Anti-B7-H3 antibody and its applications

Abstract

The present invention provides an anti-B7-H3 antibody and its application. The anti-B7-H3 antibody or antigen-binding fragment of the present invention can specifically bind to B7-H3, has high affinity and very strong internalization ability, and can be applied to the diagnosis and / or treatment of tumors (cancer and benign tumors).

Description

[Technical field]

[0001] The present invention relates to the biomedical field, and in particular to an anti-B7-H3 antibody and its application. [Background technology]

[0002] B7 homolog 3 protein (B7-H3), also known as CD276, belongs to the type I transmembrane glycoproteins of the B7 ligand family.

[0003] Currently, it has been found that B7-H3 exists in two forms, 2Ig-B7-H3 and 4Ig-B7-H3. 2Ig-B7-H3 is expressed in mouse and human cells and has an extracellular IgV-IgC structure, while 4Ig-B7-H3 is expressed only in human cells and consists of a tandem repeat IgV-IgC-IgV-IgC structure. The main form of human B7-H3 is 4IgB7-H3.

[0004] B7-H3 has a limited expression level in normal tissues, but is abnormally highly expressed in various human advanced solid tumors, including but not limited to head and neck cancer, kidney cancer, prostate cancer, lung cancer, breast cancer, gastric cancer and liver cancer. Its overexpression is usually associated with poorer prognosis and poorer clinical outcome of patients. Therefore, B7-H3 is considered to be a diagnostic marker for some tumors and an effective target for antitumor drug development. Summary of the Invention

[0005] The present invention provides an anti-B7-H3 antibody or antigen-binding fragment capable of specifically binding to B7-H3. In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment has high affinity and strong internalization ability.

[0006] Some embodiments specifically bind to B7-H3 and (a) a VH CDR1 comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:6, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:6; (b) a VH CDR2 comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO: 7; (c) a VH CDR3 comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:8, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:8; (d) a VL CDR1 comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 9 to 13, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in any one of SEQ ID NOs: 9 to 13; (e) a VL CDR2 comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 14 to 18, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in any one of SEQ ID NOs: 14 to 18; (f) Provided is an antibody or antigen-binding fragment comprising one or more amino acid sequences selected from the group consisting of the amino acid sequence shown in any one of SEQ ID NOs: 19 to 23, and a VL CDR3 comprising or consisting of an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence shown in any one of SEQ ID NOs: 19 to 23.

[0007] In some embodiments, the antibody or antigen-binding fragment specifically binds to B7-H3 and: (a) a VH CDR1 comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:6, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:6; and (b) a VH CDR2 comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:7, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:7; and (c) a VH CDR3 comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:8, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:8.

[0008] In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 set forth in SEQ ID NO:6, a VH CDR2 set forth in SEQ ID NO:7, and a VH CDR3 set forth in SEQ ID NO:8.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to B7-H3 and: (d) a VL CDR1 comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 9 to 13, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in any one of SEQ ID NOs: 9 to 13; and (e) a VL CDR2 comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 14 to 18, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in any one of SEQ ID NOs: 14 to 18; and (f) A VL CDR3 comprising, or consisting of, an amino acid sequence set forth in any one of SEQ ID NOs: 19 to 23, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in any one of SEQ ID NOs: 19 to 23.

[0010] In some embodiments, the antibody or antigen-binding fragment comprises a VL CDR1 set forth in SEQ ID NO:9, a VL CDR2 set forth in SEQ ID NO:14, and a VL CDR3 set forth in SEQ ID NO:19.

[0011] In some embodiments, the antibody or antigen-binding fragment comprises a VL CDR1 set forth in SEQ ID NO:10, a VL CDR2 set forth in SEQ ID NO:15, and a VL CDR3 set forth in SEQ ID NO:20.

[0012] In some embodiments, the antibody or antigen-binding fragment comprises a VL CDR1 set forth in SEQ ID NO:11, a VL CDR2 set forth in SEQ ID NO:16, and a VL CDR3 set forth in SEQ ID NO:21.

[0013] In some embodiments, the antibody or antigen-binding fragment comprises a VL CDR1 set forth in SEQ ID NO:12, a VL CDR2 set forth in SEQ ID NO:17, and a VL CDR3 set forth in SEQ ID NO:22.

[0014] In some embodiments, the antibody or antigen-binding fragment comprises a VL CDR1 set forth in SEQ ID NO:13, a VL CDR2 set forth in SEQ ID NO:18, and a VL CDR3 set forth in SEQ ID NO:23.

[0015] In some embodiments, the antibody or antigen-binding fragment specifically binds to B7-H3 and: (a) a VH CDR1 comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:6, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:6; and (b) a VH CDR2 comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:7, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:7; and (c) a VH CDR3 comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:8; and (d) a VL CDR1 comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 9 to 13, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in any one of SEQ ID NOs: 9 to 13; and (e) a VL CDR2 comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 14 to 18, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in any one of SEQ ID NOs: 14 to 18; and (f) A VL CDR3 comprising, or consisting of, an amino acid sequence set forth in any one of SEQ ID NOs: 19 to 23, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in any one of SEQ ID NOs: 19 to 23.

[0016] In some embodiments, the substitutions are conservative amino acid substitutions.

[0017] In some embodiments, the antibody or antigen-binding fragment specifically binds to B7-H3 and: (a) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:6, and (b) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:7, and (c) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:8, and (d) a VL CDR1 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 9 to 13; and (e) a VL CDR2 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 14 to 18; and (f) a VL CDR3 comprising an amino acid sequence shown in any one of SEQ ID NOs: 19 to 23.

[0018] In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 set forth in SEQ ID NO:6, a VH CDR2 set forth in SEQ ID NO:7, a VH CDR3 set forth in SEQ ID NO:8, a VL CDR1 set forth in SEQ ID NO:9, a VL CDR2 set forth in SEQ ID NO:14, and a VL CDR3 set forth in SEQ ID NO:19.

[0019] In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 set forth in SEQ ID NO:6, a VH CDR2 set forth in SEQ ID NO:7, a VH CDR3 set forth in SEQ ID NO:8, a VL CDR1 set forth in SEQ ID NO:10, a VL CDR2 set forth in SEQ ID NO:15, and a VL CDR3 set forth in SEQ ID NO:20.

[0020] In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 set forth in SEQ ID NO:6, a VH CDR2 set forth in SEQ ID NO:7, a VH CDR3 set forth in SEQ ID NO:8, a VL CDR1 set forth in SEQ ID NO:11, a VL CDR2 set forth in SEQ ID NO:16, and a VL CDR3 set forth in SEQ ID NO:21.

[0021] In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 set forth in SEQ ID NO:6, a VH CDR2 set forth in SEQ ID NO:7, a VH CDR3 set forth in SEQ ID NO:8, a VL CDR1 set forth in SEQ ID NO:12, a VL CDR2 set forth in SEQ ID NO:17, and a VL CDR3 set forth in SEQ ID NO:22.

[0022] In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 set forth in SEQ ID NO:6, a VH CDR2 set forth in SEQ ID NO:7, a VH CDR3 set forth in SEQ ID NO:8, a VL CDR1 set forth in SEQ ID NO:13, a VL CDR2 set forth in SEQ ID NO:18, and a VL CDR3 set forth in SEQ ID NO:23.

[0023] [Table 1(A)]

[0024] [Table 1(B)]

[0025] In some embodiments, the antibody or antigen-binding fragment further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In some embodiments, the light chain constant region is a kappa or lambda chain constant region. In some embodiments, the antibody or antigen-binding fragment is of one of the following isotypes: IgG, IgM, IgA, IgE, or IgD. In some embodiments, the isotype is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody or antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0026] In some embodiments, the Fc is a variant Fc region. In some embodiments, the variant Fc region has one or more amino acid modifications, such as substitutions, deletions, or insertions, compared to the parent Fc region. In some embodiments, the amino acid modifications of the Fc region alter the activity of effector functions, compared to the activity of the parent Fc region. In some embodiments, the variant Fc region may have altered (i.e., increased or decreased) antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, regulatory activity, or cell binding. In some embodiments, the Fc region amino acid modifications may alter the affinity of the variant Fc region for FcγR (Fcγ receptor), compared to the parent Fc region. In some embodiments, the Fc region is derived from IgG1 or IgG4. In some embodiments, the Fc region mutation is N297A, L234A, or L235A (Eu numbering). In some embodiments, the Fc region mutation is E345R or S440Y (Eu numbering).

[0027] In some embodiments, the antibody or antigen-binding fragment is an scFv, Fab, Fab' or F(ab) 2 In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody.

[0028] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence set forth in any one of SEQ ID NOs: 24 and 35 to 38, or an amino acid sequence having at least 80% or 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 24 and 35 to 38, or an amino acid sequence having one or more conservative amino acid substitutions to the amino acid sequence set forth in any one of SEQ ID NOs: 24 and 35 to 38, and / or The light chain variable region of the above antibody or antigen-binding fragment comprises an amino acid sequence set forth in any one of SEQ ID NOs: 25 to 29 and 39 to 41, or an amino acid sequence having at least 80% or 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 25 to 29 and 39 to 41, or an amino acid sequence having one or more conservative amino acid substitutions to the amino acid sequence set forth in any one of SEQ ID NOs: 25 to 29 and 39 to 41.

[0029] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence set forth in SEQ ID NO:24, or an amino acid sequence having at least 80% or 90% identity to the amino acid sequence set forth in SEQ ID NO:24, or an amino acid sequence having one or more conservative amino acid substitutions to the amino acid sequence set forth in SEQ ID NO:24; and / or The light chain variable region of the above antibody or antigen-binding fragment comprises an amino acid sequence set forth in any one of SEQ ID NOs: 25 to 29, or an amino acid sequence having at least 80% or 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 25 to 29, or an amino acid sequence having one or more conservative amino acid substitutions to the amino acid sequence set forth in any one of SEQ ID NOs: 25 to 29.

[0030] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:24, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:25.

[0031] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:24, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:26.

[0032] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:24, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:27.

[0033] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:24, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:28.

[0034] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:24, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:29.

[0035] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence set forth in any one of SEQ ID NOs: 35 to 38, or an amino acid sequence having at least 80% or 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 35 to 38, or an amino acid sequence having one or more conservative amino acid substitutions to the amino acid sequence set forth in any one of SEQ ID NOs: 35 to 38; and / or The light chain variable region of the above antibody or antigen-binding fragment comprises an amino acid sequence set forth in any one of SEQ ID NOs: 39 to 41, or an amino acid sequence having at least 80% or 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 39 to 41, or an amino acid sequence having one or more conservative amino acid substitutions to the amino acid sequence set forth in any one of SEQ ID NOs: 39 to 41.

[0036] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:35, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:39.

[0037] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:36, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:39.

[0038] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:36, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:40.

[0039] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:37, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:41.

[0040] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:38, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:41.

[0041] [Table 2(A)] [Table 2(B)]

[0042] In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence set forth in SEQ ID NO: 32 or 33, or an amino acid sequence having at least 80% or 90% identity to the amino acid sequence set forth in SEQ ID NO: 32 or 33, or an amino acid sequence having one or more conservative amino acid substitutions to the amino acid sequence set forth in SEQ ID NO: 32 or 33; and / or The light chain constant region of the above antibody or antigen-binding fragment comprises an amino acid sequence set forth in SEQ ID NO:34, or an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence set forth in SEQ ID NO:34, or an amino acid sequence having one or more conservative amino acid substitutions to the amino acid sequence set forth in SEQ ID NO:34.

[0043] Some embodiments provide an antibody that specifically binds to B7-H3, wherein the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO:24 and a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO:32, and the light chain of the antibody comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO:25 and a light chain constant region of the amino acid sequence set forth in SEQ ID NO:34.

[0044] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO:24 and a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO:32, and the light chain of the antibody comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO:26 and a light chain constant region of the amino acid sequence set forth in SEQ ID NO:34.

[0045] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO:24 and a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO:32, and the light chain of the antibody comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO:27 and a light chain constant region of the amino acid sequence set forth in SEQ ID NO:34.

[0046] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO:24 and a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO:32, and the light chain of the antibody comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO:28 and a light chain constant region of the amino acid sequence set forth in SEQ ID NO:34.

[0047] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO:24 and a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO:32, and the light chain of the antibody comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO:29 and a light chain constant region of the amino acid sequence set forth in SEQ ID NO:34.

[0048] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 35 and a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO: 32, and the light chain of the antibody comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 39 and a light chain constant region of the amino acid sequence set forth in SEQ ID NO: 34.

[0049] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 36 and a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO: 32, and the light chain of the antibody comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 39 and a light chain constant region of the amino acid sequence set forth in SEQ ID NO: 34.

[0050] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 36 and a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO: 32, and the light chain of the antibody comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 40 and a light chain constant region of the amino acid sequence set forth in SEQ ID NO: 34.

[0051] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 37 and a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO: 32, and the light chain of the antibody comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 41 and a light chain constant region of the amino acid sequence set forth in SEQ ID NO: 34.

[0052] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 38 and a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO: 32, and the light chain of the antibody comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 41 and a light chain constant region of the amino acid sequence set forth in SEQ ID NO: 34.

[0053] [Table 3]

[0054] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:50, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:52.

[0055] In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody (including a full-length monoclonal antibody), a multispecific antibody or antigen-binding fragment (eg, a bispecific antibody or antigen-binding fragment).

[0056] In some embodiments, the antibody has two heavy chains that are identical in sequence and two light chains that are identical in sequence, hi some embodiments, the Fc region pairs to form disulfide bonds.

[0057] In some embodiments, the antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment.

[0058] The present invention further provides a polynucleotide encoding the antibody or antigen-binding fragment. In some embodiments, the polynucleotide is an isolated polynucleotide. In some embodiments, the polynucleotide sequence is selected from the nucleic acid sequences set forth in SEQ ID NOs: 49 and 51.

[0059] The present invention further provides an expression carrier comprising the polynucleotide. In some embodiments, the expression carrier comprising the polynucleotide is a nucleic acid fragment, a plasmid, a phage, or a virus. In some embodiments, the expression carrier is an isolated expression carrier.

[0060] The present invention further provides a host cell comprising the polynucleotide or expression vehicle. In some embodiments, the host cell is an isolated host cell. In some embodiments, the host cell is a CHO cell, a HEK293 cell (e.g., a HEK293F cell), a BHK cell, a Cos1 cell, a Cos7 cell, a CV1 cell, or a mouse L cell.

[0061] The present invention further provides a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment, or biomaterial, and a pharma- ceutically acceptable carrier.

[0062] The invention further provides the use of the antibody or antigen-binding fragment, the biomaterial or the pharmaceutical composition in the manufacture of a medicament for diagnosing and / or treating a disease. In some embodiments, the invention provides the use of the antibody or antigen-binding fragment, the biomaterial or the pharmaceutical composition in the diagnosis and / or treatment of a disease. In some embodiments, the invention provides a method of diagnosing and / or treating a disease comprising administering to a patient in need thereof an effective amount of the antibody or antigen-binding fragment, the biomaterial or the pharmaceutical composition.

[0063] In some embodiments, the drug is a drug that inhibits B7-H3 activity. In some embodiments, the disease is selected from tumors, respiratory diseases, skin and musculoskeletal diseases, genitourinary diseases, nervous system diseases, and gastrointestinal diseases. In some embodiments, the tumor is a benign tumor or a cancer. In some embodiments, the tumor is a hematological tumor or a solid tumor. In some embodiments, the disease comprises a tumor positive for B7-H3 expression. In some embodiments, the tumor is selected from melanoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), breast cancer (e.g., triple-negative breast cancer), ovarian cancer (e.g., ovarian epithelial cancer), glioma (e.g., glioblastoma and pediatric brain stem glioma), prostate cancer (e.g., castration-resistant prostate cancer), pancreatic cancer (e.g., pancreatic ductal carcinoma), head and neck cancer, leukemia (e.g., acute myeloid leukemia (AML)), cervical cancer, renal cancer, squamous cell tumor, squamous cell carcinoma (e.g., squamous cell lung cancer or squamous cell head and neck cancer), colorectal cancer, gastric cancer, liver cancer, mesothelioma, anal cancer, skin cancer, vulvar cancer, neuroblastoma, desmoplastic small round cell tumor, medulloblastoma, meningioma, peritoneal malignancy, sarcoma, brain cancer, central nervous system tumor, brain metastasis.

[0064] The present invention further provides diagnostic methods and uses. In some embodiments, a method for detecting B7-H3 expression in a sample is provided, in which the sample is contacted with the antibody or antigen-binding fragment, whereby the antibody or antigen-binding fragment binds to B7-H3, and the binding, i.e., the content of B7-H3 in the sample, is detected. In some embodiments, the use of the antibody or antigen-binding fragment in the manufacture of a reagent kit for diagnosing or prognosing tumors (including benign tumors and cancers) is provided. In some embodiments, a diagnostic or prognostic reagent kit is provided comprising the antibody or antigen-binding fragment.

[0065] The present invention provides an anti-B7-H3 antibody or antigen-binding fragment and applications thereof. The anti-B7-H3 antibody or antigen-binding fragment of the present invention can specifically bind to B7-H3 and has high specificity, high affinity and very strong internalization ability, and can be applied to the development of ADC drugs that are expected to have superior anti-tumor activity and efficacy, and can also be applied to the diagnosis and prognosis of tumors (including benign tumors and cancers). [Brief description of the drawings]

[0066]

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[0067] 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.

[0068] definition It should be noted that the term "a" entity refers to one or more of that entity, e.g., "an antibody" should be understood as one or more antibodies, and thus the terms "a" (or "one"), "one or more" and "at least one" may be used interchangeably herein.

[0069] As used herein, the terms "comprising" or "including" mean that the antibody, composition, method, etc. includes the recited elements, such as components or steps, but does not exclude others. "Consisting essentially of" means that the antibody, composition, method, etc. excludes other elements that substantially affect the properties of the combination, but does not exclude elements that do not substantially affect the antibody, composition, method, etc. "Consisting of" means excluding elements not specifically recited.

[0070] The term "polypeptide" is intended to encompass a singular "polypeptide" and a plurality of "polypeptides" and refers to a molecule consisting of amino acid monomers linearly linked by amide bonds (also called peptide bonds). The term "polypeptide" refers to any single or multiple chains of two or more amino acids and does not refer to a specific length of the product. Thus, the definition of "polypeptide" includes peptide, dipeptide, tripeptide, oligopeptide, "protein", "amino acid chain", or any other term to indicate two or more amino acid chains, and the term "polypeptide" can be used in place of or interchangeably with any one of the above terms. The term "polypeptide" also refers to products modified after expression of a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but need not be translated from a specified nucleic acid sequence and may be produced by any method, including chemical synthesis.

[0071] An "amino acid" is an organic compound that contains both an amino group and a carboxyl group, such as an α-amino acid, and can be coded by a nucleic acid either as such or in the form of a precursor. A single amino acid is coded by a nucleic acid consisting of three nucleotides (a so-called codon or base triplet). Each amino acid is coded by at least one codon. The coding of the same amino acid by different codons is called the "degeneracy of the genetic code". Amino acids include natural and unnatural amino acids.

[0072] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Stereoisomers of the twenty conventional amino acids (e.g., D-amino acids), unnatural amino acids (such as α-, α-disubstituted amino acids), N-alkyl amino acids, lactic acid, and other unconventional amino acids may be suitable components of the polypeptides of the present disclosure. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysyl, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxy-terminal direction, consistent with standard usage and convention. Conventional (or naturally occurring) amino acids include alanine (three letter code: Ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0073] Any minor changes in the amino acid sequence of an antibody or immunoglobulin molecule are included in the present disclosure, provided that the identity of the amino acid sequence is maintained at least 75%, such as at least 80%, 90%, 95% or 99%. In some embodiments, the changes are conservative amino acid substitutions. Conservative amino acid substitutions are those that occur within a family of amino acids that are related in their side chains. Genetically encoded amino acids are broadly classified into (1) acidic amino acids, which are aspartate and glutamate; (2) basic amino acids, which are lysine, arginine, and histidine; (3) nonpolar amino acids, which are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids, which are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Other families of amino acids include (i) serine and threonine in the aliphatic-hydroxyl family, (ii) asparagine and glutamine in the amide-containing family, (iii) alanine, valine, leucine and isoleucine in the aliphatic family, and (iv) phenylalanine, tryptophan and tyrosine in the aromatic family. In some embodiments, conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. For example, the individual substitution of leucine with isoleucine or valine, the substitution of aspartate with glutamate, the substitution of threonine with serine, or similar substitutions of amino acids with structurally related amino acids can be reasonably predicted without significant effect on the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid in a binding site. Whether an amino acid change results in a functional peptide can be easily determined by measuring the specific activity of the polypeptide derivative. Fragments or analogs of antibodies or immunoglobulin molecules can be readily produced by one of skill in the art.

[0074] In some embodiments, the amino acid substitutions have the effect of (1) reducing the susceptibility of the protein to hydrolysis, (2) reducing the susceptibility to oxidation, (3) altering the binding affinity for forming protein complexes, (4) altering the binding affinity, or (5) imparting or improving other physicochemical or functional properties of such analogs. Analogs can include various mutant proteins whose sequences differ from naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in the naturally occurring sequence (preferably in the portion of the polypeptide other than the domains that form intermolecular contacts). Conservative amino acid substitutions should not significantly change the structural characteristics of the parent sequence (e.g., the substituted amino acids should not tend to disrupt the helical structure present in the parent sequence or other types of secondary structures that characterize the parent sequence).

[0075] The number of amino acids in the conservative amino acid substitutions in VL and VH is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, or about 15 conservative amino acid substitutions, or a range between any two of these values ​​(including the end values), or any value therein. The number of amino acids in the heavy chain constant region, light chain constant region, heavy chain or light chain conservative amino acid substitutions is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15, about 18, about 19, about 22, about 24, about 25, about 29, about 31, about 35, about 38, about 41, about 45 conservative amino acid substitutions, or a range between any two of these values ​​(inclusive), or any value therein.

[0076] In the present invention, the term "isolated" DNA, RNA, polypeptide, antibody, etc., as used in reference to a cell, nucleic acid, polypeptide, antibody, etc., refers to a molecule, such as DNA or RNA, that is isolated from one or more of the other components of the cell's natural environment. As used in the present invention, the term "isolated" further refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or cell culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" means to include nucleic acid fragments that are not present in the natural state and do not exist in the natural state. In the present invention, the term "isolated" is also used to mean a cell or polypeptide that is isolated from other cellular proteins or tissues. An isolated polypeptide is meant to include purified polypeptides and recombinant polypeptides. Isolated polypeptides, antibodies, etc. are generally produced by at least one purification step. In some embodiments, the purity of an isolated nucleic acid, polypeptide, antibody, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range between any two of these values ​​(endpoints), or any value therein.

[0077] The term "code", when applied to a polynucleotide, refers to a polynucleotide that is said to "encode" a polypeptide, either in its natural state or when manipulated by methods known to those of skill in the art, is capable of producing that polypeptide and / or fragments thereof by transcription and / or translation.

[0078] The term "recombinant" with respect to a polypeptide or polynucleotide refers to a form of a polypeptide or polynucleotide that does not occur in nature, a non-limiting example being one that can be combined to produce a polynucleotide or polypeptide that does not normally occur.

[0079] "Homology," "identity," or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing alignable positions in each sequence. If a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous or identical at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. "At least 80% identity" means about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or a range between any two of these values ​​(the endpoints), or any value therein. "At least 90% identity" means about 90% identity, about 91% identity, about 92% identity, about 93% identity, about 95% identity, about 96% identity, about 97% identity, about 98% identity, about 99% identity, or a range between any two of these values ​​(the endpoints), or any value therein.

[0080] A polynucleotide consists of a specific sequence of four bases, namely adenine (A), cytosine (C), guanine (G), thymine (T), or, if the polynucleotide is RNA, thymine is replaced by uracil (U). A "polynucleotide sequence" can be represented by the characters of a polynucleotide molecule. The characters can be entered into a database in a computer having a central processing unit and used for bioinformatics applications, such as functional genomics and homology searching.

[0081] The terms "polynucleotide", "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. Non-limiting examples of polynucleotides are genes or gene fragments (e.g., probes, primers, EST or SAGE labels), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, carriers, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If such modifications are present, modifications to the structure of the nucleotides may be made before or after assembly of the polynucleotide. The sequence of nucleotides may be interrupted by non-nucleotide components. After polymerization, the polynucleotide may be further modified, such as by conjugation with a labeling component. The term refers to both double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of a polynucleotide of this disclosure includes the double-stranded form and each of the two known or predicted complementarable single-stranded forms that make up the double-stranded form.

[0082] A nucleic acid or polynucleotide sequence (or a polypeptide or antibody sequence) having a certain percentage (e.g., 90%, 95%, 98% or 99%) of "identity or sequence identity" with another sequence means that, upon sequence alignment, that percentage of bases (or amino acids) are the same in the two sequences being compared. The alignment and percentage of identity or sequence identity can be determined by visual inspection or by software programs known in the art, such as those described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007). Preferably, the alignment is performed using default parameters. One alignment program is BLAST using default parameters, including BLASTN and BLASTP, both using the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sortby=HIGHSCORE; Databases=non-redundant; GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides refer to polynucleotides that have the percentage identity specified above and that encode polypeptides having the same or similar biological activity.

[0083] "Antibody" and "antigen-binding fragment" refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody may be an intact antibody and any antigen-binding fragment thereof or a single chain thereof. Thus, the term "antibody" includes any protein or peptide that contains at least a portion of an immunoglobulin molecule in the molecule that has biological activity of binding to an antigen. Antibodies and antigen-binding fragments include, but are not limited to, the complementarity determining regions (CDRs) of the heavy or light chain or ligand-binding portions thereof, the heavy chain variable region (VH), the light chain variable region (VL), the heavy chain constant region (CH), the light chain constant region (CL), the frame region (FR) or any portion thereof, or at least a portion of a binding protein. The CDR regions include the CDR regions of the light chain variable region (VL CDR1-3) and the CDR regions of the heavy chain variable region (VH CDR1-3). An antibody or antigen-binding fragment can specifically recognize and bind to one or more (e.g., two) antigen polypeptides or polypeptide complexes. An antibody or antigen-binding fragment that specifically recognizes and binds multiple (eg, two) antigens may be referred to as a multispecific (eg, bispecific) antibody or antigen-binding fragment.

[0084] The term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody. The constitution of the antibody fragment of the present invention is F(ab') of a monospecific antibody fragment. 2 , F(ab) 2 , Fab', Fab, Fv, scFv, etc. Regardless of their structure, antibody fragments bind to the same antigen that is recognized by the intact antibody. The term "antibody fragment" includes aptamers, enantiomers, and bivalent antibodies. The term "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

[0085] A "single chain variable fragment" or "scFv" is a fusion protein of an immunoglobulin heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, these regions are linked by a short linker peptide of about 10 to about 25 amino acids. The linker may be glycine-rich to increase flexibility, or serine or threonine-rich to increase solubility, and may be linked to the N-terminus of VH and the C-terminus of VL, or vice versa. The protein retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of the linker. ScFv molecules are generally known in the art, for example, as described in U.S. Pat. No. 5,892,019.

[0086] The term "antibody" includes a wide variety of polypeptides that can be biochemically distinguished. Those skilled in the art will understand that the heavy chain classes include gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), which further include several subclasses (e.g., γ1 to γ4). The nature of the chain determines the "type" of the antibody, which is IgG, IgM, IgA, IgG, or IgE, respectively. For example, immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, and IgG5, have already been fully characterized and the functional specificities conferred by them are known. Any type of immunoglobulin is within the scope of the claims disclosed in the present invention. In some embodiments, the type of immunoglobulin molecule is IgG. Two heavy chains and two light chains are linked in a "Y" configuration by disulfide bonds, in which the light chains start at the mouth of the "Y" and surround the heavy chains continuously through the variable region.

[0087] The antibodies, antigen-binding fragments or derivatives disclosed in the present invention may be polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single chain antibodies, epitope-binding fragments (e.g., class Fab, class Fab' and class F(ab')). 2 ), including, but not limited to, the class single-chain Fvs (scFv).

[0088] Light chains can be classified as kappa (κ) or lambda (λ). Each heavy chain can be bound to a κ or λ light chain. Generally, when an immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, the light and heavy chains are bound via a covalent bond, and the "tails" of the two heavy chains are bound via a covalent disulfide bond or a non-covalent bond. In the heavy chains, the amino acid sequence runs from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain. The κ light chain variable region of an immunoglobulin is V κ and the lambda light chain variable region of the immunoglobulin is V λ It is.

[0089] The terms "constant" and "variable" are used according to function. The light chain variable region (VL) and the heavy chain variable region (VH) determine antigen recognition and specificity. The light chain constant region (CL) and the heavy chain constant region (CH) confer important biological properties such as secretion, transplacental transfer, Fc receptor binding, and complement fixation. By convention, the numbering of constant regions increases with increasing distance from the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region, the C-terminal portion is the constant region, and the CH3 and CL domains actually comprise the carboxy termini of the heavy and light chains, respectively.

[0090] In naturally occurring antibodies, assuming that the antibody exhibits its three-dimensional configuration in an aqueous environment, the six "complementarity determining regions" or "CDRs" present in each antigen-binding domain are short, non-contiguous, antigen-specific binding amino acid sequences from which the antigen-binding domain is formed. The remaining other amino acids in the antigen-binding domain, called "framework" regions, exhibit relatively little intermolecular variability. Most of the framework regions adopt a β-fold conformation, forming a cyclic structure to which the CDRs are connected, or in some cases, forming part of the β-fold structure. Thus, the framework regions position the CDRs in the correct orientation in non-covalent interactions between the chains by forming a stent. An antigen-binding domain with CDRs at specific positions forms a surface complementary to the antigen epitope that promotes non-covalent binding of the antibody to the antigen epitope. For a given heavy or light chain variable region, the skilled artisan can identify the amino acids that comprise the CDRs and framework regions by known methods.

[0091] As used herein, the term "CDR" refers to a complementarity determining region in an antibody variable region. There are three CDRs in each of the heavy and light chain variable regions, which are named CDR1, CDR2 and CDR3 (or specifically VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3) for each variable region. The boundaries of the CDRs of the variable regions of the same antibody may differ according to different assignment systems. Thus, when referring to an antibody defined by a specific CDR sequence as defined in the present invention, the scope of said antibody also includes antibodies whose variable region sequences include the CDR sequences of the present invention, but whose stated CDR boundaries differ from the specific CDR boundaries defined in the present invention due to the application of different methods. The CDRs defined by Kabat and Chothia include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, it is within the scope of the present invention to use any definition to refer to the CDRs of an antibody or its variants. The exact residue numbers that include a particular CDR will vary depending on the sequence and size of the CDR. One skilled in the art can generally determine the specific residues contained in the CDRs based on the amino acid sequences of the variable regions of an antibody.

[0092] As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of the variable region minus the CDRs. The precise definition of the CDR sequence can be determined by different systems, and therefore requires different interpretations depending on the meaning of the framework sequence. The six CDRs (CDR1, CDR2, and CDR3 of the light chain and CDR1, CDR2, and CDR3 of the heavy chain) divide the framework regions of the light and heavy chains into four subregions (FR1, FR2, FR3, and FR4) of each chain, of which CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. If it is not specified that a specific subregion is FR1, FR2, FR3, or FR4, the other mentioned framework region represents the combined FRs in the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR refers to one of the four subregions, and FRs refers to two or more of the four subregions that make up a framework region.

[0093] The framework and CDR regions of a humanized antibody need not correspond exactly to the parental sequences, for example the donor antibody CDR or shared framework can be mutagenized by substitution, insertion and / or deletion of at least one amino acid residue, such that the CDR or framework residue at that site does not correspond to the donor antibody or shared framework. Usually, at least 80%, at least 85%, more preferably at least 90% or at least 95% of the humanized antibody residues correspond to those residues in the parental FR and CDR sequences. As used herein, the term "shared framework" refers to the framework region in a shared immunoglobulin sequence. As used herein, the term "shared immunoglobulin sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of related immunoglobulin sequences. In an immunoglobulin family, each position in a shared sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, the shared sequence may contain either one.

[0094] Where a term has two or more definitions as used and / or accepted in the art, the definition of the term as used herein includes all of these meanings unless expressly indicated to the contrary. A specific example uses the term "complementarity determining region" ("CDR") to describe the non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides.

[0095] Kabat et al. also defined a numbering system that can be applied to any antibody variable region sequence. One of skill in the art can apply the "Kabat numbering" system to any variable region sequence, independent of other experimental data beyond the sequence itself. "Kabat numbering" refers to the numbering system proposed by Kabat et al., USDept. of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1983). Antibodies can also use the EU or Chothia numbering systems.

[0096] The antibodies disclosed herein may be from any animal, including but not limited to fish, birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, camel, llama, horse or chicken. In other embodiments, the variable region may be from a condricthoid (e.g., shark).

[0097] A "heavy chain constant region" comprises at least one of a CH1 domain, a hinge (e.g., upper, middle and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment. The heavy chain constant region of an antibody may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide may comprise a CH1 structural domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In other embodiments, the heavy chain constant region may comprise a hinge region partially derived from an IgG1 molecule and a hinge region partially derived from an IgG3 molecule. In other embodiments, a portion of the heavy chain may comprise a chimeric hinge region partially derived from an IgG1 molecule and a chimeric hinge region partially derived from an IgG4 molecule.

[0098] The "light chain constant region" comprises a portion of the amino acid sequence derived from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain. The "light chain-heavy chain pair" refers to an assembly of a light chain and a heavy chain that can form a dimer by disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0099] The "VH domain" comprises the amino-terminal variable domain of an immunoglobulin heavy chain. The "CH1 domain" comprises the first constant region of an immunoglobulin heavy chain. The CH2 domain is not tightly paired with other domains and inserts two N-linked branched carbohydrate chains between the two CH2 domains of an intact native IgG molecule. The CH3 domain starts at the CH2 domain and extends to the C-terminus of the IgG molecule and comprises approximately 108 residues. The "hinge region" comprises a portion of the heavy chain region that connects the CH1 and CH2 domains. The hinge region comprises approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region may be subdivided into three distinct domains: the upper, middle and lower hinge domains.

[0100] "Disulfide bond" refers to a covalent bond formed between two sulfur atoms. The thiol group of a cysteine ​​can form or bridge a disulfide bond with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by a disulfide bond.

[0101] "Chimeric antibody" refers to any antibody whose variable regions are obtained or derived from a first species and whose constant regions (which may be complete, partial or modified) are derived from a second species. In some embodiments, the variable regions are non-human (e.g., murine or primate) and the constant regions are human.

[0102] "Specific binding" generally refers to an antibody or antigen-binding fragment binding to a specific antigen via its antigen-binding domain and epitope in a complementary manner to form a relatively stable complex. "Specific" can be expressed in terms of the relative affinity with which an antibody or antigen-binding fragment binds to a specific antigen or epitope. For example, if antibody "A" has a higher relative affinity for the same antigen than antibody "B", antibody "A" is considered to have a higher specificity for that antigen than antibody "B". Specific binding can be expressed in terms of an equilibrium dissociation constant (KD), where a smaller KD means a tighter binding. Methods for determining whether two molecules are specifically bound include, for example, equilibrium dialysis, surface plasmon resonance, biolayer optical interferometry, and the like, and are well known in the art. An antibody that "specifically binds" to antigen a includes an antibody with an equilibrium dissociation constant KD with antigen a of about 100 nM or less, about 10 nM or less, or about 5 nM or less.

[0103] "Treatment" refers to therapeutic and prophylactic or preventative treatment, the purpose of which is to prevent, alleviate, ameliorate or halt an undesirable physiological change or disorder, such as the progression of a disease, including but not limited to, detectable or undetectable results such as alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay or alleviation of disease progression, improvement, amelioration, reduction or elimination (partial or complete) of the disease state, or an increase in expected survival time in the absence of treatment. Patients in need of treatment include those already suffering from a disease condition or disorder, those susceptible to a disease condition or disorder, or those in need of prevention of the disease condition or disorder, those who may benefit or would benefit from administration of an antibody or pharmaceutical composition disclosed by the present invention for detection, diagnostic processes and / or treatment.

[0104] The term "cancer" is meant or intended to describe a mammalian physiological condition typically characterized by uncontrolled cell proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancer include glioblastoma, acute myeloid leukemia (AML), non-Hodgkin's lymphoma (NHL), non-small cell lung cancer, lung cancer, colon cancer, colorectal cancer, head and neck cancer, breast cancer (e.g., triple-negative breast cancer), pancreatic cancer, squamous cell tumors, squamous cell carcinoma (e.g., squamous cell lung cancer or squamous cell head and neck cancer), anal cancer, skin cancer, and vulvar cancer.

[0105] "Overexpression" or "overexpressed" may interchangeably refer to a gene, which is usually transcribed or translated at a detectably higher level in a particular cell, such as a cancer cell, compared to a normal cell. Overexpression may be protein or RNA overexpression (due to increased transcription, post-transcriptional processing, translation, post-translational processing, altered stability and altered proteolysis), as well as localized overexpression (increased nuclear localization) and enhanced functional activity due to altered protein transport mode, e.g., enzymatic hydrolysis of a substrate. Overexpression may be 50%, 60%, 70%, 80%, 90% or more compared to a normal cell or a comparison cell. In some embodiments, the anti-B7-H3 antibodies of the invention are used to treat solid tumors that may overexpress B7-H3.

[0106] As used herein, the term "administration" refers to delivering a substance (e.g., an anti-B7-H3 antibody) to achieve a therapeutic goal (e.g., treatment of a B7-H3-associated disorder). Methods of administration may be parenteral, enteral, and topical. Parenteral administration is usually by injection and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0107] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a drug, e.g., an antibody, sufficient to reduce or ameliorate the severity and / or duration of a condition (e.g., cancer) or one or more symptoms thereof, prevent progression of a condition, cause regression of a condition, prevent recurrence, progression, onset or progression of one or more symptoms associated with a condition, detect a condition, or enhance or improve the prophylactic or therapeutic effect of another therapy (e.g., a prophylactic or therapeutic agent). For example, an effective amount of an antibody can inhibit tumor growth (e.g., inhibit an increase in tumor volume), reduce tumor growth (e.g., reduce tumor volume), reduce the number of cancer cells, and / or alleviate to some extent one or more symptoms associated with cancer. For example, an effective amount can improve disease-free survival (DFS), improve overall survival (OS), or reduce the likelihood of recurrence.

[0108] The terms "patient" and "subject" are used interchangeably and refer to any mammal in need of diagnosis, prognosis or treatment, including, but not limited to, humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, etc. In some embodiments, the patient is a human patient.

[0109] As used herein, the term "in need of" refers to a patient being identified as needing a particular method or treatment. In some embodiments, this can be identified by any diagnostic method. The patient may be in need of any of the methods and treatments described herein.

[0110] As used herein, the term "administration" refers to the administration of a substance to achieve a therapeutic goal (eg, treatment of a tumor).

[0111] As used herein, the term "reagent" refers to a chemical compound, a mixture of chemical compounds, a biological polymer, or an extract made from biological materials.

[0112] The term "pharmaceutical agent" or "drug" refers to a chemical compound or composition capable of eliciting a desired therapeutic effect when properly administered to a patient.

[0113] "About" refers to the normal error range of the corresponding numerical value, which is readily known to one of ordinary skill in the art. In some embodiments, "about" as referred to herein refers to the numerical value set forth and a range of ±10%, ±5%, or ±1% thereof.

[0114] The half maximum effective concentration (EC 50 ” (concentration for 50% of maximal effect, EC 50 ) refers to the concentration that can produce 50% of the maximum effect.

[0115] As used herein, the term "label" or "labeled" refers to the incorporation of a detectable label, for example, by incorporation of a radioactively labeled amino acid or a polypeptide attached to a biotin group moiety detectable by labeled avidin (e.g., streptavidin containing a fluorescent label or having enzymatic activity detected by optical or calorimetric methods). In some cases, the marker or label may be therapeutic. A variety of methods for labeling polypeptides and glycoproteins are known and available in the art. Examples of markers for polypeptides include radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 1 25 I, 131I), fluorescent markers (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic markers (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporter genes (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags). In some embodiments, labels are attached via spacer arms of various lengths to reduce possible steric hindrance.

[0116] The term "antibody drug conjugate" or "ADC" refers to a binding protein (e.g., an antibody or antigen-binding fragment) linked to one or more chemical drugs, which may optionally be therapeutic or cytotoxic agents. In a preferred embodiment, the ADC comprises an antibody, a drug (e.g., a cytotoxic drug), and a linker that allows the drug to be attached or conjugated to the antibody. The number of bindings of an antibody to a small molecule drug in an ADC is the number of drug bindings of the antibody, which is called the drug-antibody binding ratio (DAR). In some embodiments, the value is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. When considering the average number of bindings of a small molecule drug, which is called the average number of drug bindings of an antibody, or the average drug-antibody binding ratio, the value is selected from about 0 to 10, or 2 to 8. In some embodiments, the drug-antibody binding ratio is about 3 to about 6, and in other embodiments, the drug-antibody binding ratio is about 6 to about 8, or about 7 to about 8. Non-limiting examples of drugs that can be included in an ADC include mitotic inhibitors, antitumor antibiotics, immunomodulators, gene therapy carriers, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotectants, hormones, antihormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, kinase inhibitors (e.g., TEC-family kinase inhibitors and serine / threonine kinase inhibitors), and radiosensitizers.

[0117] The terms "antibody drug conjugate" and "ADC" are used interchangeably. The terms "anti-B7-H3 antibody drug conjugate" and "anti-B7-H3 ADC" are used interchangeably and refer to an ADC comprising an antibody that specifically binds to B7-H3, in which the antibody is conjugated to one or more chemical drugs. In a preferred embodiment, the anti-B7-H3 ADC binds to human B7-H3 (hB7-H3).

[0118] Other chemical terms herein are used in accordance with conventional usage in the art.

[0119] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0120] Anti-B7-H3 antibody The present invention provides anti-B7-H3 antibodies or antigen-binding fragments. In some embodiments, the present invention provides anti-B7-H3 murine antibodies or antigen-binding fragments. In some embodiments, the present invention provides anti-B7-H3 chimeric antibodies or antigen-binding fragments. In some embodiments, the present invention provides humanized anti-B7-H3 antibodies or antigen-binding fragments.

[0121] The anti-B7-H3 antibodies or antigen-binding fragments of the present invention have, but are not limited to, the ability to bind to B7-H3 in vitro, to bind to cells expressing B7-H3, and to have strong internalization ability.

[0122] In some embodiments, the antigen-binding fragment of an anti-B7-H3 antibody is a Fab, Fab', F(ab') 2 , Fv, disulfide bond linked Fv, scFv, single domain antibody or diabody. In some embodiments, the anti-B7-H3 antibody is a multispecific antibody (e.g., a bispecific antibody).

[0123] In some embodiments, Fab, F(ab') 2Antigen-binding fragments, such as F(ab') and Fv, can be produced by complete proteolysis, for example by protease or chemical digestion. (i) Digestion of antibody molecules with pepsin to produce F(ab') 2 (ii) obtaining an F(ab') fragment; 2 These include, but are not limited to, (i) reducing the disulfide bonds of the antibody molecule to produce a Fab fragment, (ii) treating the antibody molecule with papain and a reducing agent to produce a Fab fragment, and (iv) Fv fragment.

[0124] In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment comprises an immunoglobulin heavy chain constant domain selected from a human IgG constant domain, a human IgA constant domain, a human IgE constant domain, a human IgM constant domain, and a human IgD constant domain. In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment comprises an IgG1 heavy chain constant region, an IgG2 heavy chain constant region, an IgG3 heavy chain constant region, or an IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a light chain constant region, such as a kappa light chain constant region or a lambda light chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a kappa light chain constant region.

[0125] The Fc portion of an antibody mediates several important effector functions (e.g., cytokine induction, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, complement-dependent cytotoxicity (CDC), half-life / clearance of antibody and antigen-antibody complexes). In some cases, these effector functions are necessary for therapeutic antibodies, while in other cases, they may be unnecessary or even deleterious depending on the therapeutic objectives. Some human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγRs and complement C1q, respectively. The neologous Fc receptor (FcRn) is a key component that determines the circulating half-life of an antibody. In some further embodiments, at least one amino acid residue in the antibody constant region (e.g., the Fc region of an antibody) is substituted to alter the effector function of the antibody. Substitution of amino acid residues in the Fc region of an antibody to alter antibody effector functions is described in US Pat. Nos. 5,648,260 and 5,624,821, incorporated herein by reference.

[0126] In some embodiments, the invention includes a labeled anti-B7-H3 antibody or antigen-binding fragment that is derived from or linked to one or more functional molecules (eg, another peptide or protein). For example, a labeled antibody is derived by functionally linking (by chemical bond, genetic fusion, non-covalent bonding, or other methods) an antibody or antigen-binding fragment of the invention to one or more other molecular entities, such as another antibody (e.g., a bispecific or bifunctional antibody), a detectable reagent, a drug agent, a protein or peptide capable of mediating binding of the antibody or antigen-binding fragment to another molecule (such as a streptavidin core region or a polyhistidine tag), and / or a cytotoxic or therapeutic agent selected from the group consisting of antimitotic agents, antitumor antibiotics, immunomodulatory agents, gene therapy carriers, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotectants, hormones, antihormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, kinase inhibitors, radiosensitizers, and combinations thereof.

[0127] In some embodiments, the antibodies or antigen-binding fragments of the invention are linked to a detectable reagent, such as by incorporation of a radiolabeled amino acid or attachment of a biotin moiety to the polypeptide that is detectable by labeled avidin (e.g., streptavidin that contains a fluorescent label or has enzymatic activity detected by optical or calorimetric methods). In some cases, the marker or label may be therapeutic. A variety of methods for labeling polypeptides and glycoproteins are known and available in the art. Examples of markers for polypeptides include radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent markers (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic markers (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporter genes (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags). In some embodiments, labels are attached via spacer arms of various lengths to reduce possible steric hindrance.

[0128] In some further embodiments, the antibodies or antigen-binding fragments of the present invention are used to detect the presence of B7-H3 (e.g., human B7-H3) or a fragment thereof in a sample. In some embodiments, the antibody comprises a detectable reagent. The antibody is a polyclonal antibody, or more preferably, a monoclonal antibody. An intact antibody or an antigen-binding fragment (e.g., Fab, scFv, or F(ab')2) may be used. The above detection methods may be used to detect analyte mRNA, protein, or genomic DNA in a biological sample in vitro and in vivo. For example, in vitro detection techniques for analyte mRNA include Norhtern hybridization and in situ hybridization, in vitro detection techniques for analyte protein include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence, and in vitro detection techniques for analyte genomic DNA include Southern hybridization. In addition, in vivo detection techniques for analyte protein include introducing a labeled anti-analyte protein antibody into the patient's body. For example, the antibody can be labeled with a radioactive label, whose presence and location within the patient can then be detected by standard imaging techniques.

[0129] The anti-B7-H3 antibodies disclosed herein can be monoclonal antibodies.

[0130] The binding specificity of the antibodies or antigen-binding fragments disclosed by the present invention can be detected by in vitro assays such as co-immunoprecipitation, radioimmunoassay (RIA), surface plasmon resonance, flow cytometry (Facs) or enzyme-linked immunosorbent assay (ELISA).

[0131] The present invention further includes antibodies that bind to the same epitope as the anti-B7-H3 antibodies described herein. For example, the antibodies of the present invention specifically bind to an epitope of one or more amino acid residues on human B7-H3.

[0132] Provided below is the amino acid sequence of an exemplary human B7-H3, SEQ ID NO:44 (GenBank Accession No. NP_001019907.1, incorporated herein by reference). The signal sequence (amino acids 1-28) is underlined.

[0133] SEQ ID NO:44: MLRRRGSPGMGVHVGAALGALWFCLTGA LEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKD LRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQL NLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGN VTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEALWVTVGLSVCLIALLVALAFVCWRKIKQSCEEENAGAEDQDGEGEGSKTALQPLKHSDSKEDDGQEIA

[0134] 1) Anti-B7-H3 mouse antibody Exemplary anti-B7-H3 murine antibodies of the invention include a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having suitable sequence identity, such as at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, compared to the amino acid sequence set forth in SEQ ID NO: 24, and a light chain variable region comprising an amino acid sequence set forth in any one of SEQ ID NOs: 25-29 or an amino acid sequence having suitable sequence identity, such as at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, compared to the amino acid sequence set forth in any one of SEQ ID NOs: 25-29. In some embodiments, these amino acid sequences with suitable sequence identity have at least the CDRs unchanged.

[0135] 2) Anti-B7-H3 chimeric antibody In some embodiments, the invention provides anti-B7-H3 chimeric antibodies in which the variable regions of an anti-B7-H3 murine antibody are linked to human immunoglobulin constant regions.

[0136] In some embodiments, an anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 24, or an amino acid sequence having suitable sequence identity, such as at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, these amino acid sequences with sequence identity have at least the CDRs unchanged.

[0137] In some embodiments, an anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising an amino acid sequence set forth in any one of SEQ ID NOs: 25-29, or an amino acid sequence having suitable sequence identity, such as at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, to an amino acid sequence set forth in any one of SEQ ID NOs: 25-29. In some embodiments, these amino acid sequences with sequence identity have at least the CDRs unchanged.

[0138] In some embodiments, an anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:24 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:25.

[0139] In some embodiments, an anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising (a) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO:6, (b) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO:7, and (c) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO:8, and a light chain variable region comprising (a) a VL CDR1 having the amino acid sequence set forth in SEQ ID NO:9, (b) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO:14, and (c) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO:19.

[0140] In some embodiments, an anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:24 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:26.

[0141] In some embodiments, an anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising (a) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO:6, (b) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO:7, and (c) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO:8, and a light chain variable region comprising (a) a VL CDR1 having the amino acid sequence set forth in SEQ ID NO:10, (b) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO:15, and (c) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO:20.

[0142] In some embodiments, an anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:24 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:27.

[0143] In some embodiments, an anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising (a) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO:6, (b) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO:7, and (c) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO:8, and a light chain variable region comprising (a) a VL CDR1 having the amino acid sequence set forth in SEQ ID NO:11, (b) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO:16, and (c) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO:21.

[0144] In some embodiments, an anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:24 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:28.

[0145] In some embodiments, an anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising (a) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO:6, (b) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO:7, and (c) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO:8, and a light chain variable region comprising (a) a VL CDR1 having the amino acid sequence set forth in SEQ ID NO:12, (b) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO:17, and (c) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO:22.

[0146] In some embodiments, an anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:24 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:29.

[0147] In some embodiments, an anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising (a) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO:6, (b) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO:7, and (c) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO:8, and a light chain variable region comprising (a) a VL CDR1 having the amino acid sequence set forth in SEQ ID NO:13, (b) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO:18, and (c) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO:23.

[0148] In some embodiments, an anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention further comprises a heavy chain constant region comprising an amino acid sequence set forth in SEQ ID NO: 32 or 33, or an amino acid sequence having suitable sequence identity, such as having at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, compared to the amino acid sequence set forth in SEQ ID NO: 32 or 33.

[0149] In some embodiments, an anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention further comprises a light chain constant region comprising an amino acid sequence set forth in SEQ ID NO:34, or an amino acid sequence having suitable sequence identity, such as having at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO:34.

[0150] In some embodiments, the heavy chain of the anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention further comprises a signal peptide such as MEFGLSWVFLVAILKGVQC (SEQ ID NO: 45) or MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 46). In some embodiments, the light chain of the anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention further comprises a signal peptide such as MDMRVLAQLLGLLLLCFPGARC (SEQ ID NO: 47) or MVLQTQVFISLLLWISGAYG (SEQ ID NO: 48).

[0151] In some embodiments, the binding dissociation equilibrium constant (KD) of the anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention to B7-H3 is about 1 μM or lower. In some embodiments, the KD of the anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention to B7-H3 is between about 1 μM and about 1 pM. In some embodiments, the KD of the anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention to B7-H3 is between about 100 nM and about 1 pM. In some embodiments, the KD of the anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention to B7-H3 is between about 10 nM and about 1 pM. In some embodiments, the KD of the anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention to B7-H3 is between about 1 nM and about 0.1 nM.

[0152] In some embodiments, the KD of an anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention for binding to hB7-H3-His (shown in SEQ ID NO: 4) is about 1 μM or lower. In some embodiments, the KD of an anti-B7-H3 chimeric antibody or antigen-binding fragment of the invention for binding to hB7-H3-His (shown in SEQ ID NO: 4) is between about 1 μM and about 1 pM, alternatively between about 100 nM and about 1 pM, alternatively between about 10 nM and about 1 pM, alternatively between about 1 nM and about 1 pM, alternatively between about 1 nM and about 0.1 nM, or alternatively between about 0.5 nM and about 0.3 nM.

[0153] The murine or chimeric antibodies disclosed herein can be used to generate humanized anti-B7-H3 antibodies. For example, the chimeric antibody M1 is selected for humanization.

[0154] 3) Humanized anti-B7-H3 antibody The antibodies described herein include humanized antibodies, which are suitable for administration to animals (e.g., humans) without eliciting a deleterious immune response in the treated animal (e.g., human).

[0155] In some embodiments, the humanized anti-B7-H3 antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising (a) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO:6, (b) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO:7, and (c) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO:8, and a light chain variable region comprising (a) a VL CDR1 having the amino acid sequence set forth in SEQ ID NO:9, (b) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO:14, and (c) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO:19.

[0156] In some embodiments, the humanized anti-B7-H3 antibody or antigen-binding fragment of the present invention has a heavy chain variable region comprising an amino acid sequence set forth in any one of SEQ ID NOs: 35 to 38, or an amino acid sequence having a suitable sequence identity, such as at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, compared to the amino acid sequence set forth in any one of SEQ ID NOs: 35 to 38, and / or a light chain variable region comprising an amino acid sequence set forth in any one of SEQ ID NOs: 39 to 41, or an amino acid sequence having a suitable sequence identity, such as at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, compared to the amino acid sequence set forth in any one of SEQ ID NOs: 39 to 41. In some embodiments, at least the CDRs of these amino acid sequences having sequence identity are unchanged.

[0157] In some embodiments, a humanized anti-B7-H3 antibody or antigen-binding fragment of the invention comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:35 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:39.

[0158] In some embodiments, a humanized anti-B7-H3 antibody or antigen-binding fragment of the invention comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:36 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:39.

[0159] In some embodiments, a humanized anti-B7-H3 antibody or antigen-binding fragment of the invention comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:36 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:40.

[0160] In some embodiments, a humanized anti-B7-H3 antibody or antigen-binding fragment of the invention comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:37 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:41.

[0161] In some embodiments, a humanized anti-B7-H3 antibody or antigen-binding fragment of the invention comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:38 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:41.

[0162] In some embodiments, a humanized anti-B7-H3 antibody or antigen-binding fragment of the invention further comprises a heavy chain constant region comprising an amino acid sequence set forth in SEQ ID NO: 32 or 33, or an amino acid sequence having suitable sequence identity, such as having at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 32 or 33.

[0163] In some embodiments, a humanized anti-B7-H3 antibody or antigen-binding fragment of the invention further comprises a light chain constant region comprising an amino acid sequence set forth in SEQ ID NO:34, or an amino acid sequence having suitable sequence identity, such as having at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, compared to the amino acid sequence set forth in SEQ ID NO:34.

[0164] In some embodiments, the heavy chain of a humanized anti-B7-H3 antibody or antigen-binding fragment of the invention further comprises a signal peptide such as MEFGLSWVFLVAILKGVQC (SEQ ID NO: 45) or MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 46). In some embodiments, the light chain of a humanized anti-B7-H3 antibody or antigen-binding fragment of the invention further comprises a signal peptide such as MDMRVLAQLLGLLLLCFPGARC (SEQ ID NO: 47) or MVLQTQVFISLLLWISGAYG (SEQ ID NO: 48).

[0165] In some embodiments, the binding dissociation equilibrium constant (KD) of the humanized anti-B7-H3 antibody or antigen-binding fragment of the invention to B7-H3 is about 1 μM or lower. In some embodiments, the KD of the humanized anti-B7-H3 antibody or antigen-binding fragment of the invention to B7-H3 is between about 1 μM and about 1 pM. In some embodiments, the KD of the humanized anti-B7-H3 antibody or antigen-binding fragment of the invention to B7-H3 is between about 100 nM and about 1 pM. In some embodiments, the KD of the humanized anti-B7-H3 antibody or antigen-binding fragment of the invention to B7-H3 is between about 10 nM and about 1 pM. In some embodiments, the KD of the humanized anti-B7-H3 antibody or antigen-binding fragment of the invention to B7-H3 is between about 1 nM and about 0.1 nM.

[0166] In some embodiments, the KD of a humanized anti-B7-H3 antibody or antigen-binding fragment of the invention for binding to hB7-H3-His (shown in SEQ ID NO: 4) is about 1 μM or lower. In some embodiments, the KD of a humanized anti-B7-H3 antibody or antigen-binding fragment of the invention for binding to hB7-H3-His (shown in SEQ ID NO: 4) is between about 1 μM and about 1 pM, alternatively between about 100 nM and about 1 pM, alternatively between about 10 nM and about 1 pM, alternatively between about 1 nM and about 1 pM, alternatively between about 1 nM and about 0.1 nM, or alternatively between about 0.5 nM and about 0.3 nM.

[0167] Treatment method The antibodies or antigen-binding fragments of the invention can neutralize B7-H3 activity in vivo and in vitro. Thus, the antibodies or antigen-binding fragments of the invention can be used to inhibit B7-H3 activity, for example, in cell cultures containing B7-H3, in human patients or other mammals having B7-H3 that cross-react with the antibodies of the invention. In one embodiment, the invention provides a method of inhibiting B7-H3 activity, comprising contacting B7-H3 with an antibody of the invention to inhibit B7-H3 activity. For example, in a cell culture that contains or is suspected of containing B7-H3, an antibody or antigen-binding fragment of the invention can be added to the culture medium to inhibit B7-H3 activity in the culture. In some embodiments, the B7-H3 is human B7-H3 and is a human patient.

[0168] In some embodiments, a method is provided for reducing B7-H3 activity in a patient, the patient being derived from a patient suffering from a disease or condition in which B7-H3 activity is detrimental. The invention provides a method for reducing B7-H3 activity in a patient suffering from such a disease or condition, the method comprising administering to the patient an effective amount of an antibody or antigen-binding fragment of the invention, thereby reducing B7-H3 activity in the patient. In some embodiments, the B7-H3 is human B7-H3, and the patient is a human. Alternatively, the patient may be a mammal expressing a B7-H3 to which the antibody of the invention can bind. Additionally, the patient may be a mammal into which B7-H3 has been introduced (e.g., by administration of B7-H3 or by expression of a B7-H3 transgene). The antibody or antigen-binding fragment of the invention can be administered to a human patient for therapeutic purposes. Additionally, the antibody or antigen-binding fragment of the invention can be administered to a non-human mammal expressing a B7-H3 to which the antibody can bind (for veterinary purposes or as an animal model of human disease). The above animal models of human disease can be used to evaluate the therapeutic efficacy of antibodies or antigen-binding fragments of the invention (eg, dose testing and administration time courses).

[0169] In some embodiments, a method for preventing, treating, or ameliorating diseases associated with various types of tumors (including benign tumors and cancers) and the like is provided, comprising administering an effective amount of the antibody or antigen-binding fragment to a patient. In some embodiments, the application of the antibody or antigen-binding fragment in preventing, treating, or ameliorating diseases associated with tumors (including benign tumors and cancers) and the like is provided. In some embodiments, the application of the antibody or antigen-binding fragment in the manufacture of a medicament for preventing, treating, or ameliorating diseases associated with tumors (including benign tumors and cancers) and the like is provided. In some embodiments, the tumor (including benign tumors and cancers) is a tumor (including benign tumors and cancers) that expresses B7-H3. In some embodiments, the tumor (including benign tumors and cancers) is a tumor (including benign tumors and cancers) that overexpresses B7-H3. Methods for identifying tumors that express B7-H3 (e.g., tumors that overexpress B7-H3) are known in the art. For example, expression of B7-H3 in normal and tumor tissues is detected by immunohistochemistry.

[0170] In some embodiments, the present invention provides a method of treating a disease associated with B7-H3 as a therapeutic target, which improves, alleviates, inhibits or prevents any disease or condition associated with B7-H3 overexpression, including treating a tumor (including benign tumors and cancer) in a patient, alleviating symptoms of a tumor (including benign tumors and cancer) in a patient, or avoiding recurrence of a tumor (including benign tumors and cancer) in a patient, comprising administering to the patient an effective amount of any of the antibodies or antigen-binding fragments described herein.

[0171] The antibodies or antigen-binding fragments provided by the present invention and pharmaceutical compositions comprising the same can be used as therapeutic agents for diagnosing, prognosing, monitoring, treating, mitigating and / or preventing diseases and conditions associated with abnormal B7-H3 expression, activity and / or signaling in a patient. When diseases and conditions associated with abnormal B7-H3 expression, activity and / or signaling in a patient are identified by using standard methods, the antibodies or antigen-binding fragments disclosed in the present invention and pharmaceutical compositions comprising the same can be administered.

[0172] In some embodiments, cancers to be treated and / or prevented using the antibodies or antigen-binding fragments described herein include, but are not limited to, solid tumors, hematological tumors, and metastatic lesions. Examples of cancers include, but are not limited to, carcinoma, blastoma, sarcoma, or leukemia. More specific examples of such cancers include, but are not limited to, melanoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), breast cancer (e.g., triple-negative breast cancer), ovarian cancer (e.g., ovarian epithelial cancer), glioma (e.g., glioblastoma, pediatric brain stem glioma), prostate cancer (e.g., castration-resistant prostate cancer), pancreatic cancer (e.g., pancreatic ductal carcinoma), head and neck cancer, leukemia (e.g., acute myeloid leukemia (AML)), cervical cancer, kidney cancer, squamous cell tumor, squamous cell carcinoma (e.g., squamous cell lung cancer or squamous cell head and neck cancer), colorectal cancer, gastric cancer, liver cancer, mesothelioma, anal cancer, skin cancer, vulvar cancer, neuroblastoma, desmoplastic small round cell tumor, medulloblastoma, meningioma, peritoneal malignant tumor, sarcoma, brain cancer, central nervous system tumor, brain metastasis. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with one or more tumors (including benign tumors or cancer) that overexpress B7-H3. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with a solid tumor that may overexpress B7-H3. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with squamous non-small cell lung cancer (NSCLC). In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with a solid tumor (including advanced solid tumors). In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with prostate cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with non-small cell lung cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with glioblastoma. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient with colon cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient suffering from head and neck cancer.In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient suffering from kidney cancer.In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient suffering from clear cell renal cell carcinoma. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient suffering from glioma. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient suffering from melanoma. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient suffering from pancreatic cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient suffering from gastric cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient suffering from ovarian cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient suffering from colorectal cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient suffering from small cell lung cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient suffering from hypopharyngeal squamous cell carcinoma. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient suffering from neuroblastoma. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient suffering from breast cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient suffering from endometrial cancer. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient suffering from urothelial cell carcinoma. In some embodiments, an antibody or antigen-binding fragment of the invention is administered to a patient suffering from acute myeloid leukemia (AML).

[0173] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the particular antibody or derivative used, the patient's age and weight, general health, sex and diet, and administration time, frequency of excretion, drug combination, and the severity of the particular disease being treated. These factors are determined by medical personnel within the scope of the art. The dosage will further depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage used can be determined by pharmacological and pharmacokinetic principles well known in the art. In some embodiments, the effective dosage ranges from about 0.01 mg / kg to about 100 mg / kg, and may be, for example, once a day or once a month. It should be noted that dosage values ​​can vary depending on the type and severity of the condition to be alleviated. It is further to be understood that for any particular patient, specific dosage regimens may be adjusted according to the needs of the patient and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the compositions as required.

[0174] Methods of administration of antibodies and antigen-binding fragments include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, spinal epidural and oral. Pharmaceutical compositions may be administered by any convenient route, for example, by infusion or bolus injection, may be absorbed through epithelial or mucocutaneous (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other bioactive agents. Thus, pharmaceutical compositions comprising the antibodies of the present invention can be administered orally, rectally, parenterally, intracerebrally, vaginally, intraperitoneally, topically (e.g., by powder, ointment, drop or transdermal patch), buccally, or by spray.

[0175] The term "parenteral" as used herein includes modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0176] The mode of administration may be systemic or local. Additionally, the antibodies or antigen-binding fragments of the invention may need to be introduced into the central nervous system by any suitable route, including intraventricular and intrathecal injection, which may be assisted by an intraventricular catheter connected to a reservoir such as an Ommaya reservoir. Pulmonary administration may also be used, for example, by using an inhaler or nebulizer, and by using an atomized formulation.

[0177] The antibodies or antigen-binding fragments of the invention can be administered locally to the area to be treated, including, but not limited to, local infusion during surgery, topical application in conjunction with, for example, a wound dressing after surgery, injection, catheter, suppository, or implant, which may be a porous, non-porous, or gel-like material, including a membrane (e.g., a silicone rubber membrane) or fiber. Preferably, when administering the antibodies or antigen-binding fragments of the invention, care must be taken to use a material that does not absorb proteins.

[0178] Typically, the method of performing an in vitro test to treat a disease involves administering an antibody or derivative according to the present invention, then in vivo testing for the desired therapeutic or prophylactic activity in an acceptable animal model, and finally administering to humans. Suitable animal models (including transgenic animals) are known to those skilled in the art. For example, an in vitro test to confirm the therapeutic use of an antibody, antigen-binding fragment according to the present invention includes the effect of the antibody on a cell line or a patient tissue sample. The effect of the antibody on the cell line and / or tissue sample can be detected by techniques known to those skilled in the art, such as those disclosed in other parts of the present invention. In vitro test experiments that can be used to determine whether to administer an antibody or antigen-binding fragment according to the present invention include in vitro cell culture experiments, in which a patient tissue sample is grown in culture and exposed to or otherwise administered an antibody or antigen-binding fragment, and the effect of such antibody or antigen-binding fragment on the tissue sample is observed.

[0179] A variety of known delivery systems can be used to administer the antibodies or antigen-binding fragments of the invention, or polynucleotides encoding same, including, for example, in liposomes, microparticles, microcapsules, encapsulation in recombinant cells capable of expressing the compound, receptor-mediated endocytosis, construction of the nucleic acid as part of a reverse transcribing virus or other carrier.

[0180] Combination therapy In some embodiments, the antibodies or antigen-binding fragments of the invention can be combined with other therapeutic or prophylactic regimens, including administration of one or more antibodies or antigen-binding fragments of the invention in combination with or in combination with one or more other therapeutic agents or methods. In the case of combined therapy, the antibodies or antigen-binding fragments can be administered simultaneously or separately with the other therapeutic agent. In the case of separate administration, the antibodies or antigen-binding fragments of the invention can be administered before or after administration of another other therapeutic agent.

[0181] In some embodiments, when an antibody or antigen-binding fragment of the invention is administered to a patient, the antibody molecule or pharmaceutical composition disclosed herein may also be co-administered to the patient with one or more other therapies, e.g., treatment modalities and / or other preparations (e.g., therapeutic agents).

[0182] Such combination therapy includes simultaneous administration (wherein two or more formulations are in the same preparation or in separate preparations) and separate administration, where an antibody or antigen-binding fragment of the invention can be administered before, during, and / or after administration of another therapy, e.g., a treatment modality and / or therapeutic agent. The antibody molecule and / or the other therapy, e.g., a therapeutic agent or method, can be administered during active disease or during remission or less active disease. The antibody molecule can be administered before the other treatment, during the other treatment, after the treatment, or during remission of disease.

[0183] In some embodiments, the antibodies or antigen-binding fragments of the invention are administered in combination with a therapeutic agent, including, but not limited to, cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents (e.g., systemic anti-inflammatory agents), antifibrotic agents, metabolic inhibitors, enzyme inhibitors and / or cytotoxic or cell growth inhibitors, mitotic inhibitors, antitumor antibiotics, immunomodulatory agents, gene therapy carriers, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotectants, hormones, antihormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, kinase inhibitors, or radiosensitizers.

[0184] In some embodiments, the antibody or antigen-binding fragment of the present invention is administered in combination with an anti-cancer or anti-tumor agent. The terms "anti-cancer agent" and "anti-tumor agent" refer to drugs used to treat malignant tumors, such as inhibiting cancerous growth. For example, breast cancer is often stimulated by estrogen and can be treated with drugs that inactivate estrogen. Similarly, prostate cancer can be treated with drugs that inactivate androgens. Examples of anti-cancer drugs include anti-PD1 antibodies (e.g., perilizumab), anti-PD-L1 antibodies (e.g., atezolizumab), anti-CTLA-4 antibodies (e.g., ipilimumab), MEK inhibitors (e.g., trametinib), ERK inhibitors, BRAF inhibitors (e.g., dabrafenib), ostinib, erlotinib, gefitinib, sorafenib, CDK9 inhibitors (e.g., denascillin), MCL-1 inhibitors, temozolomide, Bcl-xL inhibitors, Bcl-2 inhibitors (e.g., venetoclax), ibrutinib, mTOR inhibitors (e.g., everolimus), PI3K inhibitors (e.g., buparixib), dubelizide, idelaliside, AKT inhibitors, including, but not limited to, HER2 inhibitors (e.g., lapatinib), Herceptin, taxanes (e.g., docetaxel, paclitaxel, nanoalbumin-bound paclitaxel), aurestatin-containing ADCs, PBD (e.g., robipituzumab tesillin)-containing ADCs, maytansinoid (e.g., TDM1)-containing ADCs, TRAIL agonists, proteasome inhibitors (e.g., bortezomib), nicotinamide phosphoribosyltransferase inhibitors, Panorex, rituximab, gemtuzumab, alemtuzumab, itumomab, tositumomab, cetuximab, avastin, and herceptin.

[0185] Examples of cytokines that can be administered in combination with an antibody or antigen-binding fragment of the invention include, but are not limited to, one or more of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, IL-21, and IL-31.

[0186] Illustrative examples of other therapeutic agents that can be administered in combination with an antibody or antigen-binding fragment of the invention include one or more of inhaled steroids, beta-agonists such as short-acting or long-acting beta-agonists, leukotrienes or leukotriene receptor antagonists, combination drugs such as ADVAIR, IgE inhibitors such as anti-IgE antibodies (e.g., omalizumab), phosphodiesterase inhibitors (e.g., PDE4 inhibitors), xanthines, anticholinergics, mast cell stabilizers such as cromolyn, IL-4 inhibitors, and the like. anti-cancer drugs, IL-5 inhibitors, eotaxin / CCR3 inhibitors, histamine or its receptor (including H1, H2, H3, and H4) antagonists, and prostaglandin D or its receptor (DP1 and CRTH2) antagonists, anti-PD1 antibodies (e.g., perilizumab), anti-PD-L1 antibodies (e.g., atezolizumab), anti-CTLA-4 antibodies (e.g., ipilimumab), MEK inhibitors (e.g., trametinib), ERK inhibitors, BRAF inhibitors (e.g., dabrafenib), ostinib , erlotinib, gefitinib, sorafenib, CDK9 inhibitors (e.g., denascillin), MCL-1 inhibitors, temozolomide, Bcl-xL inhibitors, Bcl-2 inhibitors (e.g., venetoclax), ibrutinib, mTOR inhibitors (e.g., everolimus), PI3K inhibitors (e.g., buparixib), dubelizide, idelariside, AKT inhibitors, HER2 inhibitors (e.g., lapatinib), Herceptin, taxanes (e.g., docetaxel, paclitaxel , nanoalbumin-bound paclitaxel), aurestatin-containing ADCs, PBD (e.g., robipituzumab tesillin)-containing ADCs, maytansinoid (e.g., TDM1)-containing ADCs, TRAIL agonists, proteasome inhibitors (e.g., bortezomib), and nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, TNF antagonists (e.g., soluble fragments of TNF receptors, such as p55 or p75 human TNF receptors or derivatives thereof, such as 75 kD TNFR-IgG (75 kDThese include, but are not limited to, TNF enzyme antagonists such as TNF receptor-IgG fusion protein, ENBREL), TNF converting enzyme inhibitors, muscarinic receptor antagonists, TGF-β antagonists, interferon gamma, pirfenidone, leflunomide or sirolimus or analogs thereof, chemotherapeutic agents such as CCI-779, COX2 and cPLA2 inhibitors, NSAIDs, immunomodulators, p38 inhibitors, TPL-2, MK-2 and NFkB inhibitors, and the like.

[0187] In some embodiments, the antibodies or antigen-binding fragments of the invention can be used in conjunction with immune checkpoint inhibitors, hi some embodiments, the antibodies or antigen-binding fragments of the invention are administered in combination with other therapeutic or prophylactic regimens, such as radiotherapy.

[0188] Pharmaceutical Compositions The antibodies described herein, or derivatives, fragments, analogs, or homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. The principles and considerations for the preparation of such compositions, as well as guides for the selection of components, are known in the art.

[0189] Such compositions typically comprise an antibody or antigen-binding fragment and a pharma- ceutically acceptable carrier. In some embodiments, the antigen-binding fragment used is the smallest inhibitory fragment that specifically binds to the binding domain of the target protein, such as a peptide based on the variable region sequence of an antibody and that retains the ability to bind to the target protein sequence. In some embodiments, the pharmaceutical composition further comprises an anti-cancer drug (e.g., an immune checkpoint inhibitor).

[0190] In some embodiments, the term "pharmaceutically acceptable" refers to a substance approved by a government regulatory agency or listed in other generally recognized pharmacopeias for animal, and particularly human, use. Furthermore, a "pharmaceutically acceptable carrier" generally refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation additive and the like.

[0191] The term "carrier" refers to a diluent, adjuvant, excipient, or carrier with which the active ingredient can be administered to a patient. Such carriers may be sterile liquids, such as water and oils, including those of petroleum, vegetable, animal, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous glucose solutions, and glycerin solutions may also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene, ethylene glycol, water, ethanol, and the like. If desired, the composition may further contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and tonicity adjusters such as sodium chloride or dextrose are also foreseeable. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The compositions may be prepared as suppositories with conventional adhesives and carriers such as triglycerides. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable drug carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions contain a clinically effective dose of the antibody or antigen-binding fragment, preferably in purified form, combined with an appropriate amount of carrier to provide a dosage form suitable for the patient. The formulation should be adapted to the mode of administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0192] In some embodiments, the composition is prepared as a pharmaceutical composition (e.g., a pharmaceutical composition suitable for intravenous injection into humans) by conventional steps. Compositions for intravenous administration are usually a solution in a sterile or other water-permeable buffer. The composition may further include a solubilizing agent and a local anesthetic, such as lidocaine, to alleviate pain at the site of injection. The pharmaceutical composition is prepared in dosage unit form to provide ease of administration and uniformity of dose. As used herein, dosage unit form refers to physically separable units suitable as unitary doses for use in treated patients, each unit containing a predetermined amount of one or more of the above antibodies combined with the required drug carrier by calculation to produce the desired therapeutic effect. Generally, the active ingredients are supplied alone or in admixture in unit dosage form, for example in a hermetically sealed container (e.g., an ampoule or sachet) capable of indicating the amount of active agent in the form of a dried lyophilized powder or water-free concentrate. When the composition is administered by injection, the composition may be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be used so that the active ingredients may be mixed prior to administration.

[0193] In some embodiments, pharmaceutical compositions to be used for internal administration are sterile, which can be readily accomplished by filtration through sterile filtration membranes.

[0194] A pharmaceutical composition is usually compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Pharmaceutical compositions can include one or more of the following components: a sterile diluent for injection, such as water, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid (EDTA); a buffer, such as histidine hydrochloride, acetate, citrate, or phosphate; an agent for adjusting the osmolarity, such as sodium chloride or dextrose; a stabilizer, such as arginine, methionine, trehalose, sucrose, sorbitol; a surfactant, such as Tween 20 or Tween 80. The pH can be adjusted with an acid or base, e.g., hydrochloric acid or sodium hydroxide. The pharmaceutical composition can be packaged in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. In some embodiments, pharmaceutical compositions suitable for injection use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. When used, the composition must be sterile and fluid to the extent that easy injection is possible. It must be stable under the conditions of manufacture and storage and must be capable of preventing the contaminating action of microorganisms, such as bacteria and fungi. Prolonged absorption of the injectable composition can be achieved by including an agent that delays absorption in the composition, such as aluminum monostearate and gelatin.

[0195] For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, detergents, bile salts and fusidic acid derivatives used for transmucosal administration.Transmucosal administration can be achieved by using nasal drops or suppositories.For transdermal administration, one or more of the above-mentioned antibodies can be formulated into ointments, salves, gels or creams that are generally known in the art.

[0196] The pharmaceutical compositions can be contained in a container or dispenser and packaged together with instructions for administration.

[0197] The pharmaceutical compositions described herein may also contain other active ingredients depending on the specific condition requiring treatment, preferably with complementary activities that do not adversely affect each other. In some embodiments, the composition may contain an agent that enhances its function, such as a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent. Such active ingredients may be present in suitable combinations in amounts effective for the intended purpose.

[0198] The compositions of the present invention may be prepared in neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations derived from sodium, potassium, ammonium, calcium, iron hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0199] Manufacturing method Anti-B7-H3 antibodies can be produced, for example, by immunizing an animal with membrane-bound and / or soluble B7-H3 (e.g., human B7-H3 or an immunogenic fragment, derivative, or variant thereof). Alternatively, monoclonal antibodies can be produced, for example, using the hybridoma technique. In the hybridoma technique, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent such that lymphocytes produce, or are capable of producing, antibodies that specifically bind to the immunizing agent. Alternatively, antibodies can be produced by immunizing lymphocytes ex vivo.

[0200] The immunizing agent used in the hybridoma method usually contains a protein antigen, a fragment thereof, or a fusion protein thereof (e.g., hB7-H3-Fc, whose amino acid sequence is shown in SEQ ID NO:1). Usually, peripheral blood lymphocytes are used if human cells are desired, or spleen cells or lymph node cells are used if a non-human mammalian source is desired. The lymphocytes are then fused with an immortalized cell line to form hybridoma cells using a suitable fusing agent, such as polyethylene glycol. The immortalized cell line is usually a rat or mouse myeloma cell line. The hybridoma cells can be cultured in a suitable medium, which preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parent cells are deficient in hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma medium usually contains hypoxanthine, aminopterin, and thymine ("HAT medium"), which prevents the growth of HGPRT-deficient cells.

[0201] Monoclonal antibodies can also be produced by recombinant DNA methods, for example as described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies described herein can be isolated and sequenced by conventional methods (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells can act as a source of such DNA. Once isolated, the DNA can be placed into an expression carrier and then transfected into host cells such as Chinese hamster ovary (CHO) cells, human embryonic kidney 293 cells (HEK293 cells), monkey COS cells, PER.NS0 cells, SP2 / 0 cells, YB2 / 0 cells, or myeloma cells that do not otherwise produce immunoglobulins, resulting in monoclonal antibodies synthesized in the recombinant host cells. The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (see U.S. Patent No. 4,816,567), or by covalently linking the coding sequence for an immunoglobulin to the coding sequence for all or a portion of a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of the antibodies described herein, or for the variable domains of one antigen-binding site of the antibodies described herein, to generate a chimeric bivalent antibody.

[0202] For single chain Fv (scFv), refer to the technology for producing a single chain unit (U.S. Patent 4,694,778). A single chain unit is formed by amino acid bridging between the heavy and light chain fragments of the Fv region to form a single chain fusion peptide. A technology for assembling a functional Fv fragment in E. coli can be used.

[0203] Examples of techniques that can be used to produce scFvs and antibodies include those described in US Pat. Nos. 4,946,778 and 5,258,498.

[0204] Additionally, U.S. Patents 5,658,570, 5,693,780 and 5,756,096 disclose other efficient methods for producing recombinant antibodies, in particular techniques capable of producing primate antibodies containing monkey variable regions and human constant region sequences, the entire contents of each of which are incorporated herein by reference.

[0205] In some embodiments, the antibody does not provoke an adverse immune response in the treated animal (such as a human). In some embodiments, the antibody, antigen-binding fragment, or derivative disclosed by the present invention is modified by art-recognized techniques to reduce its immunogenicity. For example, the antibody may be humanized, primatized, deimmunized, or produced as a chimeric antibody. These types of antibodies are derived from non-human antibodies, usually murine or primate antibodies, and retain or essentially retain the antigen-binding properties of the parent antibody, but are less immunogenic in humans. This can be achieved by several methods, including: (a) grafting the entire non-human variable region onto a human constant region to produce a chimeric antibody. Methods for producing chimeric antibodies are known in the art, see U.S. Pat. Nos. 5,807,715, 4,816,567, and 4,816,397, the entire contents of which are incorporated herein by reference. (b) grafting at least a portion of one or more non-human complementarity determining regions (CDRs) into human framework and constant regions, with or without retaining important framework residues, or (c) grafting the entire non-human variable region, but "hiding" them by replacing surface residues with human-like portions. Usually, frame residues in the human framework regions are replaced with corresponding residues from the CDR donor antibody, residues that can improve antigen binding, etc. These frame substitutions can be identified by methods known in the art, for example, simulating the interaction of CDRs with frame residues to identify frame residues that play an important role in antigen binding, and identifying unusual frame residues at specific positions by sequence alignment (see U.S. Pat. No. 5,585,089, the entire contents of which are incorporated herein by reference). Antibodies can be humanized using a variety of techniques known in the art, such as CDR grafting (EP 239,400, WO 91 / 09967, U.S. Pat. Nos. 5,225,539, 5,530,101 and 5,585,089), repair or resurfacing (EP 592,106, EP 519,596) and chain rearrangement (U.S. Pat. No. 5,565,332), the entire contents of which are incorporated herein by reference.

[0206] Deimmunization can also be used to reduce the immunogenicity of antibodies. In the present invention, the term "deimmunization" includes altering an antibody to modify T cell epitopes (see, for example, WO / 9852976 A1 and WO / 0034317 A2). For example, the heavy and light chain variable region sequences from a starting antibody are analyzed to generate a human T cell epitope "map" from each variable region, showing the location of the epitope relative to the complementarity determining regions (CDRs) and other important residues in the sequence. By analyzing a single T cell epitope from the T cell epitope map, selectable amino acid substitutions with a low risk of altering antibody activity are identified. A set of selectable heavy and light chain variable region sequences containing combinations of amino acid substitutions are designed, and these sequences are then incorporated into a set of binding polypeptides. The complete heavy and light chain genes containing the modified variable regions and human constant regions are then cloned into an expression carrier, and the complete antibody is produced by introducing the plasmids into a cell line. The antibodies are then compared in appropriate biochemical and biological experiments to identify the most suitable antibody.

[0207] Antibodies can be produced by a variety of methods known in the art, including phage display techniques using antibody libraries derived from immunoglobulin sequences. See U.S. Patents 4,444,887 and 4,716,111, and PCT publications WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741, the entire contents of each of which are incorporated herein by reference.

[0208] Fully human antibodies that recognize a selective epitope can be produced by a technique referred to as "guided selection," in which a selected non-human monoclonal antibody (e.g., a murine antibody) is used to guide screening for fully human antibodies that recognize the same epitope (see U.S. Patent 5,565,332, the entire contents of which are incorporated herein by reference).

[0209] In another embodiment, DNA encoding the desired monoclonal antibody can be isolated and sequenced by conventional methods (e.g., using oligonucleotide probes capable of specifically binding to genes encoding mouse antibody heavy and light chains). Isolated and subcloned hybridoma cells can be the source of such DNA. Once isolated, the DNA can be placed in an expression carrier and then transfected into prokaryotic or eukaryotic host cells (e.g., E. coli cells, monkey COS cells, Chinese Hamster Ovary (CHO) cells, or other non-immunoglobulin producing myeloma cells). The isolated or synthesized DNA can be used to produce antibody constant and variable region sequences, for example, as described in U.S. Pat. No. 5,658,570, the entire contents of which are incorporated herein by reference. This method involves extracting RNA from selected cells, transforming it into cDNA, and then amplifying it by PCR techniques using Ig-specific primers. Suitable probes to be applied for this purpose have also been mentioned in U.S. Pat. No. 5,658,570.

[0210] Furthermore, one or more CDRs of the antibody of the present invention can be inserted into a frame region, for example, into a human frame region, to construct a humanized non-fully human antibody by conventional recombinant DNA techniques. The framework region can be a naturally occurring or common framework region, preferably a human framework region. Some polynucleotides can encode an antibody that specifically binds to at least one epitope of a target antigen produced by the combination of the frame region and the CDR. One or more amino acid substitutions can be made within the frame region, and an amino acid substitution that can improve the binding of the antibody to its antigen can be selected. Also, in this manner, antibody molecules can be produced in which one or more interchain disulfide bonds are deleted by substituting or deleting cysteine ​​residues in one or more variable regions involved in the formation of interchain disulfide bonds. Other modifications made to the polynucleotide within the skill of the art are also included in the present invention.

[0211] Antibodies can be produced by conventional recombinant DNA techniques. Antibody-producing carriers and cell lines, etc. can be selected, constructed and cultured by techniques known to those skilled in the art. These techniques are described in various laboratory manuals and major publications.

[0212] In some embodiments, glycosylation modifications are made to the anti-B7-H3 antibodies or antigen-binding fragments of the invention. For example, deglycosylated antibodies (i.e., antibodies lacking glycosylation) can be produced. By altering the glycosylation, the affinity of the antibody for the antigen can be increased, for example. Such modifications can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions are made to remove one or more variable region glycosylation sites, thereby removing glycosylation at those sites. Such deglycosylation can increase the affinity of the antibody for the antigen. Such methods are described in further detail in PCT Publication WO2003016466 A2 and U.S. Patents 5,714,350 and 6,350,861, each of which is incorporated herein by reference in its entirety.

[0213] Additionally, modified anti-B7-H3 antibodies or antigen-binding fragments can be produced with modified glycosylation types, such as hypofucosylated antibodies or antibodies with increased bisecting GlcNAc structures. These modifications have been shown to increase the ADCC ability of the antibody. Such modifications can be achieved, for example, by expressing the antibody in a host cell with modified glycosylation machinery. Cells with modified glycosylation machinery have been described in the art and used as recombinant antibodies of the invention to produce host cells of glycosylated antibodies. See, for example, European Patent No. EP 1,176,195, PCT Publication No. WO 03 / 035835, WO 99 / 5434280, each of which is incorporated herein by reference in its entirety.

[0214] The antibody of the present invention (e.g., anti-B7-H3 antibody) can be produced by recombinantly expressing antibody heavy and light chain genes in a host cell. For example, a host cell is transfected with one or more recombinant expression carriers carrying heavy and light chain DNA fragments encoding the antibody, whereby the heavy and light chains are expressed in the host cell, the expressed antibody can be secreted into the medium in which the host cell is cultured, and the antibody can be recovered from the medium. "Transfection" refers to various techniques, such as electroporation, lipotransfection, calcium phosphate precipitation, DEAE-glucanotransfection, that are commonly used to introduce foreign DNA into eukaryotic host cells. Standard recombinant DNA methods for obtaining antibody heavy and light chain genes, incorporating these genes into expression carriers, and introducing the carriers into host cells are known in the art and are described, for example, in U.S. Pat. No. 4,816,397. The DNA expressing the antibody heavy and light chains can be incorporated into the same carrier or different carriers, and when incorporated into different carriers, the carrier expressing the antibody heavy chain and the carrier expressing the antibody light chain can transfect the host cell in an appropriate ratio. In some embodiments, the antibody expressing carrier comprises at least one promoter element, an antibody coding sequence, a transcription termination signal and a polyA tail. Other elements can include an enhancer, a Kozak sequence (GCCACC shown in SEQ ID NO:53) and donor and acceptor sites for RNA splicing on both sides of the inserted sequence. Highly efficient transcription can be obtained by the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and the early promoter of cytomegalovirus, and some other cellular promoters such as the actin promoter can also be applied. Suitable expression vehicles may include pIRES1neo, pRetro-Off, pRetro-On, pLXSN, pLNCX, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), pSVL, pMSG, pRSVcat, pSV2dhfr, pBC12MI, pCS2, or pCHO1.0, and the like.Commonly used mammalian cells (host cells) include HEK293 cells (eg, HEK293F cells), Cos1 cells, Cos7 cells, CV1 cells, mouse L cells, CHO cells, and the like.

[0215] To express an antibody of the invention (e.g., an anti-B7-H3 antibody), DNA encoding the full-length light and heavy chains can be inserted into an expression vehicle such that the genes are operably linked to transcriptional and translational control sequences. By "operably linked" it is meant that the antibody genes are linked within the vehicle such that the transcriptional and translational control sequences within the vehicle perform their expected function of regulating the transcription and translation of the antibody genes.

[0216] The antibody (e.g., anti-B7-H3 antibody) genes can be inserted into the expression carrier by standard methods (e.g., ligating the antibody gene fragment to complementary restriction sites on the carrier, or ligating to blunt ends if no restriction sites are present). Prior to inserting the antibody-related light or heavy chain gene sequences, the expression carrier can already carry antibody constant region sequences. For example, one way to convert the VH and VL sequences related to the anti-B7-H3 antibody into full-length antibody genes is to insert them into an expression carrier that already encodes the heavy and light chain constant regions, respectively, such that the VH segment is operably linked to the CH segment in the carrier, and the VL segment is operably linked to the CL segment in the carrier. Alternatively, the recombinant expression carrier can encode a signal peptide that facilitates the secretion of the antibody heavy and light chains by the host cell. Alternatively, the antibody heavy and light chain genes can be cloned into a carrier that encodes a signal peptide that facilitates the secretion of the antibody heavy and light chains by the host cell, such that the signal peptide is linked in frame to the amino termini of the antibody heavy and light chain genes. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein), such as MEFGLSWVFLVAILKGVQC (SEQ ID NO: 45), MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 46), MDMRVLAQLLGLLLLCFPGARC (SEQ ID NO: 47), or MVLQTQVFISLLLWISGAYG (SEQ ID NO: 48).

[0217] In addition to the antibody heavy and light chain genes, the recombinant expression carrier can also carry regulatory sequences that control the expression of the antibody chain genes in a host cell. Regulatory sequences include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Those skilled in the art will recognize that the design of the expression carrier, including the selection of regulatory sequences can depend on factors such as the choice of host cell to be transformed, the level of expression of protein desired, and the like. Suitable regulatory sequences for use in mammalian host cell expression include promoters and / or enhancers derived from viral elements that direct high levels of protein expression in mammalian cells, such as cytomegalovirus (CMV) (such as the CMV promoter / enhancer), simian virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus (such as the adenovirus major processive promoter (AdMLP)) and polyoma. For further description of viral regulatory elements and sequences thereof, see, e.g., U.S. Patents 5,168,062, 4,510,245 and 4,968,615.

[0218] In addition to the antibody chain genes and regulatory sequences, the recombinant expression carrier can carry other sequences, such as sequences that regulate replication of the carrier in a host cell (e.g., origin of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the carrier has been introduced (see, e.g., U.S. Patents 4,399,216, 4,634,665 and 5,179,017). For example, one can usually select a marker gene that confers resistance to a drug (e.g., G418, hygromycin or methotrexate) on the host cells into which the carrier has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR-host cells with methotrexate selection / amplification), the neo gene (for G418 selection) and the GS gene. For expression of the heavy and light chains, the expression carriers encoding the heavy and light chains are transfected into the host cells by standard techniques.

[0219] The antibodies of the invention (e.g., anti-B7-H3 antibodies) can be expressed in eukaryotic host cells. In some embodiments, the expression of the antibodies is carried out in eukaryotic cells, such as mammalian host cells. Exemplary host cells for expressing the antibodies of the invention include Chinese hamster ovary cells (CHO cells) or modified CHO cells CHO-S, CHO-dhfr-, CHO / DG44 or ExpiCHO, NSO bone marrow cells, COS cells, SP2 cells, CV1 cells, mouse L cells, human embryonic kidney cells HEK293 or modified HEK293 cells HEK293T, HEK293F or HEK293E cells. After introducing the recombinant expression carrier encoding the antibody chain genes into the host cells, the host cells are cultured in culture medium to produce the antibody, during which the antibody is expressed in the host cells or the antibody is secreted into the culture medium. The antibody can be recovered from the culture medium using standard protein purification methods.

[0220] For recombinant expression of an antibody of the present invention (e.g., an anti-B7-H3 antibody), a host cell can be co-transfected with two recombinant expression carriers, a first recombinant expression carrier encoding an antibody heavy chain and a second recombinant expression carrier encoding an antibody light chain. The two recombinant expression carriers can contain the same selectable marker, or they can each contain a separate selectable marker. Alternatively, a host cell can be transfected with recombinant expression carriers encoding the antibody heavy and light chains.

[0221] The antibodies of the invention (eg, anti-B7-H3 antibodies) can also be produced by chemical synthesis. Cell-free platforms can also be used to generate mutant antibodies.

[0222] The antibodies of the present invention (e.g., anti-B7-H3 antibodies) produced by recombinant expression can be purified by any method known in the art for purifying immunoglobulin molecules, such as by chromatography (such as ion exchange, affinity chromatography, and fractional column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. For example, affinity chromatography with protein A or protein G provides mainly the IgG fraction in immune serum. Also, the specific antigen targeted by the immunoglobulin or its epitope can be immobilized on a column to purify the immune specific antibody by immune affinity chromatography. The antibodies of the present invention (e.g., anti-B7-H3 antibodies) can be fused to heterologous polypeptide sequences known in the art to facilitate purification. For the purification of immunoglobulins, see D. Wilkinson's article (The Scientist, The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0223] Furthermore, mutations, including but not limited to site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions, may be introduced into the nucleotide sequence encoding the antibody of the present invention by standard techniques known to those skilled in the art. Mutants (including derivatives) encode fewer than 50 amino acid substitutions, fewer than 40 amino acid substitutions, fewer than 30 amino acid substitutions, fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original heavy chain variable region VH CDR1, VH CDR2, VH CDR3 and light chain variable region VL CDR1, VL CDR2, or VL CDR3. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, for example by saturation mutagenesis, and mutants that retain activity can be identified by screening the biological activity of the resulting mutants.

[0224] In some embodiments, the substitutions described herein are conservative amino acid substitutions.

[0225] In some embodiments, the gene sequence encoding the antibody V3 heavy chain is set forth in SEQ ID NO: 49, in which the underlined portion encodes the VH CDR, and the heavy chain amino acid sequence of antibody V3 is set forth in SEQ ID NO: 50, in which the underlined portion is the VH CDR.

[0226] SEQ ID NO:49 is as follows: GAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGAGCGGCGAGTCCCTGAAGATCTCCTGTAAGGCTAGCGGCTATACCTTTACC GATTACGATATCAAC TGGGTGCGGCAGATGCCTGGCAAGGGCCTGGAGTGGATCGGC TGGATCTTTCCCGGCGATGACACCACCAAGTACAACGAGAAGTTCAAGGGCCAGGTGACCCTGAGCGCTGATAAGTCCACCAACACCGCCTACATGCAGTGGTCCTCCCTGAAGGCCTCCGACACCGCCATGTATTATTGCGCCCGG TCCCCCAGCTTCGACTAC

[0227] SEQ ID NO:50 is as follows: EVQLVQSGAEVKKSGESLKISCKASGYTFT DYDIN WVRQMPGKGLEWIG WIFPGDDTTKYNEKFKG QVTLSADKSTNTAYMQWSSLKASDTAMYYCAR SPSFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0228] In some embodiments, the gene sequence encoding the antibody V3 light chain is set forth in SEQ ID NO:51, in which the underlined portion encodes the VL CDR, and the light chain amino acid sequence of antibody V3 is set forth in SEQ ID NO:52, in which the underlined portion is the VL CDR.

[0229] SEQ ID NO:51 is as follows: CAGATCGTGCTGACCCAGAGCCCCGGCACCCTGTCCCTGAGCCCTGGAGAGAGGGCCACCCTGAGCTGT AGCGCTAGCTCCACCATCGGCTTTATGTAT TGGTATCAGCAGAAGCCTGGCCAGGCCCCCCGGAGATGGATCTAC GACACCAGCAAGCTGGCCAGC GGCGTGCCTGACAGGTTTTCCGGCAGCGGCTCCGGCACCGACTATACCCTGACCATCAGCAGGCTGGAGCCCGAGGATTTCGCCGTGTATTATTGC CACCAGCGGAGCAGCTATCCTACCTTTGGCCAGGGCACCAAGGTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAAT CGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0230] SEQ ID NO:52 is as follows: QIVLTQSPGTLSLSPGERATLSC SASSTIGFMY WYQQKPGQAPRRWIY DTSKLAS GVPDRFSGSGSGTDYTLTISRLEPEDFAVYYC HQRSSYPT FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC EXAMPLES

[0231] The technical solutions of the present invention are further described below by specific embodiments, but the specific embodiments do not limit the scope of protection of the present invention. Any non-essential modifications or adjustments made by others based on the spirit of the present invention still belong to the scope of protection of the present invention.

[0232] Materials, reagents and the like used in the following examples are all obtainable from commercial sources or known methods unless otherwise specified.

[0233] VISTA-CHO cell construction process: The gene sequence encoding VISTA (NCBI Reference Sequence: NP_071436.1) is inserted into an expression carrier, and then stably transfected into CHO cells to obtain VISTA-CHO cells.

[0234] Tim3-CHO cell construction process: The gene sequence encoding Tim3 (NCBI Reference Sequence: NP_116171.3) is inserted into an expression carrier, and then stably transfected into CHO cells to obtain Tim3-CHO cells.

[0235] Lag3-CHO cell construction process: The gene sequence encoding Lag3 (NCBI Reference Sequence: NP_002277.4) is inserted into an expression carrier, and then stably transfected into CHO cells to obtain Lag3-CHO cells.

[0236] Example 1: Antigen Production Preparation of antigen hB7-H3-Fc: A gene sequence encoding antigen hB7-H3-Fc (the amino acid sequence of antigen hB7-H3-Fc is shown in SEQ ID NO:1, and constructed by adding IgG1 Fc (shown in SEQ ID NO:3) to the C-terminus of human B7-H3 extracellular domain (shown in SEQ ID NO:2)) was cloned into an expression carrier, and then transiently transfected into HEK293F cells. After purification by Protein A affinity chromatography, antigen hB7-H3-Fc was obtained.

[0237] Antigen hB7-H3-His production: The gene sequence encoding antigen hB7-H3-His (the amino acid sequence of antigen hB7-H3-His is shown in SEQ ID NO: 4, and was constructed by adding 10×HIS tag (HHHHHHHHHH shown in SEQ ID NO: 5) to the C-terminus of human B7-H3 extracellular domain (shown in SEQ ID NO: 2)) was cloned into an expression carrier, and then HEK293F cells were transiently transfected. After purification by nickel column, antigen hB7-H3-His was obtained.

[0238] Amino acid sequence of antigen hB7-H3-Fc: (shown in SEQ ID NO:1).

[0239] Amino acid sequence of human B7-H3 extracellular domain: MLRRRGPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEG SFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTI TPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMT (shown in SEQ ID NO:2).

[0240] Amino acid sequence of IgG1 Fc: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (shown in SEQ ID NO:3).

[0241] Amino acid sequence of the antigen hB7-H3-His: MLRRRGPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFT CFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRS PTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTHHHHHHHHH (shown in SEQ ID NO: 4).

[0242] Example 2 Chimeric antibodies The amino acid sequences of the VH CDR, VL CDR, VH and VL of the mouse antibody are shown in Tables 1 to 3.

[0243] [Table 4(A)]

[0244] [Table 4(B)]

[0245] [Table 4(C)]

[0246] The construction method of the chimeric antibody is shown in Table 4, where VH and CH constitute the heavy chain of the antibody, and VL and CL constitute the light chain of the antibody, and are represented by "antibody" + antibody number. For example, the heavy chain of antibody M1 is composed of VH shown in SEQ ID NO: 24 and CH shown in SEQ ID NO: 32, and the light chain of antibody M1 is composed of VL shown in SEQ ID NO: 25 and CL shown in SEQ ID NO: 34. The control antibody M30-H1-L4 has a heavy chain shown in SEQ ID NO: 30 and a light chain shown in SEQ ID NO: 31 (see Table 5). The CDRs of the chimeric antibody are shown in Table 6, and the amino acid sequence of the IgG constant region is shown in Table 7.

[0247] The gene sequences encoding the heavy and light chains of the antibody were cloned into an expression carrier, respectively, to obtain a recombinant expression carrier, which was then transiently transfected into HEK293F cells. After cultivation, the antibody was purified by Protein A affinity chromatography to obtain an antibody, which was sequenced and found to match the desired sequence.

[0248] [Table 4(D)]

[0249] [Table 4(E)]

[0250] [Table 4(F)]

[0251] Example 3 Affinity and specificity of chimeric antibodies 1. Chimeric antibody affinity verification - 1 Biacore Detection with BiaCore T200 (GE Healthcare): Detection with Protein A chip (GE Healthcare, Cas#29127556), antibodies were added to 1× HBS-EP+ (1× HBS-EP+ production: HBS-EP+(10×) (GE The antibody was diluted to 5 μg / mL with 50 mL of glycerol (Glycer, cat#BR-1006-69) and mixed with 450 mL of ultrapure water (mixed evenly). The antibody was passed through the experimental channels (Fc2, Fc4) at a flow rate of 10 μL / min and captured for 20 seconds (s) so that the capture amount was approximately 235 RU. The flow rate was then adjusted to 30 μL / min, and different concentrations of hB7-H3-His diluted solutions (0 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, diluted with 1×HBS-EP+) were injected. The antibody was simultaneously passed through the surfaces of the experimental channels (Fc2, Fc4) and the reference channels (Fc1, Fc3) for a binding time of 180 s and a dissociation time of 600 s. Finally, the antibody was diluted with Glycine pH 1.5 (GE Healthcare, cat#BR-1006-69) and mixed with glycerol (Glycine, cat#BR-1006-69) for a binding time of 180 s and a dissociation time of 600 s. The chip was regenerated for 60 s using a 300-mL ELISA kit (Healthcare, cat#BR100354) before proceeding to the next cycle. The test results were analyzed using the data analysis software Evaluation Software 3.1, and the sensing signals collected by the test flow path of the sample were subjected to double subtraction of the reference flow path and the sample blank, and fitted with a kinetic "1:1" model to obtain the kinetic parameters (ka: binding rate, kd: dissociation rate, kD: binding dissociation equilibrium constant) for the binding of each sample to hB7-H3-His.

[0252] [Table 4(G)]

[0253] The results, as shown in Table 8, showed that both antibodies M1 and M2 were able to bind to hB7-H3-His, with antibody M1 having a higher affinity for binding to hB7-H3-His.

[0254] 2. Chimeric antibody affinity verification - 2 Flow cytometry MDA-MB-468 cells in the logarithmic growth phase were harvested, centrifuged at 1500 rpm for 5 minutes (min), the supernatant was removed, resuspended in 1x PBS, centrifuged at 1500 rpm for 5 min, the supernatant was removed, and this was repeated once. MDA-MB-468 cells were added to a V-shaped 96-well plate at 50 μL / well (200,000 cells), centrifuged at 1500 rpm for 5 min, and the PBS was discarded. Gradient-diluted chimeric antibodies (initial concentration 100 nM, 3-fold gradient dilution, solvent 1x PBS) were added at 100 μL / well, the cells were resuspended, and incubated on ice for 1 hour (h). After the incubation was completed, the cells were centrifuged at 2000 rpm for 5 min, the supernatant was removed, and the cells were resuspended in 200 μL / well of 1x PBS, centrifuged again, the supernatant was removed, and this was repeated twice. PE-labeled goat anti-human IgG Fc secondary antibody (Invitrogen, Cat. No. 12-4998-82, diluted 1:500 with 1x PBS) was added at 100μL / well, and the cells were resuspended. The cells were protected from light and incubated on ice for 30min. After incubation, the cells were centrifuged at 2000rpm for 5min, the supernatant was removed, and the cells were resuspended in 200μL / well of 1x PBS, followed by centrifugation again and removal of the supernatant, which was repeated twice. The cells were resuspended by adding 200μL / well of 1x PBS. The results were detected with a cytoflex flow cytometer (Beckman). The results are shown in Figure 1.

[0255] 3. Validation of chimeric antibody specificity - flow cytometry Cells in the logarithmic growth phase (Lag3-CHO cells, VISTA-CHO cells, Tim3-CHO cells or Raji cells) were harvested, centrifuged at 1500 rpm for 5 min, the supernatant was removed, resuspended in 1x PBS, centrifuged at 1500 rpm for 5 min, the supernatant was removed, and this was repeated once. The cells were added to a V-type 96-well plate at 50 μL / well (500,000 cells), centrifuged at 1500 rpm for 5 min, and the PBS was discarded. Antibody M1 (concentration 100 nM) was added at 100 μL / well, and the cells were resuspended. Incubated on ice for 1 h. After the incubation was completed, the cells were centrifuged at 2000 rpm for 5 min, the supernatant was removed, and the cells were resuspended in 200 μL / well of 1x PBS, followed by centrifugation again, the supernatant was removed, and this was repeated twice. PE-labeled goat anti-human IgG Fc secondary antibody (Invitrogen, Cat. No. 12-4998-82, diluted 1:500 with 1x PBS) was added at 100 μL / well, and the cells were resuspended. The cells were protected from light and incubated on ice for 30 min. After incubation, the cells were centrifuged at 2000 rpm for 5 min, the supernatant was removed, and the cells were resuspended in 200 μL / well of 1x PBS, followed by centrifugation again and removal of the supernatant, which was repeated twice. The cells were resuspended by adding 200 μL / well of 1x PBS. Detection was performed using a cytoflex flow cytometer (Beckman).

[0256] The results, as shown in FIG. 2, showed that antibody M1 did not bind to Lag3-CHO cells, VISTA-CHO cells, Tim3-CHO cells, and Raji cells.

[0257] Example 4 Humanized Antibodies The amino acid sequences of the humanized antibody VH and VL are shown in Table 9. The construction method of the humanized antibody is shown in Table 10, where VH and CH constitute the heavy chain of the antibody, and VL and CL constitute the light chain of the antibody, and are represented by "antibody" + antibody number. For example, the heavy chain of antibody V3 is composed of VH shown in SEQ ID NO: 36 and CH shown in SEQ ID NO: 32, and the light chain of antibody V3 is composed of VL shown in SEQ ID NO: 40 and CL shown in SEQ ID NO: 34. The heavy chain of the control antibody M30-H1-L4 is shown in SEQ ID NO: 30 and the light chain is shown in SEQ ID NO: 31. The heavy chain of the control antibody Trop2 is shown in SEQ ID NO: 42 and the light chain is shown in SEQ ID NO: 43 (see Table 11).

[0258] [Table 5(A)] [Table 5(B)]

[0259] [Table 6(A)]

[0260] [Table 6(B)]

[0261] The gene sequences of the heavy and light chains of the antibody were cloned into an expression carrier, respectively, to obtain a recombinant expression carrier, which was then transiently transfected into HEK293F cells. After cultivation, the humanized antibody was purified by Protein A affinity chromatography to obtain a humanized antibody, which was sequenced to match the desired sequence.

[0262] Example 5 Humanized antibody affinity, internalization ability and specificity 1. Humanized antibody affinity verification-1 ELISA The day before the experiment, the target antigen hB7-H3-Fc was diluted to 2 μg / mL with 1×PBS, then added to an ELISA plate (96-well plate) at 100 μL / well and coated overnight at 4 ° C. The next day, the coating solution was dehydrated, washed twice with washing solution PBST, tapped to dry, and then blocking solution (PBST containing 1% BSA) was added at 200 μL / well and blocked for 2 h at 37 ° C. The blocking solution was dehydrated, washed twice with washing solution PBST, tapped to dry, and then gradient-diluted humanized antibody (initial concentration 300 nM, 3-fold gradient dilution, solvent 1×PBS) was added at 100 μL / well and reacted at 37 ° C. for 1 h. The humanized antibody was dehydrated, washed five times with PBST, and tapped dry, and then 100 μL / well of HRP-labeled goat anti-human Kappa light chain secondary antibody (Invitrogen, Catalog No. A18853, diluted 1:2000 with 1×PBS) was added and reacted at 37°C for 1 h. The secondary antibody was dehydrated, washed eight times with PBST, and tapped dry, and then 100 μL / well of TMB color developing solution was added and reacted at 37°C for 5 min to 10 min. 100 μL / well of ELISA stop solution was added, and within 15 min, the absorbance value was read at a wavelength of 450 nm using a microplate reader. The results are shown in Figure 3.

[0263] 2. Humanized antibody affinity verification - 2 Flow cytometry MDA-MB-468 cells in the logarithmic growth phase were harvested, centrifuged at 1500 rpm for 5 min, the supernatant was removed, and the cells were resuspended in 1x PBS, centrifuged at 1500 rpm for 5 min, the supernatant was removed, and this was repeated once. MDA-MB-468 cells were added to a V-shaped 96-well plate at 50 μL / well (200,000 cells), centrifuged at 1500 rpm for 5 min, and the PBS was discarded. Gradient-diluted humanized antibody (initial concentration 100 nM, 3-fold gradient dilution, solvent 1x PBS) was added at 100 μL / well, and the cells were resuspended. The cells were incubated on ice for 1 h. After the incubation, the cells were centrifuged at 2000 rpm for 5 min, the supernatant was removed, and the cells were resuspended in 200 μL / well of 1x PBS, followed by centrifugation again, the supernatant was removed, and this was repeated twice. PE-labeled goat anti-human IgG Fc secondary antibody (Invitrogen, Cat. No. 12-4998-82, diluted 1:500 with 1x PBS) was added at 100 μL / well, and the cells were resuspended. The cells were protected from light and incubated on ice for 30 min. After incubation, the cells were centrifuged at 2000 rpm for 5 min, the supernatant was removed, and the cells were resuspended in 200 μL / well of 1x PBS, followed by centrifugation again and removal of the supernatant, which was repeated twice. The cells were resuspended by adding 200 μL / well of 1x PBS. The results were detected using a cytoflex flow cytometer (Beckman). The results are shown in Figure 4.

[0264] 3. Detection of humanized antibody internalization by flow cytometry A humanized antibody was prepared at 30 μg / mL using ice-cooled cell washing solution (PBS containing 2% FBS), then mixed with cells (MDA-MB-468 cells or N87 cells, 500,000 cells) at a volume ratio of 1:1 (final volume 100 μL), mixed uniformly, and then ice-bathed for 1 h. The mixture was centrifuged at 4 ° C and 1200 r / min for 5 min, and the supernatant was discarded. The cells were resuspended using ice-cooled cell washing solution, centrifuged at 4 ° C and 1200 r / min for 5 min, the supernatant was removed, and this was repeated twice. The cells were resuspended by adding 200 μL of ice-cooled cell washing solution, incubated at 37 ° C for 2 h, and the antibody bound to the cell surface was internalized, then transferred to an ice bath, and ice-cooled cell washing solution was added to stop the internalization, centrifuged at 4 ° C, 1200 r / min for 5 min, the supernatant was discarded, and 100 μL of a dilution solution of PE-labeled goat anti-human IgG Fc secondary antibody (Invitrogen, Catalog No. 12-4998-82, ice-cooled cell washing solution: PE-labeled goat anti-human IgG Fc secondary antibody = 500: 1 was used to prepare a dilution solution of PE-labeled goat anti-human IgG Fc secondary antibody) was added, the cells were resuspended, incubated at 4 ° C in the dark for 30 min, centrifuged at 4 ° C, 1200 r / min for 5 min, and the supernatant was discarded. The cells were resuspended in ice-cooled cell washing solution, centrifuged at 4°C and 1200 r / min for 5 min, the supernatant was discarded, the cells were resuspended in ice-cooled 1x PBS, centrifuged at 4°C and 1200 r / min for 5 min, the supernatant was discarded, and 200 μL of ice-cooled 1x PBS was added to resuspend the cells, and the mean fluorescence intensity was measured using a flow cytometer.

[0265] [Table 6(C)]

[0266] 4. Validation of humanized antibody specificity - flow cytometry Cells in the logarithmic growth phase (VISTA-CHO cells, Tim3-CHO cells, or Lag3-CHO cells) were harvested, centrifuged at 1500 rpm for 5 min, the supernatant was removed, and the cells were resuspended in 1x PBS, centrifuged at 1500 rpm for 5 min, the supernatant was removed, and this was repeated once. The cells were added to a V-type 96-well plate at 50 μL / well (500,000 cells), centrifuged at 1500 rpm for 5 min, and the PBS was discarded. A humanized antibody (concentration 100 nM, solvent 1x PBS) was added at 100 μL / well, and the cells were resuspended. The cells were incubated on ice for 1 h. After the incubation, the cells were centrifuged at 2000 rpm for 5 min, the supernatant was removed, and the cells were resuspended in 200 μL / well of 1x PBS, followed by centrifugation again, the supernatant was removed, and this was repeated twice. PE-labeled goat anti-human IgG Fc secondary antibody (Invitrogen, Cat. No. 12-4998-82, diluted 1:500 in 1x PBS) was added at 100 μL / well, and the cells were resuspended. The cells were protected from light and incubated on ice for 30 min. After incubation, the cells were centrifuged at 2000 rpm for 5 min, the supernatant was removed, and the cells were resuspended in 200 μL / well of 1x PBS, followed by centrifugation again, the supernatant was removed, and this was repeated twice. The cells were resuspended by adding 200 μL / well of 1x PBS. The cells were detected using a cytoflex flow cytometer (Beckman).

[0267] As a result of the detection, as shown in FIG. 5, none of the antibodies V1 to V5 bound to VISTA-CHO cells, Tim3-CHO cells, or Lag3-CHO cells.

[0268] 5. Species specificity of humanized antibodies The day before the experiment, the target antigens, cynomolgus monkey B7-H3 (novoprotein CA61), mouse B7-H3 (SinoBiological, 50973-M08H) and hB7-H3-his, were diluted to 2 μg / mL with 1×PBS, and then added to an ELISA plate (96-well plate) in two rows at 100 μL / well and coated overnight at 4 ° C. The next day, the coating solution was dehydrated, washed twice with washing solution PBST, tapped to dry, and then 200 μL / well of blocking solution was added and blocked at 37 ° C for 2 h. The blocking solution was dehydrated, washed twice with washing solution PBST, tapped to dry, and then 100 μL / well of gradient-diluted antibody V3 (initial concentration 2 μg / mL, 2-fold gradient dilution) was added and reacted at 37 ° C for 2 h. Antibody V3 was dehydrated, washed five times with PBST, tapped dry, and then anti-human kappa light chains HRP (Sigma, A7164, diluted 1:10000 with 1x PBS) was added at 100 μL / well and reacted at 37°C for 1 h. Secondary antibodies were dehydrated, washed eight times with PBST, tapped dry, and then TMB color development solution was added at 100 μL / well and reacted at 37°C for 10 min. 0.1M H 2 SO 4 Stop solution was added, and within 15 min, absorbance values ​​were read at a wavelength of 450 nm using a microplate reader.

[0269] [Table 6(D)]

[0270] As can be seen from Table 13, antibody V3 was able to bind to human and cynomolgus monkey B7-H3, but did not bind to mouse B7-H3.

Claims

1. An antibody or antigen-binding fragment, Specific binding to B7-H3 and (a) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6; (b) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7; (c) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; (d) a VL CDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 9-13; (e) a VL CDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 14-18; (f) a VL CDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 19-23; An antibody or antigen-binding fragment thereof.

2. the antibody or antigen-binding fragment comprises a VH CDR1 set forth in SEQ ID NO:6, a VH CDR2 set forth in SEQ ID NO:7, and a VH CDR3 set forth in SEQ ID NO:8; 2. The antibody or antigen-binding fragment of claim 1.

3. the antibody or antigen-binding fragment comprises a VL CDR1 set forth in any one of SEQ ID NOs: 9-13, a VL CDR2 set forth in any one of SEQ ID NOs: 14-18, and a VL CDR3 set forth in any one of SEQ ID NOs: 19-23; 2. The antibody or antigen-binding fragment of claim 1.

4. the antibody or antigen-binding fragment comprises a VH CDR1 set forth in SEQ ID NO:6, a VH CDR2 set forth in SEQ ID NO:7, a VH CDR3 set forth in SEQ ID NO:8, a VL CDR1 set forth in SEQ ID NO:9, a VL CDR2 set forth in SEQ ID NO:14, and a VL CDR3 set forth in SEQ ID NO:19; or the antibody or antigen-binding fragment comprises a VH CDR1 set forth in SEQ ID NO:6, a VH CDR2 set forth in SEQ ID NO:7, a VH CDR3 set forth in SEQ ID NO:8, a VL CDR1 set forth in SEQ ID NO:10, a VL CDR2 set forth in SEQ ID NO:15, and a VL CDR3 set forth in SEQ ID NO:20; or the antibody or antigen-binding fragment comprises a VH CDR1 set forth in SEQ ID NO:6, a VH CDR2 set forth in SEQ ID NO:7, a VH CDR3 set forth in SEQ ID NO:8, a VL CDR1 set forth in SEQ ID NO:11, a VL CDR2 set forth in SEQ ID NO:16, and a VL CDR3 set forth in SEQ ID NO:21; or the antibody or antigen-binding fragment comprises a VH CDR1 set forth in SEQ ID NO:6, a VH CDR2 set forth in SEQ ID NO:7, a VH CDR3 set forth in SEQ ID NO:8, a VL CDR1 set forth in SEQ ID NO:12, a VL CDR2 set forth in SEQ ID NO:17, and a VL CDR3 set forth in SEQ ID NO:22; or The antibody or antigen-binding fragment comprises a VH CDR1 set forth in SEQ ID NO:6, a VH CDR2 set forth in SEQ ID NO:7, a VH CDR3 set forth in SEQ ID NO:8, a VL CDR1 set forth in SEQ ID NO:13, a VL CDR2 set forth in SEQ ID NO:18, and a VL CDR3 set forth in SEQ ID NO:23; 2. The antibody or antigen-binding fragment of claim 1.

5. the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence set forth in any one of SEQ ID NOs: 24, 35-38, or an amino acid sequence having at least 80% or 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 24, 35-38, or an amino acid sequence having one or more conservative amino acid substitutions to the amino acid sequence set forth in any one of SEQ ID NOs: 24, 35-38; and / or the light chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence set forth in any one of SEQ ID NOs: 25 to 29, 39 to 41, or an amino acid sequence having at least 80% or 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 25 to 29, 39 to 41, or an amino acid sequence having one or more conservative amino acid substitutions to the amino acid sequence set forth in any one of SEQ ID NOs: 25 to 29, 39 to 41; 2. The antibody or antigen-binding fragment of claim 1.

6. An antibody or antigen-binding fragment, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:24 and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:25; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:24 and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:26; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:24 and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:27; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:24 and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:28; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:24 and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:29; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 35 and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 39; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 36 and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 39; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 36 and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 40; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 37 and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 41; or the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 38, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 41; An antibody or antigen-binding fragment thereof.

7. the antibody or antigen-binding fragment is of the IgG isotype; or the antibody or antigen-binding fragment is of the IgG1 or IgG4 isotype; 2. The antibody or antigen-binding fragment of claim 1.

8. the antibody or antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 32 or 33, or an amino acid sequence having at least 80% or 90% identity to the amino acid sequence set forth in SEQ ID NO: 32 or 33, or an amino acid sequence having one or more conservative amino acid substitutions to the amino acid sequence set forth in SEQ ID NO: 32 or 33; and / or the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 34, or an amino acid sequence having at least 80% or 90% identity to the amino acid sequence set forth in SEQ ID NO: 34, or an amino acid sequence having one or more conservative amino acid substitutions to the amino acid sequence set forth in SEQ ID NO: 34; 2. The antibody or antigen-binding fragment of claim 1.

9. An antibody, the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 24 and a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO: 32, and the light chain of the antibody comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 25 and a light chain constant region of the amino acid sequence set forth in SEQ ID NO: 34; or the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 24 and a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO: 32, and the light chain of the antibody comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 26 and a light chain constant region of the amino acid sequence set forth in SEQ ID NO: 34; or the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 24 and a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO: 32, and the light chain of the antibody comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 27 and a light chain constant region of the amino acid sequence set forth in SEQ ID NO: 34; or the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 24 and a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO: 32, and the light chain of the antibody comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 28 and a light chain constant region of the amino acid sequence set forth in SEQ ID NO: 34; or the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 24 and a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO: 32, and the light chain of the antibody comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 29 and a light chain constant region of the amino acid sequence set forth in SEQ ID NO: 34; or the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 35 and a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO: 32, and the light chain of the antibody comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 39 and a light chain constant region of the amino acid sequence set forth in SEQ ID NO: 34; or the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 36 and a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO: 32, and the light chain of the antibody comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 39 and a light chain constant region of the amino acid sequence set forth in SEQ ID NO: 34; or the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 36 and a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO: 32, and the light chain of the antibody comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 40 and a light chain constant region of the amino acid sequence set forth in SEQ ID NO: 34; or the heavy chain of the antibody comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 37 and a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO: 32, and the light chain of the antibody comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 41 and a light chain constant region of the amino acid sequence set forth in SEQ ID NO: 34; or the heavy chain of the antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 38 and a heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 32, and the light chain of the antibody comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO: 41 and a light chain constant region having the amino acid sequence shown in SEQ ID NO: 34; An antibody characterized by:

10. A biomaterial comprising: a polynucleotide encoding part or all of the antibody or antigen-binding fragment according to any one of claims 1 to 8, or the antibody according to claim 9; or an expression carrier comprising a polynucleotide encoding part or all of the antibody or antigen-binding fragment according to any one of claims 1 to 8, or the antibody according to claim 9; or A cell characterized in that it is a cell comprising a polynucleotide encoding part or all of the antibody or antigen-binding fragment according to any one of claims 1 to 8, or the antibody according to claim 9. Biomaterials.

11. 1. A pharmaceutical composition comprising: A composition comprising part or all of the antibody or antigen-binding fragment of any one of claims 1 to 8 or the antibody of claim 9, and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising:

12. 10. The use of part or all of an antibody or antigen-binding fragment according to any one of claims 1 to 8 or an antibody according to claim 9 in the manufacture of a medicament for diagnosing and / or treating a disease, comprising: Alternatively, the disease is selected from tumors, respiratory diseases, skin and musculoskeletal diseases, genitourinary diseases, nervous system diseases, and digestive diseases; alternatively, the tumor is a benign tumor or cancer; alternatively, the tumor is a blood tumor or a solid tumor; alternatively, the tumor is a B7-H3 expression-positive tumor; alternatively, the tumor is selected from melanoma, lung cancer such as non-small cell lung cancer, colorectal cancer, head and neck cancer, kidney cancer, prostate cancer such as castration-resistant prostate cancer, breast cancer, gastric cancer, liver cancer such as hepatocellular carcinoma, cervical cancer, ovarian cancer such as ovarian epithelial cancer, glioma such as pediatric brainstem glioma, pancreatic cancer such as pancreatic ductal carcinoma, leukemia, mesothelioma, squamous cell carcinoma, neuroblastoma, desmoplastic small round cell tumor, medulloblastoma, meningioma, peritoneal malignant tumor, sarcoma, brain cancer, central nervous system tumor, and brain metastasis; application.

13. The use of the pharmaceutical composition of claim 11 in the manufacture of a drug for diagnosing and / or treating a disease, comprising: Alternatively, the disease is selected from tumors, respiratory diseases, skin and musculoskeletal diseases, genitourinary diseases, nervous system diseases, and digestive diseases; alternatively, the tumor is a benign tumor or cancer; alternatively, the tumor is a blood tumor or a solid tumor; alternatively, the tumor is a B7-H3 expression-positive tumor; alternatively, the tumor is selected from melanoma, lung cancer such as non-small cell lung cancer, colorectal cancer, head and neck cancer, kidney cancer, prostate cancer such as castration-resistant prostate cancer, breast cancer, gastric cancer, liver cancer such as hepatocellular carcinoma, cervical cancer, ovarian cancer such as ovarian epithelial cancer, glioma such as pediatric brainstem glioma, pancreatic cancer such as pancreatic ductal carcinoma, leukemia, mesothelioma, squamous cell carcinoma, neuroblastoma, desmoplastic small round cell tumor, medulloblastoma, meningioma, peritoneal malignant tumor, sarcoma, brain cancer, central nervous system tumor, and brain metastasis; application.

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