent bifunctional antibody or a bivalent single-chain antibody (BsFv), containing two dimerized VH-VL (connected by a polypeptide linker) groups, wherein the VH of one group cooperates with the VL of the other group to form two binding sites, and the two binding sites can target the same antigen (or antigenic epitope) or different antigens (or antigenic epitopes). In other embodiments, "scFv dimer" is a bispecific bifunctional antibody, containing mutually connected VL1-VH2 (connected by a polypeptide linker) and VH1-VL2 (connected by a polypeptide linker), wherein VH1 and VL1 cooperate, VH2 and VL2 cooperate, and each cooperative pairing has a different antigen specificity.

[0198] As used herein, the term "multimeric antibody" refers to an antibody comprising four or more (e.g., six, eight, or ten) immunoglobulin variable domains. In some embodiments, the multimeric antibody is capable of cross-linking a target molecule (e.g., B7-H4) with at least one second target molecule on the surface of a mammalian cell (e.g., a human T cell).

[0199] scFv-Fc Antibody

[0200] In 1988, Skerra and Better first expressed active antibody fragments using E. coli, and genetic engineering antibody technology began to emerge. Genetically engineered antibodies either eliminate some unfavorable characteristics of natural antibodies or add new biological functions, expanding the application range of antibodies and improving the application performance of antibodies. To date, genetically engineered antibodies have been widely used in many fields, among which single-chain antibodies and their different forms of fusion proteins are favored by researchers. Studies have confirmed that single-chain antibody fragment fusion proteins have good specificity and sensitivity as in vivo and in vitro detection and diagnostic reagents. However, ScFv often shows aggregation tendency, especially at 37°C, ScFv has poor stability. This is related to the fact that the light and heavy chain variable regions are connected together by non-covalent bonds with weaker forces. The emergence of ScFv-Fc fusion antibodies has solved this problem. Since this fusion antibody retains the hinge region, it can self-assemble into ScFv-Fc dimers in vitro, which enhances stability. In addition, the bivalent ScFv-Fc dimer also enhances the affinity with specific antigens. The presence of the Fc fragment enables this fusion antibody to be purified using affinity chromatography such as protein A or protein G, which greatly simplifies the production process and makes it possible for ScFv-Fc fusion antibodies to be widely used in clinical practice.

[0201] The variable region (V) of an antibody is the site that specifically recognizes the antigen, while the constant region (C) has nothing to do with antigen recognition. In the late 1980s, in order to solve the problem of HAMA (human anti-mouse antibody) produced when mouse-derived monoclonal antibodies were used in humans, scientists replaced the gene sequence of the constant region of mouse-derived monoclonal antibodies with human gene sequences to prepare human-mouse chimeric antibodies. Human-mouse chimeric anti-CD20 monoclonal antibodies (SCT400) have achieved good results in the treatment of non-Hodgkin's lymphoma. Metuzumab is a human-mouse chimeric monoclonal antibody against CD147, which has anti-non-small cell lung cancer effects in mice, rats and crab-eating monkeys.

[0202] Complementarity determining region (CDR)

[0203] The hypervariable regions in antibodies, called "complementarity determining regions (CDRs)", form loops that comprise the major antigen binding surface of the antibody. The four framework regions adopt primarily a β-sheet conformation, and the CDRs form loops that connect and in some cases form part of the β-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, together with the CDRs from the other chain, contribute to the formation of the antigen binding region.

[0204] According to certain embodiments of the present application, the antibody comprises at least one (e.g., one, two, three, four, five or six) complementarity determining regions (CDRs) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is any antigen binding molecule that is capable of specifically binding to an epitope.

[0205] The CDR boundaries of the antibodies and antigen-binding fragments disclosed in the present invention can be named or identified by Kabat, Chothia or Al-Lazikani nomenclature. The Kabat definition is based on sequence variability, and the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described in, for example, Martin, "Protein sequence and structure analysis of antibody variable domains," Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan, et al. "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecular immunology 45.14 (2008): 3832-3839; Wu, TT and Kabat, EA (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991); Morea et al., Biophys Chem. 68 (1-3): 9-16 (Oct. 1997); Morea et al., J Mol Biol. 275 (2): 269-94 (Jan. 1998); Chothia et al. al., Nature 342(6252):877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); each of which is incorporated herein by reference in its entirety. Unless otherwise specified in the present disclosure, Kabat numbering is used by default in the present disclosure.

[0206] Epitope

[0207] CDR is important for identifying antigen epitopes. As used herein, "epitope" is the smallest part of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be about 3, 4, 5, 6 or 7 amino acids, but these amino acids need not be located in a continuous linear sequence of the primary structure of the antigen, because the epitope can depend on the three-dimensional conformation of the antigen based on the secondary and tertiary structures of the antigen. If two antibodies show competitive binding to antigens, the same epitope on the antigen may be bound. If the blocking of an antibody or its antigen-binding fragment to a reference antibody is bound to an antigen by at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90%, the antibody or its antigen-binding fragment can be considered to be bound to the same / closely related epitope with the reference antibody, so

[0208] According to certain embodiments of the present application, the B7-H4 antibody XF04-4 binds to the B7-H4 epitope SEQDEMFRGRT, which has no mutations in colon cancer and other tumors.

[0209] Humanization

[0210] The term "humanization" used in this application, when used for antibodies or antigen-binding fragments, refers to antibodies or antigen-binding fragments including CDRs derived from non-human animals, FR regions derived from humans, and constant regions derived from humans (when applicable). Because humanized antibodies or antigen-binding fragments have reduced immunogenicity, they can be used as therapeutic agents for people in certain embodiments. In some embodiments, the non-human animal is a mammal such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster. In some embodiments, the humanized antibody or antigen-binding fragment is substantially composed of human sequences except that the CDR sequence is non-human. In some embodiments, the FR region derived from humans may include the same amino acid sequence as the human antibody from which it comes, or it may include some amino acid changes, for example, no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid changes. In some embodiments, the amino acid change may be present only in the heavy chain FR region, only in the light chain FR region, or in both chains at the same time. In some preferred embodiments, the humanized antibody includes human FR1-3 and human JH and Jκ.

[0211] Humanized Antibodies

[0212] The term "humanized antibody" used in the present application refers to a non-human antibody, which includes a minimal sequence derived from a non-human (e.g., mouse) immunoglobulin and includes a sequence derived from a human immunoglobulin. In some non-limiting examples, a humanized antibody is a human antibody (acceptor antibody), wherein the hypervariable (e.g., CDR) region residues of the acceptor antibody are replaced by hypervariable (e.g., CDR) region residues from a non-human antibody (e.g., donor antibody) (e.g., mouse, rat or rabbit antibody) with desired specificity, affinity and ability. In some embodiments, the Fv framework residues of a human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, a humanized antibody may include residues that are not present in an acceptor antibody or a donor antibody. These modifications may be performed to further improve antibody performance. In some embodiments, a humanized antibody includes at least one and generally substantially all of two variable domains, wherein all or substantially all of the hypervariable loops (CDR) correspond to those of a non-human (e.g., mouse) immunoglobulin, and all or substantially all of the framework regions are those of a human immunoglobulin. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.Humanized antibodies can be produced using molecular biology methods known in the art.

[0213] Percentage of humanization

[0214] Humanization percentage refers to the percentage identity of the heavy chain or light chain variable region sequence compared with the human antibody sequence in the International Immunogenetics Information System (IMGT) database. The highest hit (top hit) refers to that the heavy chain or light chain variable region sequence is closer to a specific species relative to other species. For example, the highest hit in people refers to that the sequence is closer to people relative to other species. The highest hit in people and cynomolgus monkeys (Macaca fascicularis) means that the sequence has the same percentage identity as the human sequence and the cynomolgus monkey sequence, and compared with the sequences of other species, these percentage identities are the highest. In some embodiments, the humanization percentage is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95%. Detailed descriptions of how to determine the humanization percentage and how to determine the top hits are known in the art and are described, for example, in Jones, Tim D., et al. "The INNs and outs of antibody nonproprietary names" MAbs. Vol. 8. No. 1. Taylor & Francis, 2016, which is incorporated herein by reference in its entirety. A high humanization percentage generally has multiple advantages, for example, being safer and more effective in humans, being more easily tolerated by human subjects and / or being less likely to have side effects.

[0215] Human Antibodies

[0216] The term "human antibody" as used herein refers to an antibody encoded by endogenous nucleic acids present in humans (e.g., rearranged human immunoglobulin heavy chain or light chain loci). In some embodiments, human antibodies are collected from humans or produced in human cell cultures (e.g., human hybridoma cells). In some embodiments, human antibodies are produced in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies are produced in bacteria or yeast cells. In some embodiments, human antibodies are produced in transgenic non-human animals (e.g., cattle) containing unrearranged or rearranged human immunoglobulin loci (e.g., heavy chain or light chain human immunoglobulin loci).

[0217] Chimerism

[0218] The term "chimeric" used in this application refers to an antibody or antigen-binding fragment having a portion of a heavy chain and / or a light chain derived from one species, and the remainder of the heavy chain and / or the light chain derived from a different species. In an illustrative example, a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human animal such as a mouse.

[0219] The term "chimeric antibody" as used herein refers to an antibody comprising a sequence present in at least two different antibodies (e.g., antibodies from two different mammalian species, such as human antibodies and mouse antibodies). A non-limiting example of a chimeric antibody is an antibody comprising a variable domain sequence (e.g., all or part of a light chain and / or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and a constant domain of a human antibody. Other examples of chimeric antibodies are described herein and are known in the art.

[0220] Conservative modification

[0221] The term "conservative modification" refers to nucleotide and amino acid sequence modifications that do not significantly affect or change the antibody binding properties of the amino acid sequence encoded by the nucleotide sequence or comprising the amino acid sequence. Such conservative sequence modifications include nucleotide and amino acid substitutions, additions and deletions. Modifications can be introduced into the sequence by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0222] In the present application, when "conservative modification" is used for amino acid sequences, it means that an amino acid residue is replaced with another amino acid residue with a side chain of similar physicochemical properties. It is known in the art that conservative substitutions usually do not cause significant changes in the conformational structure of the protein, and therefore the biological activity of the protein can be retained. Families of amino acid residues with similar side chains are known to those skilled in the art. These families include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0223] Anti-B7-H4 Antibody

[0224] As used herein, "anti-B7-H4 antibody" refers to an antibody that can bind to B7-H4 (eg, human, monkey or mouse B7-H4) with affinity and specificity sufficient to provide diagnostic and / or therapeutic uses.

[0225] According to certain embodiments of the present application, the anti-B7-H4 antibody does not include a mutation.

[0226] According to certain embodiments of the present application, the anti-B7-H4 antibody includes conservative modifications.

[0227] Antibodies and Tumor Therapy

[0228] Because antibody therapy has high targeting and selectivity, and its therapeutic effect is obvious, it can not only effectively inhibit the growth and reproduction of tumor cells, but also avoid adverse effects on normal tissues or cells, thereby ensuring the patient's normal life. Therefore, it is widely used in hematopoietic tumors such as leukemia, lymphoma, and myeloma, and solid tumors such as gastric cancer, colorectal cancer, breast cancer, ovarian cancer, lung cancer, melanoma, Hodgkin's lymphoma, and nasopharyngeal carcinoma.

[0229] Combination

[0230] The term "binding" as used herein refers to an antibody affinity for a portion of B7-H4 that is greater than its affinity for other related polypeptides. The term "substantially greater affinity" refers to a measurable increase in affinity for a portion of B7-H4 compared to the affinity for other related polypeptides.

[0231] "Specific binding" or "specific binding" in this application refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen. In certain embodiments, the antibody or antigen-binding fragment thereof of the present application specifically binds to human B7-H4, and its binding affinity (KD) is ≤10 -6 M(eg: ≤5x10 -7 M, ≤2x10 -7 M, ≤10 -7 M, ≤5x10 -8 M, ≤2x10 -8 M, ≤10 -8 M, ≤5x10 -9 M, ≤2x10 -9 M, ≤10 -9 M, ≤10 -10 KD in the present application refers to the ratio of the dissociation rate to the association rate (koff / kon), which can be determined by surface plasmon resonance, for example, using an instrument such as Biacore.

[0232] According to certain embodiments of the present invention, the affinity of the B7-H4 antibody for a specific portion of B7-H4 is at least 1.5 times, 2 times, 5 times, 10 times, 100 times, 10 times greater than that for other proteins. 3 times, 10 4 times, 10 5times, 10 6 According to certain embodiments of the present invention, the binding affinity is determined by enzyme-linked immunosorbent assay (ELISA), or by fluorescence activated cell sorting (FACS) analysis or surface plasmon resonance (SPR). More preferably, the binding specificity is obtained by fluorescence activated cell sorting (FACS) analysis.

[0233] As used herein, the phrases "specifically bind to" and "specifically binds to" when referring to antibodies refer to an antibody that preferentially interacts with its target molecule (e.g., B7-H4) relative to other molecules because the interaction depends on the presence of a specific structure (i.e., antigenic determinant or epitope) on the target molecule; in other words, the agent recognizes and binds to molecules containing a specific structure, rather than all molecules in general. An antibody that specifically binds to a target molecule may be referred to as a target-specific antibody. For example, an antibody that specifically binds to a B7-H4 molecule may be referred to as a B7-H4-specific antibody or an anti-B7-H4 antibody.

[0234] Cross-reactivity

[0235] The term "cross-reactivity" as described herein refers to binding to antigenic fragments of the same target molecule of human, monkey, and / or murine (mouse or rat) origin. Therefore, "cross-reactivity" should be understood as interspecies reaction with the same molecule expressed in different species. The cross-reactivity specificity of antibodies recognizing human, monkey, and / or murine (mouse or rat) B7-H4 can be determined by FACS analysis.

[0236] Blocking Binding

[0237] The ability to "block binding" or "compete for the same epitope" in this application refers to the ability of an antibody or antigen-binding fragment thereof to inhibit the binding interaction between two molecules (e.g., human B7-H4 and anti-B7-H4 antibody) to any detectable extent. In certain embodiments, the antibody or antigen-binding fragment that blocks the binding between two molecules can inhibit the binding interaction between the two molecules by at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%. In certain embodiments, such inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0238] The term "bivalent" in this application refers to an antibody or antigen-binding fragment having two antigen-binding sites; the term "monovalent" refers to an antibody or antigen-binding fragment having only a single antigen-binding site; and the term "multivalent" refers to an antibody or antigen-binding fragment having multiple antigen-binding sites. In some embodiments, the antibody or antigen-binding fragment described in this application is bivalent.

[0239] Homologous

[0240] The terms "homologous" and "homologous" as used herein are used interchangeably and refer to a nucleic acid sequence (or its complementary strand) or an amino acid sequence that is at least 70%, at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to another sequence when optimally aligned.

[0241] When "percent sequence identity" is used for amino acid sequences (or nucleic acid sequences), it refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence after the sequences are aligned and, if necessary, intervals are introduced to maximize the number of identical amino acids (or nucleic acids). Conservative substitutions of the amino acid residues may or may not be considered identical residues. The sequences can be aligned to determine the percentage sequence identity of the amino acid (or nucleic acid) sequence by tools disclosed in the art, such as BLASTN, BLASTp (National Center for Biotechnology Information website (NCBI), see also Altschul SF et al., J. Mol. Biol., 215: 403-410 (1990); Stephen F et al., Nucleic Acids Res., 25: 3389-3402 (1997)), ClustalW2 (European Bioinformatics Institute website, see Higgins DG et al., Methods in Enzymology, 266: 383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, UK), 23 (21): 2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters of the tools or appropriately adjust the parameters according to the needs of the alignment, such as by selecting a suitable algorithm.

[0242] According to some embodiments of the present invention, the homologous sequence refers to a nucleotide sequence that is at least 70% identical to the sequence. According to some embodiments of the present invention, the homologous sequence has a nucleotide sequence that is at least 70% identical, such as a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98% or at least 99% identical.

[0243] According to certain embodiments of the present invention, the homologous sequence has substantially the same activity as the sequence disclosed in the present invention.

[0244] According to certain embodiments of the present invention, the homologous sequence refers to an amino acid sequence that is at least 70% identical to the sequence. According to certain embodiments of the present invention, the homologous sequence has an amino acid sequence that is at least 70% identical, such as an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98% or at least 99% identical.

[0245] According to certain embodiments of the present invention, the homologous sequence has substantially the same activity as the sequence disclosed in the present invention.

[0246] Antibody characteristics

[0247] The antibodies or antigen-binding fragments thereof described herein can block the binding between B7-H4 and its interacting proteins.

[0248] In some embodiments, by binding to B7-H4, the antibody can promote the B7-H4 signaling pathway. In some embodiments, by binding to B7-H4, the antibody can inhibit the B7-H4 signaling pathway. Thus, in some embodiments, the antibodies or antigen-binding fragments thereof described herein are B7-H4 agonists. In some embodiments, the antibodies or antigen-binding fragments thereof are B7-H4 antagonists.

[0249] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can increase the activity or number of B7-H4 by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can decrease the activity or number of B7-H4 by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

[0250] In some embodiments, the antibody (or antigen-binding fragment thereof) specifically binds to B7-H4 (e.g., human B7-H4, monkey B7-H4, mouse B7-H4, and / or chimeric B7-H4) with an off rate (koff): less than 0.1 s -1 Less than 0.01s -1 , less than 0.001s -1 , less than 0.0001s -1 or less than 0.0001s -1 In some embodiments, the dissociation rate (koff) is greater than 0.01 s -1 , greater than 0.001s -1 , greater than 0.0001s -1, greater than 0.0001s -1 or greater than 0.00001s -1 .

[0251] In some embodiments, the kinetic association rate (kon) is greater than 1x10 2 / Ms, greater than 1x10 3 / Ms, greater than 1x10 4 / Ms, greater than 1x10 5 / Ms or greater than 1x10 6 / Ms. In some embodiments, the kinetic association rate (kon) is less than 1x10 5 / Ms, less than 1x10 6 / Ms or less than 1x10 7 / Ms.

[0252] Affinity can be derived from the quotient of the kinetic rate constants (KD = koff / kon). In some embodiments, KD is less than 1x10 -6 M, less than 1x10 -7 M, less than 1x10 -8 M, less than 1x10 -9 M or less than 1x10 -10 In some embodiments, the KD is less than 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is greater than 1 x 10 -7 M, greater than 1x10 -8 M, greater than 1x10 -9 M, greater than 1x10 -10 M, greater than 1x10 -11 M or larger than 1x10 -12 M.

[0253] General techniques for measuring the affinity of an antibody for an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the antibody binds to human B7-H4. In some embodiments, the antibody binds to monkey B7-H4. In some embodiments, the antibody binds to mouse B7-H4. In some embodiments, the antibody binds to chimeric B7-H4.

[0254] In some embodiments, thermal stability is determined. The Tm of antibody or antigen binding fragment described herein can be greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95 ° C. Since IgG can be described as a multi-domain protein, the melting curve sometimes shows two transitions, i.e., the first denaturation temperature TmD1 and the second denaturation temperature TmD2. The presence of these two peaks usually indicates the denaturation of Fc domain (TmD1) and Fab domain (TmD2) respectively. When there are two peaks, Tm usually refers to TmD2.

[0255] In some embodiments, the TmD1 of an antibody or antigen binding fragment as described herein is greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C. In some embodiments, the TmD2 of the antibodies or antigen-binding fragments described herein is greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C.

[0256] In some embodiments, Tm, TmD1, TmD2 is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95°C.

[0257] In some embodiments, the tumor growth inhibition percentage (TGI%) of the antibody is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200%. In some embodiments, the tumor growth inhibition percentage of the antibody is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200%. The TGI% can be determined, for example, at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the start of treatment, or at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the start of treatment. As used herein, the tumor growth inhibition percentage (TGI%) is calculated using the following formula:

[0258] TGI(%)=[I-(Ti-TO) / (Vi-V0)]×100

[0259] Wherein, Ti is the mean tumor volume of the treatment group on day i. T0 is the mean tumor volume of the treatment group on day 0. Vi is the mean tumor volume of the control group on day i. V0 is the mean tumor volume of the control group on day 0.

[0260] Effector function

[0261] As used in this application, "effector function" refers to the biological activity of the Fc region of an antibody binding to its effector, such as the C1 complex and the Fc receptor. Exemplary effector functions include complement-dependent cytotoxicity (CDC) induced by the interaction of the antibody with C1q on the C1 complex, antibody-dependent cell-mediated cytotoxicity (ADCC) induced by the binding of the Fc region of the antibody to the Fc receptor on the effector cell, and phagocytosis. According to certain embodiments of the present invention, the antibody described in this application has the effector function.

[0262] Internalization

[0263] According to certain embodiments of the present invention, the antibodies described herein are capable of internalization or of being internalized. As used herein, an "internalizing" or "capable of being internalized" antibody is an antibody that is taken up by a cell after binding to its antigen on the cell surface. In some embodiments, the antibodies and fragments thereof described herein can be internalized by cells expressing B7-H4 on their surface, at least to a certain extent. For example, in some embodiments, the anti-B7-H4 antibodies described herein can be internalized by cells after binding to B7-H4 expressed on the cell surface.

[0264] Internalization can occur in vitro or in vivo. For therapeutic applications, internalization can occur in vivo. Whether an antigen is internalized after binding to a mammalian cell can be determined by various tests, including those described in the following examples (e.g., Fab-Zap methods). Methods for detecting whether an antibody is internalized into a cell are also described in U.S. Patent No. 7,619,068, which is incorporated herein by reference in its entirety. In some embodiments, antibodies and antigen-binding fragments thereof that can be internalized can be connected or conjugated to an anticancer agent (e.g., a cytotoxic portion that kills cells after internalization). Depending on the efficacy of the antibody or antibody conjugate, in some cases, the uptake of a single antibody molecule into a cell is sufficient to kill the target cell bound to the antibody. For example, some toxins have a high killing efficacy, and internalization of one molecule of the toxin conjugated to the antibody is sufficient to kill tumor cells.

[0265] Conjugate

[0266] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are linked to one or more conjugates. In certain embodiments, the conjugates include chemotherapeutic agents, toxins, radioisotopes, lanthanides, luminescent labels, fluorescent labels, or enzyme substrate labels. In certain embodiments, the conjugates are toxins.

[0267] The terms "treat," "treatment," or "treatment method" include preventing or alleviating a condition, reducing the rate of onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination of the above. Those in need of treatment include those already with the particular medical condition, as well as those who may eventually acquire that condition.

[0268] As used herein, the terms "object", "subject" and "patient" are used interchangeably in this application, and describe the human or non-human animal for which the method according to the invention provides treatment. Veterinary and non-veterinary applications are contemplated by the present invention. Human subjects can be adults or minors (e.g., people less than 18 years old). In addition to humans, subjects also include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs and primates. Including, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pigs, miniature pigs), horses, dogs, cats, cattle and other domestic, farm and zoo animals.

[0269] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to amino acid polymers of any length of at least two amino acids.

[0270] As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein to refer to nucleotide polymers of any length of at least two nucleotides, and include but are not limited to DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0271] As used herein, "isolated" means that the substance has been artificially altered or separated from its natural state. If a "separated" substance or component occurs in nature, it has been altered or separated from its original state, or both. For example, a polynucleotide or polypeptide naturally present in a living animal is not isolated, but if the substance with which these polynucleotides or polypeptides coexist in nature is sufficiently separated and present in a sufficiently pure state, it can be considered "isolated." Isolated "nucleic acid" and "polynucleotide" are used interchangeably to refer to the sequence of an isolated nucleic acid molecule. In certain embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment thereof that is at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% pure as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis), or chromatography (e.g., ion exchange chromatography or reversed phase HPLC).

[0272] IC50 and EC50

[0273] The term "half inhibitory concentration (IC50)", or "half inhibition rate", refers to the concentration of the inhibitor when the activity of the target substance is inhibited by half during the process of the inhibitor inhibiting the target substance. Generally, the stronger the inhibitory ability of the inhibitor, the lower the IC50. According to certain embodiments of the present application, the inhibitor is a B7-H4 inhibitor. According to certain embodiments of the present application, the inhibitor is a B7-H4 antibody. According to certain embodiments of the present application, the IC50 described in the present application is the concentration of the B7-H4 antibody when the activity of B7-H4 is inhibited by 50% by the B7-H4 antibody.

[0274] The term "half maximal effect concentration (EC50)" refers to the concentration at which an inhibitor can cause 50% of the maximum effect. EC50 is an indicator of drug efficacy. According to certain embodiments of the present application, the inhibitor is a B7-H4 inhibitor. According to certain embodiments of the present application, the inhibitor is a B7-H4 antibody. According to certain embodiments of the present application, EC50 determines the intensity of the effect of the B7-H4 antibody on cells.

[0275] Nucleic acid molecules, vectors, host cells

[0276] According to one aspect of the present invention, the present invention provides a nucleic acid molecule encoding the anti-B7-H4 antibody XF04-4 or an antigen-binding fragment thereof.

[0277] According to some embodiments of the present application, the nucleic acid molecule comprises the sequence of SEQ ID NO: 1. According to some embodiments of the present application, the nucleic acid molecule is the sequence of SEQ ID NO: 1. According to some embodiments of the present application, the nucleic acid molecule encodes the sequence of SEQ ID NO: 2.

[0278] According to one aspect of the present invention, the present invention provides a nucleic acid molecule encoding the variable region sequence of the anti-B7-H4 antibody XF04-4.

[0279] According to certain embodiments of the present application, the nucleic acid molecule comprises any one of SEQ ID NOs: 3-8. According to certain embodiments of the present application, the nucleic acid molecule comprises the SEQ ID NO: 3 sequence. According to certain embodiments of the present application, the nucleic acid molecule comprises the SEQ ID NO: 4 sequence. According to certain embodiments of the present application, the nucleic acid molecule comprises the SEQ ID NO: 5 sequence. According to certain embodiments of the present application, the nucleic acid molecule comprises the SEQ ID NO: 6 sequence. According to certain embodiments of the present application, the nucleic acid molecule comprises the SEQ ID NO: 7 sequence. According to certain embodiments of the present application, the nucleic acid molecule comprises the SEQ ID NO: 8 sequence.

[0280] According to certain embodiments of the present application, the nucleic acid molecule is any one of SEQ ID NOs: 3-8. According to certain embodiments of the present application, the nucleic acid molecule is a SEQ ID NO: 3 sequence. According to certain embodiments of the present application, the nucleic acid molecule is a SEQ ID NO: 4 sequence. According to certain embodiments of the present application, the nucleic acid molecule is a SEQ ID NO: 5 sequence. According to certain embodiments of the present application, the nucleic acid molecule is a SEQ ID NO: 6 sequence. According to certain embodiments of the present application, the nucleic acid molecule is a SEQ ID NO: 7 sequence. According to certain embodiments of the present application, the nucleic acid molecule is a SEQ ID NO: 8 sequence.

[0281] In the present invention, "vector" refers to a vehicle into which a polynucleotide encoding a protein can be operatively inserted and the protein can be expressed. The vector can be used to transform, transduce or transfect host cells so that the genetic material elements it carries are expressed in the host cells. According to certain embodiments of the present application, the vector includes: plasmid, phagemid, cosmid, artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC), bacteriophage such as λ phage or M13 phage, and animal virus, etc. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). The vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site. The vector may also include components that assist it in entering the cell, including but not limited to viral particles, liposomes or protein shells. The vector can be an expression vector or a cloning vector. The vector (e.g., expression vector) provided herein contains a nucleic acid sequence encoding an antigen or its antigen-binding fragment as described herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably connected to the nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retroviruses (including slow viruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), λ phages and M13 phages, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQDHyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p1 5TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0282] According to one aspect of the present invention, the present invention provides a vector comprising the nucleic acid molecule. According to certain embodiments of the present application, the encoding of the anti-B7-H4 antibody XF04-4 or its antigen-binding fragment. According to certain embodiments of the present application, the nucleic acid molecule encodes the variable region sequence of the anti-B7-H4 antibody XF04-4. According to certain embodiments of the present application, the nucleic acid molecule includes the sequence of SEQ ID NO: 1. According to certain embodiments of the present application, the nucleic acid molecule is the sequence of SEQ ID NO: 1. According to certain embodiments of the present application, the nucleic acid molecule encodes the sequence of SEQ ID NO: 2.

[0283] According to certain embodiments of the present application, the nucleic acid molecule comprises any one of SEQ ID NOs: 3-8. According to certain embodiments of the present application, the nucleic acid molecule is any one of SEQ ID NOs: 3-8.

[0284] In the present invention, "host cell" refers to a cell into which an exogenous polynucleotide and / or vector is introduced.

[0285] According to one aspect of the present invention, the present invention provides a host cell, wherein the host cell comprises the vector.

[0286] Transgenic animals

[0287] According to one aspect of the present invention, the present invention provides a transgenic animal, wherein the transgenic animal expresses the anti-B7-H4 antibody XF04-4. According to certain embodiments of the present application, the transgenic animal is a transgenic mouse. According to certain embodiments of the present application, the transgenic animal is a transgenic rat.

[0288] Method for preparing anti-B7-H4 antibodies

[0289] Isolated fragments of human B7-H4 can be used as immunogens to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be generated in animals by multiple injections (e.g., subcutaneous or intraperitoneal) of antigenic peptides or proteins. In some embodiments, the antigenic peptides or proteins are injected with at least one adjuvant. In some embodiments, the antigenic peptides or proteins can be conjugated with an agent that is immunogenic in the species to be immunized. The antigenic peptides or proteins can be injected into the animal more than once (e.g., twice, three times, or four times).

[0290] The full-length polypeptide or protein can be used, or alternatively, an antigenic peptide fragment thereof can be used as an immunogen. The antigenic peptide of the protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of B7-H4, and comprises an epitope of the protein such that antibodies raised against the peptide form a specific immune complex with the protein. As described above, the full-length sequence of human OX40 is known in the art.

[0291] Immunogens are typically used to prepare antibodies by immunizing a suitable subject (e.g., a human or a transgenic animal expressing at least one human immunoglobulin locus). Suitable immunogenic preparations may include, for example, recombinantly expressed or chemically synthesized polypeptides (e.g., fragments of human B7-H4). The preparation may further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulant.

[0292] Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a B7-H4 polypeptide or antigenic peptide thereof (e.g., a portion of B7-H4) as an immunogen. Antibody titers in the immunized subject can be monitored over time by standard techniques, such as an enzyme-linked immunosorbent assay (ELISA) using immobilized B7-H4 polypeptide or peptide. If desired, the antibody molecules can be isolated from a mammal (e.g., from blood) and further purified by known techniques such as protein A or protein G chromatography to obtain the IgG fraction. At an appropriate time after immunization, for example, when specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature 256: 495-497, 1975), human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72, 1983), EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985) or trioma technique. Techniques for producing hybridomas are well known (see generally, Current Protocols in Immunology, 1994, Coliganet al. (Eds.), John Wiley & Sons, Inc., New York, NY). Hybridoma cells producing monoclonal antibodies are detected by screening hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest, for example, using a standard ELISA assay.

[0293] Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding the human antibodies, humanized antibodies or chimeric antibodies, or antigen-binding fragments thereof described herein or by peptide synthesis. Such variants include, for example, deletions, insertions or substitutions of residues in the amino acid sequence that constitutes the antigen binding site of the antibody or antigen-binding domain. In a population of such variants, some antibodies or antigen-binding fragments will have an increased affinity for the target protein (e.g., B7-H4). Any combination of deletions, insertions and / or combinations can be performed to obtain an antibody or antigen-binding fragment thereof with an increased binding affinity for the target. Amino acid changes introduced into the antibody or antigen-binding fragment can also change the antibody or antigen-binding fragment or introduce new post-translational modifications therein, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence to allow attachment of a different sugar by an enzyme present in the cell), or introducing a new glycosylation site.

[0294] The antibodies disclosed herein can be derived from any animal species, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates (e.g., monkeys and apes), cattle, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents that have been genetically modified to produce human antibodies.

[0295] Human antibodies and humanized antibodies include antibodies with variable and constant regions derived from human germline immunoglobulin sequences (or with the same amino acid sequence as those derived therefrom). Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), such as in CDRs.

[0296] Humanized antibodies generally have a human framework (FR) grafted with non-human CDRs. Therefore, humanized antibodies have one or more amino acid sequences introduced therein from a non-human source. These non-human amino acid residues are generally referred to as "import" residues, which are generally taken from the "import" variable domain. Humanization can be performed essentially by, for example, replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. These methods are described, for example, in Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988); each of which is incorporated herein by reference in its entirety. Therefore, a "humanized" antibody is a chimeric antibody in which substantially less than a complete human V domain has been replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are typically mouse antibodies in which some CDR residues and some FR residues are substituted by residues from analogous sites in human antibodies.

[0297] The selection of human VH and VL domains for preparing humanized antibodies is very important for reducing immunogenicity. According to the so-called "best fit" method, the sequence of the V domain of mouse antibodies is screened for the entire library of known human domain sequences. The human sequence closest to the sequence of the mouse is then used as the human FR of the humanized antibody (Sims et al., J.Immunol., 151: 2296 (1993); Chothia et al., J.Mol.Biol., 196: 901 (1987)).

[0298] It is further important that the antibody is humanized while retaining high specificity and avidity and other favorable biological properties to the antigen. To achieve this purpose, humanized antibodies can be prepared by using the three-dimensional model of the parental sequence and the humanized sequence to analyze the parental sequence and various conceptual humanized products. The three-dimensional immunoglobulin model is generally available and is familiar to those skilled in the art. The computer program that illustrates and displays the possible three-dimensional conformational structure of selected candidate immunoglobulin sequences is available. Inspection of these displays allows analysis of the possible effects of residues in the function of candidate immunoglobulin sequences, that is, analysis affects the residues of the ability of candidate immunoglobulin and its antigen binding. In this way, FR residues can be selected from acceptor sequence and input sequence and combined, thereby obtaining desired antibody characteristics, for example, the avidity to the raising of the target antigen.

[0299] Generally, amino acid sequence variants of human, humanized or chimeric anti-B7-H4 antibodies will comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% percent identity to the sequence present in the light chain or heavy chain of the original antibody.

[0300] Identity or homology relative to the original sequence is typically the percentage of amino acid residues present in the candidate sequence that are identical to the sequence present in the human, humanized or chimeric anti-B7-H4 antibody or fragment, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity.

[0301] The anti-B7-H4 antibody or antigen-binding fragment may be further modified. For example, cysteine ​​residues may be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in this region. The homodimeric antibody thus produced may have any increased in vitro and / or in vivo half-life. Homodimeric antibodies with extended in vitro and / or in vivo half-lives may also be prepared using heterobifunctional cross-linkers, as described, for example, in Wolff et al. (Cancer Res. 53:2560-2565, 1993). Alternatively, antibodies with two Fc regions may be engineered (see, for example, Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).

[0302] In some embodiments, the anti-B7-H4 antibodies or antigen-binding fragments thereof may be covalently modified. These covalent modifications may be performed by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of the antibodies or antibody fragments are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatizing agent that is capable of reacting with selected side chains or N- or C-terminal residues.

[0303] According to one aspect of the present invention, the present invention provides a method for producing the anti-B7-H4 antibody, the method comprising culturing the host cell and isolating the B7-H4 antibody.

[0304] According to one aspect of the present invention, the present invention provides a hybridoma, which produces the B7-H4 antibody.

[0305] According to one aspect of the present invention, the present invention provides a method for producing the anti-B7-H4 antibody, the method comprising purifying the B7-H4 antibody from the hybridoma.

[0306] According to certain embodiments of the present application, the hybridoma is a mouse hybridoma. According to certain embodiments of the present application, the hybridoma is a rat hybridoma.

[0307] Treatment methods, pharmaceutical uses and detection methods

[0308] The antibodies or antigen-binding fragments thereof described herein can be used for a variety of therapeutic purposes. On the one hand, the disclosure provides methods for treating cancer in a subject, methods for reducing the rate at which the volume of a tumor in a subject increases over time, methods for reducing the risk of metastasis, or methods for reducing the risk of additional metastasis in a subject. In some embodiments, treatment can stop, slow down, delay or inhibit the progression of cancer. In some embodiments, treatment can result in a reduction in the number, severity and / or duration of one or more symptoms of cancer in a subject.

[0309] In one aspect, the disclosure features a method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to a subject in need thereof (e.g., a subject having cancer or identified or diagnosed as having cancer). In some embodiments, the cancer is colorectal cancer, such as ascending colon cancer, descending colon cancer, sigmoid colon cancer, and rectal cancer. In some embodiments, the colon cancer includes squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, and undifferentiated carcinoma.

[0310] In some embodiments, cancer is breast cancer (e.g., triple-negative breast cancer), carcinoid, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematological malignancies. In some embodiments, cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the subject suffers from a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), or colorectal cancer.

[0311] According to one aspect of the present invention, the present invention provides a method for treating cancer, wherein the method comprises administering the anti-B7-H4 antibody to a subject.

[0312] According to certain aspects of the present invention, B7-H4 is highly expressed in the cancer.

[0313] According to some aspects of the invention, the cancer is colorectal cancer.

[0314] According to certain aspects of the present invention, the colorectal cancer is colorectal adenocarcinoma.

[0315] According to certain aspects of the present invention, the colorectal adenocarcinoma cells are HT29 and LS174T cells.

[0316] According to one aspect of the present invention, the present invention provides a method for inhibiting tumor cell growth in a subject, the method comprising administering a therapeutically effective amount of the B7-H4 antibody or an antigen-binding fragment thereof to the subject to inhibit tumor cell growth.

[0317] According to certain aspects of the present invention, B7-H4 is highly expressed in the cancer.

[0318] According to some aspects of the invention, the cancer is colorectal cancer.

[0319] According to certain aspects of the present invention, the colorectal cancer is colorectal adenocarcinoma.

[0320] According to certain aspects of the present invention, the colorectal adenocarcinoma cells are HT29 and LS174T cells.

[0321] According to one aspect of the present invention, the present invention provides use of the anti-B7-H4 antibody in the preparation of a drug for treating cancer.

[0322] According to certain aspects of the present invention, B7-H4 is highly expressed in the cancer.

[0323] According to some aspects of the invention, the cancer is colorectal cancer.

[0324] According to certain aspects of the present invention, the colorectal cancer is colorectal adenocarcinoma.

[0325] According to certain aspects of the present invention, the colorectal adenocarcinoma cells are HT29 and LS174T cells.

[0326] According to one aspect of the present invention, the present invention provides a method for treating a B7-H4-related disease, wherein the method comprises administering the anti-B7-H4 antibody to a subject.

[0327] According to certain aspects of the present invention, B7-H4 is highly expressed in the B7-H4-related disease.

[0328] According to certain aspects of the present invention, the B7-H4-related disease is colorectal cancer.

[0329] According to certain aspects of the present invention, the colorectal cancer is colorectal adenocarcinoma.

[0330] According to certain aspects of the present invention, the colorectal adenocarcinoma cells are HT29 and LS174T cells.

[0331] According to one aspect of the present invention, the present invention provides use of the anti-B7-H4 antibody in the preparation of a medicament for treating a B7-H4-related disease.

[0332] According to certain aspects of the present invention, B7-H4 is highly expressed in the B7-H4-related disease.

[0333] According to certain aspects of the present invention, the B7-H4-related disease is cancer.

[0334] According to some aspects of the invention, the cancer is colorectal cancer.

[0335] According to certain aspects of the present invention, the colorectal cancer is colorectal adenocarcinoma.

[0336] According to certain aspects of the present invention, the colorectal adenocarcinoma cells are HT29 and LS174T cells.

[0337] According to one aspect of the present invention, the present invention provides a method for regulating B7-H4 activity in a cell expressing B7-H4, the method comprising contacting the cell expressing B7-H4 with the anti-B7-H4 antibody or an antigen-binding fragment thereof.

[0338] According to certain aspects of the present invention, the cell expressing B7-H4 is a cancer cell.

[0339] According to some aspects of the invention, the cancer is colorectal cancer.

[0340] According to certain aspects of the present invention, the colorectal cancer is colorectal adenocarcinoma.

[0341] According to certain aspects of the present invention, the colorectal adenocarcinoma cells are HT29 and LS174T cells.

[0342] According to one aspect of the present invention, the present invention provides a method for killing cells expressing B7-H4 in vivo or in vitro, the method comprising contacting the cells expressing B7-H4 with the anti-B7-H4 antibody or an antigen-binding fragment thereof.

[0343] According to certain aspects of the present invention, the cell expressing B7-H4 is a cancer cell.

[0344] According to some aspects of the invention, the cancer is colorectal cancer.

[0345] According to certain aspects of the present invention, the colorectal cancer is colorectal adenocarcinoma.

[0346] According to certain aspects of the present invention, the colorectal adenocarcinoma cells are HT29 and LS174T cells.

[0347] According to one aspect of the present invention, the present invention provides a method for detecting the presence or content of B7-H4 in a sample, the method comprising contacting the sample with an anti-B7-H4 antibody or an antigen-binding fragment thereof, and determining the presence or content of B7-H4 in the sample.

[0348] According to certain aspects of the present invention, the sample is a cell expressing B7-H4.

[0349] According to certain aspects of the present invention, the cell expressing B7-H4 is a cancer cell.

[0350] According to some aspects of the invention, the cancer is colorectal cancer.

[0351] According to certain aspects of the present invention, the colorectal cancer is colorectal adenocarcinoma.

[0352] According to certain aspects of the present invention, the colorectal adenocarcinoma cells are HT29 and LS174T cells.

[0353] According to one aspect of the present invention, the present invention provides a method for diagnosing a disease or condition associated with B7-H4 in a subject, the method comprising: a) obtaining a sample from the subject; b) contacting the sample with the anti-B7-H4 antibody or its antigen-binding fragment; c) determining the presence or amount of B7-H4 in the sample; d) correlating the presence or amount of B7-H4 with the disease or condition of the subject.

[0354] According to certain aspects of the present invention, the subject has the B7-H4-related disease

[0355] According to certain aspects of the present invention, B7-H4 is highly expressed in the subject.

[0356] According to certain aspects of the present invention, the B7-H4-related disease is cancer.

[0357] According to some aspects of the invention, the cancer is colorectal cancer.

[0358] According to certain aspects of the present invention, the colorectal cancer is colorectal adenocarcinoma.

[0359] According to certain aspects of the present invention, the colorectal adenocarcinoma cells are HT29 and LS174T cells.

[0360] The terms "administration", "administering" or "administering" refer to administering a dose of a compound or pharmaceutical composition to a subject via a suitable administration method.

[0361] The "administration method" includes, but is not limited to, oral administration, intravenous administration, intrarespiratory administration, sublingual administration, topical administration, intramuscular administration, intraocular administration, transdermal absorption, parenteral administration, intraperitoneal administration, vaginal administration, buccal administration, rectal administration, and any other administration methods known in the art. It should be understood by those skilled in the art that the administration method of the subject depends on a number of factors, including the location of the disease, the age of the subject, the severity of the disease, and the ingredients of the pharmaceutical composition.

[0362] In some embodiments, the compositions and methods disclosed herein can be used to treat a subject at risk for cancer.A subject having cancer can be identified by a variety of methods known in the art.

[0363] As used herein, "effective amount" means an amount or dosage sufficient to produce a beneficial or desired result, including stopping, slowing, delaying or inhibiting the progression of a disease, such as cancer. The effective amount will vary, for example, according to the age and weight of the individual to whom the antibody, antigen-binding fragment, polynucleotide encoding the antibody, vector comprising the polynucleotide, and / or a composition thereof is to be administered, the severity of the symptoms, and the route of administration, and thus administration may be determined on an individual basis.

[0364] An effective amount may be administered in one or more administrations. For example, an effective amount of an antibody or antigen binding fragment is an amount sufficient to improve, stop, stabilize, reverse, inhibit, slow down and / or delay cancer progression in a patient, or an amount sufficient to improve, stop, stabilize, reverse, slow down and / or delay proliferation of cells (e.g., biopsy cells, any cancer cell described herein, or cell line (e.g., cancer cell line)) in vitro. As understood in the art, an effective amount of an antibody or antigen binding fragment may vary, depending in particular on the patient's medical history and other factors, such as the type (and / or dosage) of the antibody used.

[0365] The effective amount and schedule for administering the antibodies, polynucleotides encoding the antibodies, and / or compositions disclosed herein can be determined empirically, and it is within the skill of the art to make such determinations. It will be appreciated by those skilled in the art that the dosage that must be administered will vary, for example, depending on the mammal to which the antibodies, polynucleotides encoding the antibodies, and / or compositions disclosed herein will be administered, the route of administration, the specific type of antibodies, polynucleotides encoding the antibodies, antigen-binding fragments, and / or compositions disclosed herein used, and other drugs administered to the mammal. Guidance for selecting the appropriate dosage of an antibody or antigen-binding fragment can be found in the literature on the therapeutic use of antibodies and antigen-binding fragments, for example, Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, 1985, ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pp. 365-389.

[0366] The typical daily dose of the effective amount of the antibody is 0.01 mg / kg to 100 mg / kg. In some embodiments, the dosage can be less than 100 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg or 0.1 mg / kg. In some embodiments, the dosage can be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg or 0.01 mg / kg. In some embodiments, the dosage is about 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0367] In any of the methods described herein, at least one antibody, antigen binding fragment, or pharmaceutical composition thereof (e.g., any antibody, antigen binding fragment, or pharmaceutical composition described herein) and optionally at least one additional therapeutic agent may be administered to a subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies and / or antigen binding fragments are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen binding fragment and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition comprising at least one antibody or antigen binding fragment and a solid oral composition comprising at least one additional therapeutic agent. In some embodiments, at least one antibody or antigen binding fragment and at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition comprising at least one antibody or antigen binding fragment and a solid oral composition comprising at least one additional therapeutic agent).

[0368] In some embodiments, the at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is a sustained release oral formulation.

[0369] In some embodiments, one or more additional therapeutic agents may be administered to a subject before or after administration of at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any antibody, antigen-binding antibody fragment, or pharmaceutical composition described herein). In some embodiments, one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any antibody, antigen-binding antibody fragment, or pharmaceutical composition described herein) are administered to a subject such that there is overlap in the biological activity period of the one or more additional therapeutic agents and at least one antibody or antigen-binding fragment (e.g., any antibody or antigen-binding fragment described herein) in the subject.

[0370] In some embodiments, at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) may be administered to a subject over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or tracking the effectiveness of treatment (e.g., observing at least one symptom of cancer). As described herein, based on an assessment of the effectiveness of treatment (e.g., using any of the methods described herein and known in the art), a skilled medical professional may also change the identity and amount (e.g., increase or decrease) of the antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to the subject, and may also adjust (e.g., increase or decrease) the dose or frequency of administering at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) to the subject.

[0371] In some embodiments, one or more additional therapeutic agents may be administered to a subject. The additional therapeutic agent may include one or more selected from the group consisting of B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3 kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, PI3K / mTOR dual inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and inhibitors of isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2).

[0372] In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of HER3 inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, inhibitors of activated hedgehog signaling pathways, and agents that selectively degrade estrogen receptors.

[0373] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votr ient, pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, Hsp90 inhibitors, Ad-GM-CSF, Temozolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

[0374] In some embodiments, the additional therapeutic agents may include one or more therapeutic agents selected from the following: adjuvants, TLR agonists, tumor necrosis factor (TNF) α, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, CX3CL1-targeted therapies, CXCL9-targeted therapies, CXCL10-targeted therapies, CCL5-targeted therapies, LFA-1 agonists, ICAM1 agonists, and selectin agonists.

[0375] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to the subject.

[0376] In some embodiments, the additional therapeutic agent is an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody.

[0377] Pharmaceutical compositions and kits

[0378] Also provided herein are pharmaceutical compositions comprising at least one (e.g., one, two, three, or four) antibody or antigen-binding fragment described herein. Two or more (e.g., two, three, or four) of any antibody or antigen-binding fragment described herein may be present in the pharmaceutical composition in any combination. The pharmaceutical composition may be formulated in any manner known in the art.

[0379] According to one aspect of the present invention, the present invention provides a pharmaceutical composition, comprising a therapeutically effective amount of the anti-B7-H4 antibody and a pharmaceutically acceptable carrier.

[0380] According to one aspect of the present invention, the present invention provides a method for preparing the pharmaceutical composition, characterized in that the method comprises mixing the anti-B7-H4 antibody with a pharmaceutically acceptable carrier for preparation.

[0381] According to one aspect of the present invention, the present invention provides a drug kit comprising the anti-B7-H4 antibody.

[0382] The term "pharmaceutically acceptable" means that the referred carrier, vehicle, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

[0383] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable component or medium, including but not limited to solvents, excipients, diluents, adjuvants, carriers, fillers, etc. Common pharmaceutically acceptable carriers include physiological saline, buffers, sugars, gelatin, starch, Ringer's solution, cellulose, etc. The pH of the carrier is usually 3-11, preferably 5-9, and more preferably 7-8.

[0384] The term "effective amount" or "therapeutically effective amount" of the compound refers to an amount sufficient to achieve the desired therapeutic effect after administration to a subject, such as effectively inhibiting the activity of B7-H4 and effectively improving or treating a B7-H4-related disease in a subject.

[0385] The term "pharmaceutical composition" refers to a mixture containing a therapeutically effective amount of one or more of the compounds and their pharmaceutically acceptable tautomers, solvates, hydrates or salts, and other pharmaceutically acceptable carriers. The purpose of preparing the compounds into a pharmaceutical composition is to more conveniently administer them to a subject.

[0386] The pharmaceutical composition described in the present application can be a tablet, a pill, a capsule, a granule, a powder, a suppository, a powder, an ointment, a patch, an injection, a solution, a suspension, a spray, a lotion, a drop, or an ointment. The pharmaceutical composition can be prepared in the form of a dry powder and mixed with sterile water or a buffer solution before administration to prepare a solution form. The pH of the buffer solution is generally 3-11, preferably 5-9, and more preferably 7-8.

[0387] Various dosage forms of the composition of the present invention can be prepared by conventional methods in the pharmaceutical field, wherein the content of the antibody is 0.1%-99.9% by weight of the composition (by weight), for example, 0.1%-99%, 0.1%-90%, 0.1%-80%, 0.1%-60%, 0.1%-40%, 0.1%-20%, 0.1%-10%, 10%-99.9%, 10%-99%, 10%-90%, 10%-80%, 10%-60%, 10%- 40%, 10%-20%, 20%-99.9%, 20%-99%, 20%-90%, 20%-80%, 20%-60%, 20%-40%, 40%-99.9%, 40%-99%, 40%-90%, 40%-80%, 40%-60%, 60%-99.9%, 60%-99%, 60%-90%, 60%-80%, 80%-99.9%, 80%-99%, 80%-90% or 90%-99.9%. Various dosage forms of the composition of the present invention can be prepared by conventional methods in the pharmaceutical field, wherein the content of the antibody is 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5.%, 99.6%, 99.7%, 99.8% or 99.9% by weight of the composition.

[0388] The terms "kit" or "test kit" are used interchangeably in this application. The present application discloses a kit comprising a therapeutically effective amount of the therapeutic agent or pharmaceutical composition. According to certain embodiments of the present application, the kit further comprises one or more other therapeutic agents. According to certain embodiments of the present application, the kit further comprises instructions for use. According to certain embodiments of the present application, the kit further comprises a device for a corresponding administration method, such as but not limited to a needle.

[0389] Route of administration

[0390] The pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The composition may include a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvent; an antibacterial or antifungal agent, such as benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate, or phosphate; and an isotonic agent, such as a sugar (e.g., glucose), a polyol (e.g., mannitol or sorbitol), or a salt (e.g., sodium chloride); or any combination thereof.

[0391] Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811). The formulation of the composition can be prepared and packaged in ampoules, disposable syringes, or multiple dose vials. Where necessary (as in, e.g., injectable formulations), appropriate fluidity can be maintained by, e.g., using a coating (e.g., lecithin) or a surfactant. The absorption of the antibody or its antigen-binding fragment can be extended by including an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by an implant and a microencapsulated delivery system that can include a biodegradable biocompatible polymer (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

[0392] Compositions containing one or more of any of the antibodies or antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined amount of the active compound for ease of administration and uniformity of dosage).

[0393] The toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical procedures in cell culture or experimental animals (e.g., monkeys). For example, the LD50 (the dose that is lethal to 50% of the population) and the ED50 (the dose that is therapeutically effective in 50% of the population) can be determined: the therapeutic index is the ratio of LD50:ED50. Agents that exhibit a high therapeutic index are preferred. If the agent exhibits undesirable side effects, care should be taken to minimize potential hazards (i.e., reduce unwanted side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0394] The data obtained from cell culture assays and animal studies can be used to formulate any given agent in a suitable dose for use in an object (e.g., a human). The therapeutically effective amount of one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any antibody or antibody fragment described herein) is such an amount that it treats the disease in the object (e.g., killing cancer cells) in an object (e.g., a human object determined to have cancer), or in an object determined to be at risk of disease occurrence (e.g., an object that previously had cancer but is now cured), and reduces the severity, frequency, and / or duration of one or more symptoms of the disease in the object (e.g., a human). The effectiveness and dosage of any antibody or antigen-binding fragment described herein can be determined by a health care professional or veterinary professional using methods known in the art and by observing one or more symptoms of the disease in the object (e.g., a human). Certain factors may affect the dosage and timing required for effective treatment of an object (e.g., the severity of the disease or condition, previous treatment, the overall health and / or age of the object, and the presence of other diseases).

[0395] Exemplary dosages include milligrams or micrograms of any antibody or antigen-binding fragment described herein per kilogram of subject body weight (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 10 μg / kg to about 0.5 mg / kg; or about 1 μg / kg to about 50 μg / kg). Although these dosages cover a wide range, it will be appreciated by those of ordinary skill in the art that therapeutic agents, including antibodies and antigen-binding fragments thereof, vary in efficacy and that effective amounts can be determined by methods known in the art. Typically, a relatively low dose is first administered, and the attending health care professional or veterinary professional (in the case of therapeutic applications) or researcher (when still in the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it should be understood that the specific dosage level for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the age, weight, general health, sex and diet of the subject, the time of administration, the route of administration, the rate of excretion and the half-life of the antibody or antibody fragment in vivo.

[0396] When the subject is a human, common doses for systemic administration include, but are not limited to, 10 ng / kg-100 mg / kg, such as 10-100 ng / kg, 10-500 ng / kg, 10-1000 ng / kg, 50-500 ng / kg, 50-1000 ng / kg, 1-10 μg / kg, 5-50 μg / kg, 10-100 μg / kg, 100-500 μg / kg, 250-750 μg / kg, 10-1000 μg / kg, 100-1000 0μg / kg, 500-1000μg / kg, 1-10mg / kg, 5-50mg / kg, 20-50mg / kg, 25-75mg / kg, 1-100mg / kg, 20-100mg / kg, 50-10 0mg / kg, 75-100mg / kg, 1mg / kg, 5mg / kg, 10mg / kg, 20mg / kg, 30mg / kg, 40mg / kg, 50mg / kg, 75mg / kg, 100mg / kg, etc.

[0397] The compound or pharmaceutical composition described in the present application can be administered at any time. For example, the compound or pharmaceutical composition can be administered before, during or after the onset of the disease in the subject, for example, it can be administered about 1 hour, about 2 hours, about 4 hours, about 5 hours, about 8 hours, about 12 hours, about 24 hours, about 2 days, about 4 days, about 8 days, about 16 days, about 30 days, or 1 month, about 2 months, about 4 months, or about 6 months before or after the onset of the disease.

[0398] The compound or pharmaceutical composition described in the present application can be administered once or multiple times. When multiple times are administered, the dosage can be at any interval, for example, the interval between the subsequent dose and the previous dose is about 8 weeks, about 4 weeks, about 2 weeks, about 1 week, about 5 days, about 3 days, about 2 days, about 24 hours, about 12 hours, about 8 hours, about 6 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 30 minutes or less.

[0399] The compounds or pharmaceutical compositions described herein can be administered alone or in combination with other suitable drugs. The combination can be, for example, administered simultaneously with other drugs or administered sequentially. When administered sequentially, the drugs can be administered at any interval.

[0400] The pharmaceutical composition may be included in a container, pack, or dispenser together with instructions for administration. The present disclosure also provides methods for preparing antibodies or antigen-binding fragments thereof for the various uses described herein.

[0401] In this application, when "about" is used to modify a numerical value, it means that the numerical value can fluctuate within the range of ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1%.

[0402] Unless otherwise specified in this application or clearly contradicted by the context, the terms "a", "an", "said", "the", "at least one" and similar references used in the context of describing this application (including the context of the claims) are interpreted to cover the singular and the plural. Unless otherwise specified in this application or clearly contradicted by the context, the terms "comprising", "having", "including" and "containing" used in this application are interpreted as open terms (i.e., "including but not limited to"). Unless otherwise specified in this application or clearly contradicted by the context, all methods described in this application can be performed in any suitable order according to the understanding of those skilled in the art.

[0403] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; however, other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety to the same extent as if each document were cited individually as a reference. If there is a conflict between the present application and the documents provided herein, the content in the present application shall prevail.

[0405] This application describes preferred implementations and embodiments, and those skilled in the art may make appropriate changes to the implementations and embodiments described in this application based on reading this application. Therefore, the content of this application for protection includes all equivalent modifications and changes to the subject matter in the claims of this application within the scope permitted by law.

[0406] Example 1: Method for preparing anti-B7-H4 antibodies

[0407] This example discloses a method for preparing an anti-B7-H4 ScFv-FC antibody.

[0408] (1) Mouse immunization

[0409] (i) Experimental mouse immunization

[0410] The recombinant human B7-H4 protein is used to immunize mice, and the specific steps include: (1) selecting 20 6-8 week old BALB / c female mice, feeding them normally for 3 days to allow them to fully adapt to the environment; (2) emulsifying the recombinant human B7-H4 protein with an equal volume of Freund's complete adjuvant, resonating for 30 minutes to fully mix them, and injecting them subcutaneously at multiple points in the mouse body; (3) two weeks after the first immunization, the mouse titer is tested, and the immunized mice with high titer are selected for intraperitoneal shock immunization; (4) emulsifying the B7-H4 protein with an equal volume of Freund's incomplete adjuvant, resonating for 30 minutes to fully mix them, and injecting them intraperitoneally into the mouse; (5) repeating step (4) 3 times to strengthen the immune stress response.

[0411] (ii) RNA extraction

[0412] After the mice were anesthetized to death, they were immersed in pure alcohol for 3 minutes for disinfection. The spleen was removed under sterile conditions, precooled in GBSS buffer and residual fat and connective tissue were removed. The spleen was quickly cut into pieces and divided into 2mL centrifuge tubes treated with DEPC, placed in liquid nitrogen for 2 minutes, ground with a tissue grinder, and 1mL Trizol was added to each tube. The tube was left standing at room temperature for 5 minutes and centrifuged at 12000r / min for 5 minutes. The supernatant was added to 200μL chloroform, mixed thoroughly, left standing for 15 minutes, and centrifuged at 12000g for 15 minutes. The upper aqueous phase was aspirated, 500μL isopropanol was added, mixed thoroughly, left standing for 10 minutes, centrifuged at 12000g for 10 minutes, and the supernatant was discarded. The precipitate was resuspended with 1mL 75% ethanol, centrifuged at 8000g for 5 minutes, and the supernatant was discarded. After drying at room temperature, the precipitate was dissolved with 30μL RNase-free water. The concentration of RNA was determined by measuring A260 / A280 with an ultra-micro spectrophotometer, and its purity was detected by agarose gel electrophoresis.

[0413] (iii) Reverse transcription of cDNA

[0414] The RNA obtained in the previous step was reverse transcribed into cDNA according to the instructions of the reverse transcription kit (Thermo, K1622).

[0415] (iv) Amplification of Antibody Fragments Specific antibody fragments were amplified from the reverse transcribed cDNA by PCR amplification using Taq DNA Polymerase Hot Start enzyme.

[0416] The PCR reaction system was: cDNA template 2ul, upstream primer (TGGAATTGTGAGCGGATAACAATT) 2ul, downstream primer (GTAAATGAATTTTCTGTATGAGG) 2ul, 10X Taq Buffer 5ul, dNTP 4ul, Taq (HS) 0.25ul, and ddH2O to make up to 50ul.

[0417] The reaction conditions of PCR were as follows: 95°C for 3 min; 95°C for 15 s, 60°C for 15 s, 72°C for 15 s, with an increase of 2 s in each cycle, repeated for 35 cycles, and 72°C for 5 min.

[0418] (v) Cloning into phage plasmid: The antibody gene sequence and phage vector obtained by amplification in the previous step were digested with BglI. The digestion system was: 12ug of amplified gene or 3ug of vector, 3ul of 10×BglI Buffer, 4.5ul of BglI, and water was added to 30ul. The digestion was allowed to react at 37 degrees for 3-4 hours. After digestion, the vector and amplified gene were recovered using a DNA purification recovery kit according to the instructions, and then ligation was performed. The ligation reaction system was: 200ng of vector, 80ng of amplified gene, 2ul of T4 ligase, 5ul of 10X ligation buffer, and water was added until 50ul. The reaction was allowed to react overnight at 4 degrees. The ligation product was recovered using a DNA purification recovery kit according to the instructions, and dissolved in ultrapure water.

[0419] (vi) Transformation of SS320: Pre-cool the electroporation cuvette on ice. After the SS320 competent cells have thawed, add 1 ul of the recovered ligation product. Transfer the mixed competent cells and ligation product to the pre-cooled electroporation cuvette and use the preset Bacteria transformation program of the electroporator for electroporation. Immediately after electroporation, add 1 mL of SOC medium to the electroporation cuvette. Resuscitate the cells at 37 degrees for 60 minutes and then spread them on LB culture plates containing tetracycline and ampicillin resistance for overnight growth.

[0420] The cells on the culture plate grown overnight in the previous step were washed and scraped with LB medium and a coating rod, and then stored at -80 degrees after adding 20% ​​glycerol.

[0421] (vii) Amplification and purification of phage library: Transfer about 10^9 cells scraped from the previous step into 100 mL of 2×YT culture medium pre-added with tetracycline and ampicillin antibiotics, and culture at 37 degrees at 220 rpm until OD600 reaches 0.5. Add helper phage at a ratio of 20:1 of helper phage to bacterial cells and continue to culture at 37 degrees for 30 minutes. Add kanamycin at a final concentration of 0.5 and 0.2 mM IPTG, and culture overnight at 30 degrees.

[0422] Centrifuge the cells cultured overnight at 4 degrees and 13,000 rpm for 5 minutes, transfer the supernatant to a new centrifuge tube, add 1 / 4 volume of pre-cooled 5×PEG8000 / NaCl, and incubate on ice for 30 minutes. Centrifuge at 4 degrees and 13,000 rpm for 10 minutes, remove the supernatant, and add 1 mL of PBS buffer to dissolve the precipitate. Add 250 ul of 5×PEG8000 / NaCl again, incubate on ice for 10 minutes, centrifuge at 4 degrees and 16,000 g for 15 minutes, remove the supernatant, and dissolve the precipitate in 1 mL of PBS to obtain the phage library.

[0423] (2) Phage library screening

[0424] The KN14 mouse phage library was used for phage display antibody screening. After two rounds of screening, 11 antibodies were initially screened out and constructed in the PNFCG2 human IgG2 plasmid.

[0425] (3) Antibody expression

[0426] Before transient transfection of cells, the cell density and survival rate must be determined. To ensure the transfection effect, use cells in the exponential growth phase (density of about 2-4×10 6 Cells can be transfected with a cell density of 2×10 6 If the cell density is >2×10 6 cells / mL, fresh KOP293 culture medium should be added to dilute the cell density to 2×10 6 Pieces / mL.

[0427] Place the vial in a 5% CO2 constant temperature shaker, 37°C, 120rpm constant temperature shaking culture for 10 minutes before starting transfection. Prepare two 15mL sterile centrifuge tubes, add 1mL KPM (transfection reagent) and 20μg sterile plasmid DNA to one of them, and gently blow to mix; take another centrifuge tube, add 1mL KPM and 100μL TA-293 transfection reagent, and gently blow to mix. Transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid, and gently blow to mix. Let it stand at room temperature for 10 minutes to prepare the plasmid-vector complex. Remove the cells from the constant temperature shaker, add the prepared plasmid-vector complex while shaking, and return to the CO2 constant temperature shaker for shaking culture. Add 120μL antibiotics after 3h. 24 hours after transfection, 120 μL 293 cell protein expression enhancer (KE-293) and 400 μL nutrient additive (KT-Feed 50×) can be added to increase product expression. The culture supernatant was collected on the 5th day after transfection.

[0428] (4) Antibody purification

[0429] The AKTA system (Cytiva, Purification of whole antibody pro 25M1

[0430] Install the Protein-A purification column (Cytiva) into System; set system parameters according to the specifications of the purification column. Use 5 column volumes of Binding (20mM sodium phosphate, 0.5M sodium chloride, PH7.2) to balance the purification column; wait for the baseline level, and start injection. After the injection is completed, use 5 column volumes of Binding buffer (20mM sodium phosphate, 0.5M sodium chloride, PH 7.2) to wash away the impurities; wait for the baseline level, and adjust the UV to zero. Pre-add a certain volume of 1M pH 8.8Tris-Hcl to the collection tube, and elute with 5 column volumes of 0.1M sodium citrate (pH 3.0), collect the sample in the collection tube, and mix it continuously. Wash the purification column with 5 column volumes of PBS, put the injection port in 20% ethanol, wait for the loop to be filled, remove the purification column, and store it at 4°C.

[0431] (5) Purified protein verification

[0432] The antibodies before and after purification were added to 5× loading buffer, and after protein denaturation at 100°C, polyacrylamide gel electrophoresis was performed and stained with Coomassie Brilliant Blue to compare the changes before and after protein purification. The experimental steps and results are as follows:

[0433] (i) Preparation of cell protein samples

[0434] Add the purified XF04-4, XF04-5, XF04-9, XF04-10, and XF04-12 antibodies to 5× Loading Buffer and vortex to mix, boil in a 100°C metal bath for 8 minutes, and store in a -20°C refrigerator after the samples have cooled.

[0435] (ii) Western blotting

[0436] Prepare separation gel and concentration gel: clean and dry the glass plate, align it tightly and clamp it on the glass plate clamp, then place it on the bracket and shake it gently to ensure it is clamped.

[0437] Prepare separation gel: Add various reagents according to the ingredient list, mix well, and slowly inject the separation gel between the two glass plates from one side with a pipette. Stop injecting after adding to the predetermined height, and use a pipette to draw 200μL of isopropanol to flatten the liquid surface. After about 30 minutes, the colloid solidifies and then pour out the isopropanol and dry it with filter paper. In the same way, inject the prepared concentrated gel, and gently insert the comb to ensure that there are no bubbles, and keep the comb horizontal, and place it at room temperature. After 20 minutes, the concentrated gel is completely solidified and the gel plate can be removed. If not used in time, wrap it with soaked absorbent paper and store it in a refrigerator at 4°C.

[0438] 10% separation gel components: 4mL ultrapure water; 3.3mL 30% acrylamide; 2.5mL 1.5M Tris HCl PH8.8; 100μL 10% SDS; 100μL 10% ammonium persulfate (AP); 10μL TEMED

[0439] 5% stacking gel components: 4.1 mL ultrapure water; 1 mL 30% acrylamide; 0.75 mL 1 M Tris HCl PH 6.8; 60 μL 10% SDS; 60 μL 10% ammonium persulfate (AP); 6 μL TEMED.

[0440] Add 20μL B7-H4 antigen sample to each well. Markers are placed on both sides. Install the electrophoresis equipment correctly, set the electrophoresis parameters, run the concentrated gel at 80V first, and when the red protein marker appears, adjust the voltage to 120V, and end when the sample reaches the target protein at the desired position.

[0441] (iii) Protein before and after purification by Coomassie Brilliant Blue staining

[0442] The polyacrylamide gel after electrophoresis was placed in Coomassie Brilliant Blue staining solution and stained on a shaker for 2 h. The stained gel was added to water and placed on a shaker overnight.

[0443] The experimental results are as follows Figure 1As shown in the figure, the protein bands before purification are more complex, and there is only one protein band after purification. Because our antibody is ScFv-Fc, after purification with protein A, only the antibody binds to protein A, so the result after purification is single, and the protein size is correct, which is the antibody we need.

[0444] Example 2: Binding of anti-B7-H4 antibodies to B7-H4 antigen in vitro

[0445] This example discloses the use of Western experiments to verify the binding of anti-B7-H4 antibodies to B7-H4 antigen in vitro.

[0446] (1) Expression of anti-B7-H4 antibodies

[0447] In this example, the expression of eleven plasmids (XF04-4, XF04-5, XF04-9, XF04-10, XF04-12, XF04-1, XF04-20, XF04-27, XF04-33, XF04-54, and XF05-56) was detected.

[0448] (i) Preparation of cell protein samples

[0449] XF04-4, XF04-5, XF04-9, XF04-10, XF04-12, XF04-1, XF04-20, XF04-27, XF04-33, XF04-54, and XF05-56 plasmids were transfected into 293T cells, and the culture supernatant was collected by centrifugation after 5 days. 100 μL of culture supernatant was added to 5× Loading Buffer, vortexed and mixed, boiled in a 100℃ metal bath for 8 minutes, and stored in a -20℃ refrigerator after the sample cooled.

[0450] (ii) Western blotting

[0451] Prepare separation gel and concentration gel: clean and dry the glass plate, align and clamp it on the glass plate clamp, then place it on the bracket and shake it gently to ensure it is clamped.

[0452] Prepare separation gel: Add various reagents according to the ingredient list, mix well, and slowly inject the separation gel between the two glass plates from one side with a pipette. Stop injecting after adding to the predetermined height, and use a pipette to draw 200μL of isopropanol to flatten the liquid surface. After about 30 minutes, the colloid solidifies and then pour out the isopropanol and dry it with filter paper. In the same way, inject the prepared concentrated gel, and gently insert the comb to ensure that there are no bubbles, and keep the comb horizontal, and place it at room temperature. After 20 minutes, the concentrated gel is completely solidified and the gel plate can be removed. If not used in time, wrap it with soaked absorbent paper and store it in a refrigerator at 4°C.

[0453] 10% separation gel components: 4mL ultrapure water; 3.3mL 30% acrylamide; 2.5mL 1.5M Tris HCl PH8.8; 100μL 10% SDS; 100μL 10% ammonium persulfate (AP); 10μL TEMED

[0454] 5% stacking gel components: 4.1 mL ultrapure water; 1 mL 30% acrylamide; 0.75 mL 1 M Tris HCl PH 6.8; 60 μL 10% SDS; 60 μL 10% ammonium persulfate (AP); 6 μL TEMED.

[0455] Add 20μL transfection supernatant to each well. Markers are placed on both sides. Install the electrophoresis equipment correctly and set the electrophoresis parameters. Run the concentrated gel at 80V first. When the red protein marker appears, adjust the voltage to 120V and end when the sample reaches the target protein at the desired position. Immobilize the protein onto the PVDF membrane. After transfer, use TBST to prepare 5% skim milk powder for blocking and incubate on a shaker at room temperature for 1 hour. Goat anti Human IgG Fc Cross-Adsorbed Secondary Antibody.HRP (invitrogen, 31413) was added to the antibody incubation box as the primary antibody and incubated on a shaker overnight (4°C). Take out the membrane treated with the primary antibody and wash it with TBST for 3×10 minutes. Use ECL luminescent solution (a solution and a 1:1 mixture of solution A and solution B) for development and collect image information.

[0456] Figure 2 A shows the expression of antibodies after plasmid transfection into 293F cells. The experimental results show that after the constructed plasmids were transfected into cells, five plasmids (XF04-4, XF04-5, XF04-9, XF04-10 and XF04-12) can express B7-H4 antibodies. However, the other six plasmids (XF04-1, XF04-20, XF04-27, XF04-33, XF04-54 and XF05-56) could not express B7-H4 antibodies after transfection into cells.

[0457] Five plasmids with higher expression levels (XF04-4, XF04-5, XF04-9, XF04-10 and XF04-12) were screened for subsequent experiments.

[0458] (2) Binding of anti-B7-H4 antibodies to B7-H4 antigen in vitro

[0459] (i) Preparation of cell protein samples

[0460] The purchased B7-H4 antigen (Acro BIOSYSTEMS, B74-H5222) was added to PBS to prepare a 10 μg / mL solution. 100 μL of B7-H4 antigen solution was added to 5× Loading Buffer and vortexed to mix well. The sample was boiled in a 100°C metal bath for 8 minutes. After the sample was cooled, it was stored in a -20°C refrigerator.

[0461] (ii) Western blotting

[0462] Prepare separation gel and concentration gel: clean and dry the glass plate, align it tightly and clamp it on the glass plate clamp, then place it on the bracket and shake it gently to ensure it is clamped.

[0463] Prepare separation gel: Add various reagents according to the ingredient list, mix well, and slowly inject the separation gel between the two glass plates from one side with a pipette. Stop injecting after adding to the predetermined height, and use a pipette to draw 200μL of isopropanol to flatten the liquid surface. After about 30 minutes, the colloid solidifies and then pour out the isopropanol and dry it with filter paper. In the same way, inject the prepared concentrated gel, and gently insert the comb to ensure that there are no bubbles, and keep the comb horizontal, and place it at room temperature. After 20 minutes, the concentrated gel is completely solidified and the gel plate can be removed. If not used in time, wrap it with soaked absorbent paper and store it in a refrigerator at 4°C.

[0464] 10% separation gel components: 4mL ultrapure water; 3.3mL 30% acrylamide; 2.5mL 1.5M Tris HCl PH8.8; 100μL 10% SDS; 100μL 10% ammonium persulfate (AP); 10μL TEMED

[0465] 5% stacking gel components: 4.1 mL ultrapure water; 1 mL 30% acrylamide; 0.75 mL 1 M Tris HCl PH 6.8; 60 μL 10% SDS; 60 μL 10% ammonium persulfate (AP); 6 μL TEMED.

[0466] Add 20μL B7-H4 antigen sample to each well. Markers are placed on both sides. Install the electrophoresis equipment correctly, set the electrophoresis parameters, run the concentrated gel at 80V first, and when the red protein marker appears, adjust the voltage to 120V, and stop when the sample reaches the target protein at the desired position. Immobilize the protein onto the PVDF membrane. After transfer, use TBST to prepare 5% skim milk powder for blocking, and incubate on a shaker at room temperature for 1 hour.

[0467] Purified B7-H4 antibodies XF04-4, XF04-5, XF04-9, XF04-10, and XF04-12 were added to the antibody incubation box as primary antibodies and incubated on a shaker overnight (4°C). The membrane treated with the primary antibody was removed and washed with TBST for 3×10 minutes. The secondary antibody was Goat anti Human IgG Fc Cross-Adsorbed Secondary Antibody.HRP (invitrogen, 31413). The secondary antibody was diluted with TBST according to the recommended dilution multiple, 1% BSA was added, and incubated on a shaker at room temperature for 1 hour. The secondary antibody was recovered and placed at 4°C for reuse. The membrane was washed with TBST for 3×10 minutes. ECL luminescent solution (A solution and B solution mixed 1:1) was used for development and image information was collected.

[0468] (iii) Coomassie blue staining of B7-H4 antigen.

[0469] The polyacrylamide gel after electrophoresis was placed in Coomassie Brilliant Blue staining solution and stained on a shaker for 2 h. The stained gel was added to water and placed on a shaker overnight.

[0470] Figure 2 B shows Coomassie Brilliant Blue staining and Western experiments to verify the binding of antibodies to B7-H4 antigen. The experimental results show that XF04-4, XF04-9 and XF04-12 can bind to B7-H4 antigen. XF04-5 and XF04-10 cannot bind to B7-H4 antigen, which is consistent with the immunofluorescence results in the subsequent Example 3.

[0471] Example 3: Inhibition of cell proliferation by anti-B7-H4 antibodies

[0472] This example uses four types of cells: 293T, DLD1, LS174T, and HT29. This example shows that LS174T and HT29 cells have a large amount of B7-H4 protein, and anti-B7-H4 antibodies have an inhibitory effect on cell proliferation.

[0473] (1) Western blotting

[0474] 20 million 293T, DLD1, LS174T, and HT29 cells were plated in large dishes and cultured for 24 hours before extracting cell membrane proteins. 5× Loading Buffer was added to 200 μL of the sample and vortexed and mixed, then boiled in a 100°C metal bath for 8 minutes. After the sample cooled, it was stored in a -20°C refrigerator.

[0475] Prepare separation gel and concentration gel: clean and dry the glass plate, align and clamp it on the glass plate clamp, then place it on the bracket and shake it gently to ensure it is clamped.

[0476] Prepare separation gel: Add various reagents according to the ingredient list, mix well, and slowly inject the separation gel between the two glass plates from one side with a pipette. Stop injecting after adding to the predetermined height, and use a pipette to draw 200μL of isopropanol to flatten the liquid surface. After about 30 minutes, the colloid solidifies and then pour out the isopropanol and dry it with filter paper. In the same way, inject the prepared concentrated gel, and gently insert the comb to ensure that there are no bubbles, and keep the comb horizontal, and place it at room temperature. After 20 minutes, the concentrated gel is completely solidified and the gel plate can be removed. If not used in time, wrap it with soaked absorbent paper and store it in a refrigerator at 4°C.

[0477] 10% separation gel components: 4mL ultrapure water; 3.3mL 30% acrylamide; 2.5mL 1.5M Tris HCl PH8.8; 100μL 10% SDS; 100μL 10% ammonium persulfate (AP); 10μL TEMED

[0478] 5% stacking gel components: 4.1 mL ultrapure water; 1 mL 30% acrylamide; 0.75 mL 1 M Tris HCl PH 6.8; 60 μL 10% SDS; 60 μL 10% ammonium persulfate (AP); 6 μL TEMED.

[0479] Add 20μL protein sample to each well. Markers are placed on both sides. Install the electrophoresis equipment correctly, set the electrophoresis parameters, run the concentrated gel at 80V first, and when the red protein marker appears, adjust the voltage to 120V, and end when the sample reaches the target protein at the desired position.

[0480] Transfer the protein to the PVDF membrane. Remove the membrane from the device, put it in 5% skim milk powder prepared with TBST, and incubate it on a shaker at room temperature for 1 hour. Rabbit Anti-B7H4 Polyclonal Antibody (ZIKER, ZIKER-0673R) was added to the antibody incubation box as the primary antibody and incubated on a shaker at 4°C overnight. Take out the membrane treated with the primary antibody and wash it with TBST for 3×10 minutes. The secondary antibody is Goat Anti-rabbit IgG H&L / HRP (Bioss, bs-0295G-HRP). Dilute the secondary antibody with TBST according to the recommended dilution multiple, add 1% BSA, and incubate it on a shaker at room temperature for 1 hour. Recover the secondary antibody and place it at 4°C, and it can be used again. Wash the membrane with TBST for 3×10 minutes. Prepare ECL luminescent solution (mix A solution and B solution 1:1) for development and collect image information.

[0481] Figure 3 A and B show the results of western blotting. Figure 3A is a Western experiment to detect the expression of B7-H4 protein on the cell membrane of 293T, DLD1, HT29, and LS174T cells. Figure 3 B. Statistical methods were used to quantitatively analyze the expression of B7-H4 protein on the cell membrane of 293T, DLD1, HT29, and LS174T cells.

[0482] Figure 3 A and B show that in Western experiments, there is no B7-H4 protein on the surface of 293T cells, and there is a very small amount of B7-H4 protein on the surface of DLD1 cells. However, there are a large amount of B7-H4 protein on the surface of LS174T and HT29 cells.

[0483] (2) Cell inhibition experiment

[0484] 293T and colon cancer cell lines LS174T, HT29 and DLD1 were selected as research objects, and the killing effect was detected by CCK-8 method. The specific methods include:

[0485] The cells were seeded in 24-well plates at a cell seeding volume of 1×10 4 / well, with 3 replicates per group. After the cells grow to the logarithmic growth phase, fresh medium is replaced. Each well is treated with 1) 10 μg / mL anti-B7-H4 antibody, 2) no antibody but only medium as a blank control, or 3) 10 μg / mL IgG antibody as an antibody IgG control.

[0486] Incubate in a 37°C cell culture incubator for 72 hours. Add CCK-8 (ZETA, USA) reagent (250 μL / well) to each well, incubate in a 37°C cell culture incubator for 30 minutes, and measure the absorbance at 450 nm. Count the cell viability according to the instructions of the CCK-8 kit (ZETA, USA).

[0487] Figure 3 CF shows the cell proliferation of four cell lines, 293T, LS174T, HT29 and DLD1, detected by CCK8 experiment after treatment with anti-B7-H4 antibodies (XF04-4, XF04-5, XF04-9, XF04-10, XF04-12), blank control with only culture medium added (control) and IgG antibody treatment.

[0488] Figure 3D and E show that the anti-B7-H4 antibody XF04-4 has an inhibitory effect on the proliferation of LS174T and HT29 cells, but the anti-B7-H4 antibodies XF04-5, XF04-9, XF04-10 and XF04-12 have no inhibitory effect on the proliferation of LS174T and HT29 cells. The reason why XF04-5 and XF04-10 cannot inhibit the proliferation of LS174T and HT29 cells may be that XF04-5 and XF04-10 cannot bind to the B7-H4 protein and have no immunological activity. The reason why XF04-9 and XF04-12 cannot inhibit the proliferation of LS174T and HT29 cells is that they have no biological activity.

[0489] Figure 3 C and F show that anti-B7-H4 antibodies (XF04-4, XF04-5, XF04-9, XF04-10, XF04-12) had no inhibitory effect on the proliferation of 293T and DLD1. This result is consistent with the Western experiment.

[0490] The results of this example show that LS174T and HT29 cells have a large amount of B7-H4 protein, and the B7-H4 antibody XF04-4 can inhibit the proliferation of LS174T and HT29 cells, that is, it is a neutralizing antibody, but has no effect on the proliferation of 293T and DLD1 cells.

[0491] Example 4: Binding of anti-B7-H4 antibodies to cell surface antigens

[0492] This example discloses immunofluorescence experiments to verify the binding of anti-B7-H4 antibodies to B7-H4 antigens on the surfaces of 293T, LS174T, HT29 and DLD1 cells.

[0493] (1) XF04-4, XF04-9, XF04-12 and LS174T cell membrane binding

[0494] In this example, LS174T cells were used to test the binding of five antibodies, XF04-4, XF04-5, XF04-9, XF04-10, and XF04-12, to the LS174T cell membrane.

[0495] Soak a 13 mm cover glass in alcohol, air dry and place in a 24-well plate. 5 LS174T cells (ATCC, Manassas, VA, USA) were treated with Fc receptor blocker (FXP064-050, Sizhengbai Biotechnology) (5 μL blocker added to 100 μL cell suspension) for 10 min, resuspended in 1640 medium, and plated in a 24-well plate with a slide, with 2 × 10 cells per well. 5Cells were cultured in a 5% CO2 cell culture incubator for 24 hours. The culture medium was discarded the next day. Washed 3 times with 1×PBS. Use 1% paraformaldehyde fixative to fix, 300μL per well for 30min, and air-dried for 5min. Centrifuge, remove 1% paraformaldehyde fixative, and wash 3 times with PBS. Use 10% normal goat serum for serum incubation, 300μL per well, and block at room temperature for 30min. Then, different treatments were used with PBS, IgG antibody (Beyotime, A7001), B7-H4 antibody Rabbit Anti-B7H4 Polyclonal Antibody (ZIKER, ZIKER-0673R) and five purified antibodies (XF04-4, XF04-5, XF04-9, XF04-10, XF04-12) as primary antibodies, 150mL per well, 4℃ overnight. Return to temperature for 30min the next day, recover primary antibodies, and wash 3 times with PBS.

[0496] Dilute (dilution ratio 1:200000) Goat Anti-Human IgG Fc Antibody, Fluorescein (FITC) Conjugate, affinity purified (invitrogen, A18818) and goat anti-rabbit fluorescent secondary antibody (Bioss, bs-0295G-AF488), 150mL per well, room temperature and dark for 1h, wash 3 times with PBS. Add DAPI, 100μL per well, room temperature for 10min for nuclear staining. Then drop a drop of sealing agent on the coverslip. Use high-content imaging system (Molecular Devices, Qpix420) for observation.

[0497] Figure 4 The results showed that XF04-4, XF04-9 and XF04-12 could bind to the LS174T cell membrane and had immune activity, while XF04-5 and XF04-10 could not bind to the LS174T cell membrane.

[0498] (2) Anti-B7-H4 antibody XF04-4 binds to the cell membrane of XF04-4 and LS174T

[0499] The results of Example 3 show that the anti-B7-H4 antibody XF04-4 can inhibit the proliferation of LS174T and HT29 cells. This example tests the binding of the anti-B7-H4 antibody XF04-4 to the cell membranes of XF04-4 and LS174T.

[0500] Soak a 13 mm cover glass in alcohol, air dry and place in a 24-well plate. 5LS174T, HT29, DLD1, and 293T cells (ATCC, Manassas, VA, USA) were treated with Fc receptor blockers (FXP064-050, Sizhengbai Biotechnology) (5 μL of blocker added to 100 μL of cell suspension) for 10 min, resuspended in 1640 medium, and plated in 24-well plates with slides, with 2 × 10 cells per well. 5 Cells were cultured in a 5% CO2 cell culture incubator for 24 hours. The culture medium was discarded the next day. Washed 3 times with 1×PBS. Use 1% paraformaldehyde fixative to fix, 300μL per well for 30min, and air-dried for 5min. Centrifuge, remove 1% paraformaldehyde fixative, and wash 3 times with PBS. Use 10% normal goat serum for serum incubation, 300μL per well, and block at room temperature for 30min. Then, different treatments were used with PBS, IgG antibody (Beyotime, A7001), B7-H4 antibody Rabbit Anti-B7H4Polyclonal Antibody (ZIKER, ZIKER-0673R) and purified XF04-4 antibody as primary antibodies, 150mL per well, overnight at 4℃. Return to temperature for 30min the next day, recover primary antibody, and wash 3 times with PBS.

[0501] Dilute (dilution ratio 1:200000) Goat Anti-Human IgG Fc Antibody, Fluorescein (FITC) Conjugate, affinity purified (invitrogen, A18818) and goat anti-rabbit fluorescent secondary antibody (Bioss, bs-0295G-AF488), 150mL per well, room temperature and dark for 1h, wash 3 times with PBS. Add DAPI, 100μL per well, room temperature for 10min for nuclear staining. Then drop a drop of sealing agent on the coverslip. Use high-content imaging system (Molecular Devices, Qpix420) for observation.

[0502] Figure 5 The immunofluorescence assays to verify the antibody and 293T ( Figure 5 A), LS174T( Figure 5 B), HT29( Figure 5 C), DLD1( Figure 5 D) Cell surface antigen binding.

[0503] Figure 5The results showed that the B7-H4 fluorescence on the cell membrane surface of colon cancer cells LS174T and HT29 was high, indicating that the B7-H4 protein content on the cell membrane surface of LS174T and HT29 was high. As controls, 293T cells and DLD1 cells did not have B7-H4 protein on their cell membrane surfaces, so they could not bind to the B7-H4 protein antibody XF04-4. The lack of fluorescence on the B7-H4 on the cell membrane surface of 293T cells and DLD1 cells also proved the reason why the B7-H4 antibody XF04-4 could not inhibit the proliferation of 293T and DLD1 cells.

[0504] B7-H4 antibody XF04-4 can bind to B7-H4 protein on the cell membrane surface of colon cancer cells LS174T and HT29, but cannot bind to the cell membrane surface of 293T cells and DLD1 cells.

[0505] The results of Example 2, the binding of the anti-B7-H4 antibody to the B7-H4 antigen in vitro, and Example 4, the binding of the anti-B7-H4 antibody to the cell surface antigen, indicate that the anti-B7-H4 antibody XF04-4 can bind to both the B7-H4 protein in vitro and the cell membranes of colon cancer cells LS174T and HT29, thereby demonstrating that the anti-B7-H4 antibody XF04-4 has immunological activity.

[0506] Example 5: Six epitopes bound to VL and VH of B7-H4 antibody XF04-4

[0507] In this example, molecular docking was used to find out six epitopes that bind to VL and VH of the B7-H4 antibody XF04-4.

[0508] In order to predict possible epitopes on the surface of B7-H4 protein, the protein structure of anti-B7-H4 antibody XF04-4 was constructed by molecular modeling, and then the binding model of anti-B7-H4 antibody XF04-4 and B7-H4 protein was predicted by molecular docking.

[0509] The pymol software was used to predict the six antigenic epitopes that the anti-B7-H4 antibody XF04-4 binds to the B7-H4 protein, which are the six polypeptide chains EGVL, QLTDAGTY, SEQDEMFRGRT, HSITVTTVA, IVGN, and SCTFEPDI.

[0510] Figure 6 The results of docking between the VL and VH of B7-H4 antibody XF04-4 and B7-H4 molecule are shown in Figure 1. Figure A is the binding model of the docking results between the VL region of B7-H4 antibody XF04-4 and B7-H4 molecule; Figure B is the binding model of the docking results between the VH region of B7-H4 antibody XF04-4 and B7-H4 molecule. Figure C is the analysis of the molecular docking results.

[0511] Figure 6 The results show that the six antigenic epitopes that the anti-B7-H4 antibody XF04-4 binds to the B7-H4 protein are the six polypeptide chains EGVL, QLTDAGTY, SEQDEMFRGRT, HSITVTTVA, IVGN, and SCTFEPDI.

[0512] Example 6: Binding of anti-B7-H4 antibodies to B7-H4 epitopes

[0513] This example tests the binding of anti-B7-H4 antibodies to the B7-H4 epitope.

[0514] In order to further verify whether the anti-B7-H4 antibody XF04-4 binds to the predicted epitope, a competition experiment between the anti-B7-H4 antibody and the predicted epitope was performed using ELISA to detect the binding of the anti-B7-H4 antibody XF04-4 to the six polypeptide chains of the predicted epitope.

[0515] Dissolve B7-H4 antigen (Acro BIOSYSTEMS, B74-H5222) in PBS at an antigen concentration of 10μg / mL. Add the dissolved antigen to a 96-well plate and place in a 4°C refrigerator overnight. Take it out the next day and recover the antigen. Wash three times with PBST (0.05% Tween). Add 100μL of 2% MPBS (2% milk) and culture in the greenhouse for 1h. Remove MPBS and wash 5 times with PBST. Mix the peptides with concentrations of 0.000001μg / mL, 0.00001μg / mL, 0.01μg / mL, 0.1μg / mL, 1μg / mL, and 10μg / mL with anti-B7-H4 antibody (10μg / mL), add 100μL to each well, and incubate at 4°C overnight. Aspirate the mixture the next day and wash 5 times with PBST. Add 100 μL of Goat anti Human IgG Fc Cross-Adsorbed Secondary Antibody.HRP (Invitrogen, 31413) and incubate on a shaker at room temperature for 1 hour.

[0516] Remove the secondary antibody, wash 5 times with PBST, add 50 μL of TMB colorimetric solution, incubate at 37°C in the dark for 30 min, terminate the reaction with 1M H2SO4, and read the OD450 using a microplate reader.

[0517] Figure 7 The binding of the anti-B7-H4 antibody XF04-4 to six polypeptide chains of the predicted epitope is shown in FIG.

[0518] Figure 7 Figures AF show the competitive binding experiment between the predicted epitope peptide and the XF04-4 antibody, wherein the abscissa is the concentration of the peptide used. Figure 7A is the epitope polypeptide EGVL, Figure 7 B is the epitope polypeptide QLTDAGTY, Figure 7 C is the epitope polypeptide SEQDEMFRGRT, Figure 7 D is the epitope peptide SCTFEPDI, Figure 7 E is the epitope peptide HSITVTTVA, Figure 7 F is the epitope polypeptide IVGN.

[0519] Figure 7 Figures AB and DF show that these five peptides cannot compete with B7-H4 protein for binding to B7-H4 antibody XF04-4.

[0520] Figure 7 C As the concentration of the polypeptide increases, the binding of the polypeptide to the B7-H4 antibody XF04-4 increases, thereby inhibiting the binding of the B7-H4 antibody XF04-4 to the B7-H4 protein, and the results present an S curve.

[0521] Figure 7 C The results prove that SEQDEMFRGRT may be the binding epitope of B7-H4 antibody XF04-4 and B7-H4 protein.

[0522] B7-H4 is a member of the B7 family of immunomodulatory ligands that negatively regulates T cell function; it contains an IgV domain and an IgC domain. The B7-H4 epitope SEQDEMFRGRT is located in the B7-H4 IgV domain. The B7-H4 IgV domain is located outside the cell, and binding to an unknown receptor can inhibit TCR-mediated T cell proliferation, cell cycle progression, and IL-2 production.

[0523] Example 7: Affinity of anti-B7-H4 antibodies

[0524] This example detects the affinity of the anti-B7-H4 antibody XF04-4 to human B7-H4.

[0525] This experiment uses a molecular interaction instrument (SARTORIUS, OCTETR2) for detection. The detection steps are as follows:

[0526] (1) B7-H4 antibody XF04-4 and B7-H4 antigen were diluted to 10 μg / mL with PBST.

[0527] (2) Add PBST, B7-H4 antibody XF04-4, PBST, and 200 μL of B7-H4 antigen into a 96-well plate.

[0528] (3) The sensor is a protein A sensor. The sensor and B7-H4 antigen are placed in a molecular interaction instrument for detection.

[0529] (4) The results were analyzed using Octet Analysis Studio 12.2 software.

[0530] The data results of the detection of the affinity between the anti-B7-H4 antibody XF04-4 and human B7-H4 in this example are shown in the following table:

[0531] Anti-B7-H4 Antibody Affinity (M) Binding constant (1 / Ms) Dissociation constant (1 / s) XF04-4 3.06E-08 1.63E+05 4.97E-03

[0532] This affinity constant is relatively high for the B7-H4 antibody XF04-4, demonstrating that the B7-H4 antibody XF04-4 has a good affinity.

[0533] In the art, antibody affinity values ​​are between 10 -8 to 10 -10 This shows that the antibody has a good affinity.

[0534] This example also detects the affinity value of XF04-12, and the affinity of XF04-4 is better than that of XF04-12.

[0535] The results of XF04-12 are shown in the figure below. The affinity coefficient of B7-H4 antibody XF04-4 is smaller than that of XF04-12, indicating that XF04-4 has better affinity than XF04-12.

[0536] Anti-B7-H4 Antibody Affinity (M) Binding constant (1 / Ms) Dissociation constant (1 / s) XF04-12 1.49E-07 3.38E+06 5.04E-01

[0537] Example 8: IC50, EC50 of B7-H4 Antibody Inhibition

[0538] This example examines the IC50 and EC50 of B7-H4 antibody inhibition.

[0539] (1) IC50

[0540] This example detected the competitive blocking experiment between B7-H4 antibody XF04-4 and the predicted antigen epitope VCAM-1 protein. GraphPad Prism 9.3.0 was used to analyze the data and draw the curve. The IC50 of the B7-H4 antibody competition inhibition experiment was calculated by this software to be 0.5520 μg / mL.

[0541] The ligand of B7-H4 has not yet been discovered. Researchers have identified some molecules that may be ligands of B7-H4. For example, molecules such as ICOSL, VCAM-1, NOTCH-1, NOTCH-2, and STEAP4 have all been shown to interact with B7-H4.

[0542] In this example, VCAM-1 was selected as the target protein in the competitive blocking experiment between B7-H4 antibody XF04-4 and B7-H4 ligand.

[0543] First, fix the antigen VCAM-1. Dissolve the VCAM-1 antigen (Acro BIOSYSTEMS, VC1-H5224) in PBS to a concentration of 10 μg / mL. Add the dissolved antigen to a 96-well plate and place it in a 4°C refrigerator overnight.

[0544] Take out the next day and recover the antigen. Wash three times with PBST (0.05% Tween). Add 100μL of 2% MPBS (2% milk) and culture in the greenhouse for 1h. Remove MPBS and wash 5 times with PBST. Use anti-B7-H4 antibody XF04-4 with concentrations of 0μg / mL, 0.0001μg / mL, 0.001μg / mL, 0.01μg / mL, 0.1μg / mL, 1μg / mL, and 10μg / mL, mix with B7-H4 antigen (10μg / mL), add 100μL to each well, and incubate overnight at 4°C.

[0545] The next day, aspirate the mixture and wash 5 times with PBST. Add 100μL of Rabbit Anti-B7H4 PolyclonalAntibody (ZIKER, ZIKER-0673R) and incubate at room temperature on a shaker for 1h. Remove the B7-H4 antibody and wash 5 times with PBST. Add 100μL of secondary antibody Goat Anti-rabbit IgG H&L / HRP (Bioss, bs-0295G-HRP) and incubate at room temperature on a shaker for 1h. Remove the secondary antibody and wash 5 times with PBST. Add 50μL of TMB colorimetric solution. Incubate at 37℃ in the dark for 30min. Terminate the reaction with 1M H2SO4. Read the OD450 of the plate using an enzyme reader.

[0546] Figure 8 A shows a competition blocking experiment, where the abscissa represents the concentration of B7-H4 protein. Figure 8 The experimental results of A showed that as the concentration of B7-H4 antibody XF04-4 increased, the binding of B7-H4 protein to VCAM-1 protein decreased.

[0547] This indicates that the B7-H4 antibody XF04-4 can compete with VCAM-1 for binding to the B7-H4 protein, thereby blocking the binding of VCAM-1 to the B7-H4 protein. GraphPad Prism 9.3.0 was used to analyze the data and draw the curve. This software was used to analyze the results and the IC50 of the competitive binding was calculated to be 1.384 μg / mL.

[0548] There is no literature report of a B7-H4 antibody that can bind to this epitope. This application first discovered this epitope. The IC50 value of the B7-H4 antibody XF04-4 is low, proving that the predicted epitope has a strong binding affinity with XF04-4 and can effectively prevent the binding of B7-H4 protein to the XF04-4 antibody.

[0549] (2)EC50

[0550] The experimental steps and results of EC50 of B7-H4 antibody XF04-4 binding to antigen are as follows:

[0551] Dissolve B7-H4 antigen (Acro BIOSYSTEMS, B74-H5222) in PBS at an antigen concentration of 10 μg / mL. Add the dissolved antigen to a 96-well plate and place in a 4°C refrigerator overnight. Take it out the next day and recover the antigen. Wash three times with PBST (0.05% Tween). Add 100 μL of 2% MPBS (2% milk) and culture in the greenhouse for 1 hour. Remove MPBS and wash 5 times with PBST. Add 100 μL of anti-B7-H4 antibody XF04-4 with concentrations of 0.000001 μg / mL, 0.00001 μg / mL, 0.0001 μg / mL, 0.001 μg / mL, 0.01 μg / mL, 0.1 μg / mL, and 1 μg / mL to each well and incubate overnight at 4°C. Aspirate the antibody the next day and wash 5 times with PBST. Add 100 μL of Goat anti Human IgG Fc Cross-Adsorbed Secondary Antibody.HRP (invitrogen, 31413) and incubate on a shaker at room temperature for 1 hour.

[0552] Remove the secondary antibody, wash 5 times with PBST, add 50 μL of TMB colorimetric solution, incubate at 37°C in the dark for 30 min, terminate the reaction with 1M H2SO4, and read the OD450 using a microplate reader.

[0553] GraphPad Prism 9.3.0 was used to analyze the data and draw the curve. The EC50 of the binding of B7-H4 antibody XF04-4 to B7-H4 antigen was calculated to be 14.75 ng / mL.

[0554] The EC50 value of B7-H4 antibody XF04-4 is small, which proves that B7-H4 antibody XF04-4 has good affinity to B7-H4 antigen.

[0555] The EC50 value of the B7-H4 antibody CH17 reported in the literature (Development of a novel anti-B7-H4 antibody enhances anti-tumor immune response of human T cells. Biomedicine & Pharmacotherapy 141 (2021) 111913) is 13.94 ng / mL, and this antibody has good affinity. The EC50 of the antibody in this example is 14.75 ng / mL, which is close to the value of the control, so it shows that the antibody has good affinity.

[0556] Example 9: Experiment on the inhibitory activity of peptide against XF04-4 antibody on cells

[0557] This example tests the inhibitory activity of polypeptide against XF04-4 antibody on cells. LS174T, HT29 and DLD1 cells are selected in this example.

[0558] LS174T, HT29, and DLD1 cells were seeded in 24-well plates at a cell seeding volume of 1×10 4 / well, with 3 replicates per group. After the cells grow to the logarithmic growth phase, fresh medium is replaced. 10 μg / mL B7-H4 antibody XF04-4 is incubated with 10 μg / mL epitope SEQDEMFRGRT polypeptide chain and then added to the cells.

[0559] 1) 10 μg / mL of anti-B7-H4 antibody XF04-4 was added to each well, 2) no antibody was added as a blank control, or 3) 10 μg / mL IgG antibody was added as an antibody IgG control. Incubate in a 37°C cell culture incubator for 72 hours. CCK-8 (ZETALIFE, K009) reagent (250 μL / well) was added to each well, incubated in a 37°C cell culture incubator for 30 minutes, and the absorbance was measured at 450 nm. According to the instructions of the CCK-8 kit (ZETA, USA), the cell viability was counted.

[0560] Fig. 9 The results of the experiment of the polypeptide of this example antagonizing the cell inhibitory activity of XF04-4 antibody are shown. Fig. 9 The results of A and B show that for LS174T cells and HT29 cells, the blank control and IgG antibody control had no effect on cell survival. 10μg / mL B7-H4 antibody XF04-4 treatment of LS174T and HT29 cells caused a significant decrease in cell survival. After 10μg / mL polypeptide chain SEQDEMFRGRT was incubated with 10μg / mL B7-H4 antibody XF04-4, the cell survival rate of LS174T and HT29 cells was significantly increased.

[0561] Fig. 9 C shows that for DLD1 cells, after incubation with the blank control, IgG antibody control, 10 μg / mL B7-H4 antibody XF04-4, and 10 μg / mL polypeptide chain SEQDEMFRGRT and 10 μg / mL B7-H4 antibody XF04-4, there was no change in the survival rate of DLD1 cells.

[0562] The results of this example show that the B7-H4 antibody XF04-4 has an inhibitory effect on LS174T and HT29 cells, and the polypeptide chain SEQDEMFRGRT can antagonize the inhibitory effect of the B7-H4 antibody XF04-4 on LS174T and HT29 cells.

[0563] The results of this example further demonstrate that the binding epitope of the anti-B7-H4 antibody to B7-H4 is SEQDEMFRGRT.

[0564] Example 10: Anti-B7-H4 Antibody Non-Specific Binding Assay

[0565] This example detects the binding specificity of the anti-B7-H4 antibody, demonstrating that the anti-B7-H4 antibody does not bind to the TIM3 protein.

[0566] TIM3 protein is also expressed in tumor cells and is one of the most studied immunotherapy targets. TIM3 consists of three parts: the extracellular region, the transmembrane region, and the intracellular region. Its extracellular region also has an IgV region, which is similar to the B7-H4 protein. TIM3 protein was selected for nonspecific testing.

[0567] Dissolve TIM3 antigen (Acro BIOSYSTEMS, TIM3-H5229) and B7-H4 antigen (Acro BIOSYSTEMS, B74-H5222) in PBS at an antigen concentration of 10 μg / mL. Add the dissolved antigen to a 96-well plate and place in a 4°C refrigerator overnight. PBS is used as the antigen group as a control. Take it out the next day and recover the antigen. Wash three times with PBST (0.05% Tween). Add 100 μL of 2% MPBS (2% milk) and incubate in the room for 1 hour. Then remove MPBS and wash 5 times with PBST. Add 100 μL of B7-H4 antibody XF04-4 (10 μg / mL) to each well and incubate at 4°C overnight. Aspirate the mixture the next day and wash 5 times with PBST. Add 100 μL of FC secondary antibody (Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody.HRP) (invitrogen, 31413). Shake at room temperature for 1 hour. Remove the secondary antibody, wash 5 times with PBST, and add 50 μL of TMB colorimetric solution. Incubate at 37°C in the dark for 30 minutes. Terminate the reaction with 1M H2SO4. Read the OD450 using a microplate reader.

[0568] Fig.10 The figure shows that the B7-H4 antibody XF04-4 can specifically bind to the B7-H4 antigen, but not to TIM3 and PBS control. ** in the figure indicates statistical significance, P = 0.001.

[0569] The results of this example show that the B7-H4 antibody XF04-4 does not bind to the TIM3 protein, while XF04-4 has a high binding specificity to the B7-H4 antigen.

[0570] Example 11: Anti-B7-H4 antibody inhibits phosphorylation of JAK2 protein

[0571] This example discloses that the phosphorylation of JAK2 protein can be inhibited by using anti-B7-H4 antibody. In this example, DLD1, LS174T and HT29 cells were selected.

[0572] After treating LS174T, HT29 and DLD1 cells with 10ug / mL anti-B7-H4 antibody XF04-4, Western blot was used to detect the phosphorylation of JAK2 protein in the cells.

[0573] (1) Preparation of cell protein samples

[0574] Take LS174T, HT29, and DLD1 cells in the logarithmic growth phase, digest them with trypsin for 1-2 minutes, adjust the cell concentration, and inoculate the cells in a 6-well plate with 1×10 cells per well. 6 The cells were cultured in a 5% CO2 cell culture incubator for 24 hours.

[0575] The cells were 1) treated with 10 μg / mL B7-H4 antibody XF04-4; 2) treated with only medium as a blank control, or 3) treated with 10 μg / mL IgG as an antibody IgG control, and cultured in a 5% CO2 cell culture incubator for 72 hours.

[0576] Take out the treated cells from the cell room, carefully remove the culture medium with a pipette, put them into pre-cooled PBS and shake gently to wash, repeat twice to ensure that there is no culture medium residue.

[0577] (2) Western blotting

[0578] Prepare separation gel and concentration gel: clean and dry the glass plate, align and clamp it on the glass plate clamp, then place it on the bracket and shake it gently to ensure it is clamped.

[0579] Prepare separation gel: Add various reagents according to the ingredient list, mix well, and slowly inject the separation gel between the two glass plates from one side with a pipette. Stop injecting after adding to the predetermined height, and use a pipette to draw 200μL of isopropanol to flatten the liquid surface. After about 30 minutes, the colloid solidifies and then pour out the isopropanol and dry it with filter paper. In the same way, inject the prepared concentrated gel, and gently insert the comb to ensure that there are no bubbles, and keep the comb horizontal, and place it at room temperature. After 20 minutes, the concentrated gel is completely solidified and the gel plate can be removed. If not used in time, wrap it with soaked absorbent paper and store it in a refrigerator at 4°C.

[0580] 10% separation gel components: 4mL ultrapure water; 3.3mL 30% acrylamide; 2.5mL 1.5M Tris HCl PH8.8; 100μL 10% SDS; 100μL 10% ammonium persulfate (AP); 10μL TEMED

[0581] 5% stacking gel components: 4.1 mL ultrapure water; 1 mL 30% acrylamide; 0.75 mL 1 M Tris HCl PH 6.8; 60 μL 10% SDS; 60 μL 10% ammonium persulfate (AP); 6 μL TEMED.

[0582] Add 20μL protein sample to each well. Markers are placed on both sides. Install the electrophoresis equipment correctly, set the electrophoresis parameters, run the concentrated gel at 80V first, and when the red protein marker appears, adjust the voltage to 120V, and end when the sample reaches the target protein at the desired position.

[0583] Transfer the protein to the PVDF membrane. Remove the membrane from the device and place it in 5% skim milk powder prepared with TBST, and incubate it on a shaker at room temperature for 1 hour. Add the primary antibodies JAK2 Rabbit Monoclonal Antibody (Beyotime, AF1489), Phospho-JAK2 (Tyr1007 / 1008) Rabbit Monoclonal Antibody (Beyotime, AF1486), STAT3 Rabbit Monoclonal Antibody (Beyotime, AF1492), and Phospho-STAT3 (Tyr705) Rabbit Polyclonal Antibody (Beyotime, AF5941) to the antibody incubation box and incubate it on a shaker at 4°C overnight. Remove the membrane treated with the primary antibody and wash it with TBST for 3×10 minutes. The secondary antibody is Goat Anti-rabbit IgG H&L / HRP (Bioss, bs-0295G-HRP). Dilute the secondary antibody with TBST according to the recommended dilution multiple, add 1% BSA, and incubate on a shaker at room temperature for 1 hour. Recover the secondary antibody and place it at 4°C before reuse. Wash the membrane with TBST for 3×10 minutes. Prepare ECL luminescent solution (mix A solution and B solution 1:1) for development and collect image information.

[0584] Fig.11 The figure shows the phosphorylation of JAK2 protein in HT29 and LS174T cells after treatment with 10ug / mL anti-B7-H4 antibody XF04-4. Fig.11 Actin is the control. B, D, and F are grayscale statistics of the western of A, C, and E, i.e., quantification.

[0585] Fig.11 Figures A and C show that after treatment with B7-H4 antibody XF04-4, compared with the blank control and IgG treatment control, the total JAK2 and STAT3 proteins in LS174T and HT29 cells did not change, but the phosphorylated JAK2 (p-JAK2) and STAT3 (p-STAT3) proteins decreased.

[0586] Fig.11 The results of A and C indicate that the B7-H4 antibody XF04-4 can inhibit the phosphorylation of JAK2 and STAT3 proteins in LS174T and HT29 cells, thereby inhibiting the proliferation of LS174T and HT29 cells.

[0587] Fig.11Figure E shows that after treatment with B7-H4 antibody XF04-4, there was no change in the total JAK2 and STAT3 proteins in DLD1 cells compared with the blank control and IgG treatment control, and there was no change in the phosphorylated JAK2 (p-JAK2) and STAT3 (p-STAT3) proteins.

[0588] Fig.11 E shows that B7-H4 antibody XF04-4 cannot inhibit the phosphorylation of JAK2 and STAT3 proteins in DLD1 cells. Because the content of B7-H4 protein on the surface of DLD1 cells is very low, B7-H4 antibody XF04-4 has no effect on DLD1 cells. The results of DLD1 cells are shown in the figure below.

[0589] The results of this example show that the anti-B7-H4 antibody XF04-4 can inhibit the phosphorylation of JAK2 protein in HT29 and LS174T cells after treating HT29 and LS174T cells.

[0590] Example 12: Inhibition of cell proliferation by anti-B7-H4 antibodies at different concentrations

[0591] This example discloses the inhibition of cell proliferation by anti-B7-H4 antibodies. In this example, colon cancer cell lines LS174T, HT29 and DLD1 were selected as research subjects, and the concentrations of anti-B7-H4 antibody XF04-4 were 0.1 μg / mL, 1 μg / mL and 10 μg / mL, respectively.

[0592] LS174T, HT29 and DLD1 cells were seeded in 24-well plates at a cell seeding density of 1×10 4 / well, with 3 replicate wells in each group. After the cells grow to the logarithmic growth phase, fresh medium is replaced. (1) 10 μg / mL anti-B7-H4 antibody, (2) no antibody as a blank control (control), and (3) 10 μg / mL IgG antibody as an antibody IgG control are added to the 3 replicate wells.

[0593] Incubate at 37°C for 72 hours. Add CCK-8 (ZETA LIFE, K009) reagent (250 μL / well) to each well, incubate at 37°C for 30 minutes, and measure the absorbance at 450 nm. Count the cell viability according to the instructions of the CCK8 kit (ZETA, USA).

[0594] Fig.12 AC shows the results of inhibition of cell proliferation by different concentrations of anti-B7-H4 antibody. Fig.12 A shows that for DLD1 cells, the anti-B7-H4 antibody XF04-4 at three concentrations of 0.1 μg / mL, 1 μg / mL, and 10 μg / mL had no inhibitory effect on cell proliferation.

[0595] Fig.12 B and C show that the blank control (control) and antibody IgG control have no inhibitory effect on the proliferation of LS174T and HT29 cells, while the anti-B7-H4 antibody XF04-4 has an inhibitory effect on the proliferation of LS174T and HT29 cells, and as the antibody concentration increases, the inhibition of the proliferation of HT29 and LS174T cells by the B7-H4 antibody XF04-4 increases. The 10ug / mL anti-B7-H4 antibody XF04-4 has the strongest inhibition on the cell proliferation of HT29 and LS174T cells.

[0596] The results of this example show that the inhibitory effect of B7-H4 antibody XF04-4 on HT29 and LS174T cells has a dose-dependent effect.

[0597] Example 13: Effect of anti-B7-H4 antibodies on cell cycle

[0598] This example detects the effect of anti-XF04-4 antibody on cell cycle. LS174T, HT29, and DLD1 cells were selected in this example.

[0599] 3×10 6 LS174T, HT29, and DLD1 cells were plated in 6-well plates, with 1×10 cells per well. 6 cells.

[0600] Add 1) 10μg / mL of anti-B7-H4 antibody XF04-4 to each well, 2) no antibody as a blank control, or 3) add 10μg / mL IgG antibody as an antibody IgG control. Antibody treatment for 3 days. Collect cells, centrifuge and discard supernatant. Wash once with PBS, centrifuge and discard supernatant. Add 400μL DNA Staining Solution (Lianke Bio, 70-CCS012) and 4μLPermeabilization solution (Lianke Bio, 70-CCS012), vortex for 5-10 seconds to mix. Incubate at room temperature in the dark for 30 minutes. Select the lowest loading speed and detect on the flow cytometer.

[0601] Fig.13 LS174T( Fig.13 A), HT29( Fig.13 B), DLD1( Fig.13 C) Cell cycle analysis of cells showing the effect of XF04-4 antibody on LS174T ( Fig.13 A), HT29( Fig.13 B), DLD1( Fig.13 C) Role of the cell cycle.

[0602] and Fig.13Compared with DLD1T cells in C, after treatment with 10 μg / mL XF04-4 antibody, Fig.13 A shows the LS174T and Fig.13 In HT29 cells shown in B, the S phase of the cell cycle increases.

[0603] The results of this example demonstrate that XF04-4 antibody can arrest the cell cycle of LS174 and HT29 cells at the S phase, but has no effect on the cell cycle of DLD1 cells.

[0604] The embodiments disclosed in the present invention are only for detailed description of the present invention, but are not sufficient to limit the present invention. A person skilled in the art may replace the preparations described herein with obvious similar substitutes or modifications, or replace the preparations with certain chemically or biologically structurally related preparations, or make changes to the relevant contents of the present invention, but they shall not exceed the spirit, scope and idea of ​​the present invention, and shall fall within the scope of protection claimed by the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that binds to B7-H4, the antibody or antigen-binding fragment thereof comprising: 1) a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and 2) a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence; The selected VH CDR 1, 2 and 3 amino acid sequences are shown by SEQ ID NOs: 3, 4 and 5, respectively, and the selected VL CDR 1, 2 and 3 amino acid sequences are shown by SEQ ID NOs: 6, 7 and 8, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDRs 1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 3, 4, and 5, respectively, and the VL comprises CDRs 1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 6, 7, and 8, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the amino acid sequence comprised by the antibody or antigen-binding fragment is at least 80% identical to the amino acid sequence of SEQ ID NO:

2.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment specifically binds to human B7-H4.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment inhibits B7-H4; and the antibody or antigen-binding fragment binds to the epitope of SEQ ID NO:

11.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment is a single-chain variable fragment (scFv).

8. A nucleic acid comprising a polynucleotide encoding a polypeptide comprising: (1) an immunoglobulin heavy chain or a fragment thereof having a heavy chain variable region (VH), wherein the heavy chain variable region (VH) comprises complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 3, 4, and 5, respectively, and wherein the VH binds to B7-H4 when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 13; or (2) an immunoglobulin light chain or a fragment thereof having a light chain variable region (VL), wherein the VL comprises CDRs 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NOs: 6, 7 and 8, respectively, and wherein the VL binds to B7-H4 when paired with a VH comprising the amino acid sequence shown in SEQ ID NO:

12.

9. The nucleic acid of claim 8, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NOs: 3, 4 and 5, respectively.

10. The nucleic acid of claim 8, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NOs: 6, 7 and 8, respectively.

11. The nucleic acid of any one of claims 8-10, wherein the VH specifically binds to human B7-H4 when paired with the VL, or the VL specifically binds to human B7-H4 when paired with the VH.

12. The nucleic acid according to any one of claims 8 to 11, wherein the immunoglobulin heavy chain or fragment thereof is a humanized immunoglobulin heavy chain or fragment thereof, and the immunoglobulin light chain or fragment thereof is a humanized immunoglobulin light chain or fragment thereof.

13. The nucleic acid according to any one of claims 8 to 12, wherein the nucleic acid encodes a single-chain variable fragment (scFv).

14. A vector comprising one or more of the nucleic acids according to any one of claims 8 to 13.

15. A vector comprising two of the nucleic acids of any one of claims 8-13, wherein the vector encodes a VL region and a VH region that together bind to B7-H4.

16. A vector pair, wherein each vector comprises one of the nucleic acids of any one of claims 8-13, wherein the vector pair collectively encodes a VL region and a VH region that together bind to B7-H4.

17. A cell comprising the vector according to claim 14 or 15, or the vector pair according to claim 16.

18. A cell comprising one or more of the nucleic acids according to any one of claims 8-13.

19. A cell comprising two of the nucleic acids according to any one of claims 8 to 13.

20. The cell of claim 19, wherein the two nucleic acids together encode a VL region and a VH region that together bind B7-H4.

21. A method for producing an antibody or an antigen-binding fragment thereof, the method comprising (a) culturing the cell according to any one of claims 17 to 20 under conditions sufficient for the cell to produce an antibody or antigen-binding fragment; and (b) collecting the antibodies or antigen-binding fragments produced by the cells.

22. A transgenic animal, wherein: The transgenic animal expresses the antibody of any one of claims 1-7.

23. An antibody or antigen-binding fragment thereof that binds to B7-H4, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) comprising an amino acid sequence that is at least 90% identical to a selected VH sequence, and the light chain variable region (VL) comprising an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 12, and the selected VL sequence is SEQ ID NO:

13.

24. An antibody or antigen-binding fragment thereof comprising VH CDRs 1, 2, 3 and VL CDRs 1, 2, 3 of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 and 23.

25. An antibody or antigen-binding fragment thereof that cross-competes for binding to B7-H4 with the antibody or antigen-binding fragment thereof of any one of claims 1-7 and 23-24.

26. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 and 23 to 25, and a pharmaceutically acceptable carrier.

27. An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-7 and 23-25 ​​covalently bound to a therapeutic agent.

28. The antibody-drug conjugate of claim 27, wherein the therapeutic agent is a cytotoxic agent or a cytostatic agent.

29. A pharmaceutical composition comprising the antibody-drug conjugate according to claim 27 or 28, and a pharmaceutically acceptable carrier.

30. A method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective dose of a composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-7 and 23-25, or the pharmaceutical composition or antibody-drug conjugate according to any one of claims 26-29.

31. The method of claim 30, wherein B7-H4 is highly expressed in the cancer.

32. The method of claim 30 or 31, wherein the cancer is colorectal cancer; the colorectal cancer is colorectal adenocarcinoma.

33. A method for treating a B7-H4-related disease, the method comprising administering to a subject an anti-B7-H4 antibody or antigen-binding fragment thereof according to any one of claims 1-7 and 23-25, or a pharmaceutical composition or antibody-drug conjugate according to any one of claims 26-29.

34. The method according to claim 33, wherein B7-H4 is highly expressed in the B7-H4-related disease.

35. The method of claim 34, wherein the B7-H4-related disease is colorectal cancer; and the colorectal cancer is colorectal adenocarcinoma.

36. A method for reducing tumor growth rate, the method comprising administering to a subject in need thereof an effective dose of a composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-7 and 23-25, or the pharmaceutical composition or antibody-drug conjugate according to any one of claims 26-29.

37. A method of killing tumor cells, the method comprising administering to a subject in need thereof an effective dose of a composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-7 and 23-25, or the pharmaceutical composition or antibody-drug conjugate according to any one of claims 26-29.

38. A method for detecting the presence or amount of B7-H4 in a sample, the method comprising contacting the sample with the anti-B7-H4 antibody or antigen-binding fragment thereof according to any one of claims 1-7 and 23-25, and determining the presence or amount of B7-H4 in the sample.

39. The method of claim 38, wherein: The sample is a cell expressing B7-H4; the cell expressing B7-H4 is a cancer cell; the cancer is colorectal cancer; the colorectal cancer is colorectal adenocarcinoma.

40. A method of diagnosing a disease or condition associated with B7-H4 in a subject, the method comprising: a) obtaining a sample from the subject; b) contacting the sample with the anti-B7-H4 antibody or antigen-binding fragment thereof; c) determining the presence or amount of B7-H4 in the sample according to claim 38 or 39; d) correlating the presence or amount of B7-H4 with a disease or condition in the subject.

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