Anti-b7-h3 antibodies and methods of making and using the same

Abstract

Provided are an anti-B7-H3 antibody and preparation and application thereof, and a drug conjugate and recombinant protein comprising the antibody. The antibody of the present application can be effectively endocytosed by cells, and the antibody conjugate prepared using the antibody of the present application exhibits a tumor inhibition effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, more particularly to an anti-B7-H3 antibody and preparation and application thereof. BACKGROUND

[0002] Tumor growth and metastasis depend to a large extent on its ability to evade host immune surveillance and overcome host defenses. While most tumors express antigens that can be recognized by the host immune system to varying degrees, in many cases, an inadequate immune response is elicited due to inefficient activation of effector T cells.

[0003] CD4+ T-lymphocytes are the major orchestrators of most mammalian immune and autoimmune responses. Activation of CD4+ helper T-cells has been found to be mediated by costimulation between antigen presenting cells and naive CD4+ T-lymphocytes, a process that requires interaction between the two cells. In the first interaction, the antigen presenting cell must display the relevant target antigen via the major histocompatibility complex (MHC) on the cell surface so that it can bind to the T-cell receptor ("TCR") of the naive CD4+ T-lymphocyte. In the second interaction, a ligand of the antigen presenting cell must bind to the CD28 receptor on the surface of the CD4+ T-lymphocyte. Upon receiving the costimulatory signal, the CD4 helper T-cell is able to produce cytokines, such as interleukin-2 and interleukin-12, which further develop into Thl cells. These cells are able to produce interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α), which further mediate inflammatory responses to target cells expressing the target antigen. B-cell activation and proliferation also occurs, which in turn produces specific antibodies against the target antigen. During T-cell activation, the absence of either costimulatory signal will result in the T-cell entering a state of clonal anergy. In pathological conditions, Thl cells are key players in a variety of organ-specific autoimmune diseases, such as type I diabetes, rheumatoid arthritis, and multiple sclerosis.

[0004] B7 family members are members of the immunoglobulin superfamily with immunoglobulin-V-like and immunoglobulin-C-like domains (e.g., IgV-IgC). The IgV and IgC domains of B7 family members are each encoded by a single exon, with additional exons encoding a leader sequence, a transmembrane domain, and a cytoplasmic domain. The cytoplasmic domain is short, ranging in length from 19 to 62 amino acid residues and can be encoded by multiple exons.

[0005] B7-H3 is unique in that its predominant human form contains two extracellular, tandem IgV-IgC domains (i.e., IgV-IgC-IgV-IgC). Members of the B7 family are predicted to form back-to-back, non-covalent homodimers on the cell surface, and such dimers have been found to be associated with B7-1 (CD80) and B7-2 (CD86). A variant of four immunoglobulin extracellular domains ("4Ig-B7-H3") has been identified and found to be the more common human protein form. Because the native mouse form (2Ig) and the human 4Ig form show similar functionality, no functional differences between the two forms have been observed. The 4Ig-B7-H3 molecule inhibits natural killer cell-mediated lysis of cancer cells). Human B7-H3 (2Ig form) has been found to promote T-cell activation and IFN-γ production by binding to specific receptors on activated T cells.

[0006] The mode of action of B7-H3 is complex when it mediates T-cell costimulation and co-inhibition. B7-H3 binds to (TREM)-like transcript 2 (TLT-2) and costimulates T-cell activation, and to a hitherto unidentified receptor to mediate T-cell co-inhibition. In addition, through interaction with unknown receptors, B7-H3 becomes an inhibitor of natural killer cells and osteoblasts, and this inhibition can occur through interaction with members of most signaling pathways that T-cell receptors (TCRs) use to regulate gene transcription (e.g., NFAT, NF-κB, or AP-1 factors). B7-H3 costimulates the proliferation of CD4+ and CD8+ T-cells. B7-H3 also stimulates IFN-γ production and CD8+ lytic activity. However, the protein can also inhibit T-cell activation through NFAT (nuclear factor for activated T-cells), NF-κB (nuclear factor kappa B), and AP-1 (activator protein-1) factors. B7-H3 is also believed to inhibit Th1, Th2, or Th17 in vivo.

[0007] Multiple independent studies have shown that human malignancy cells exhibit a significant increase in B7-H3 protein expression and that this increase in expression is associated with an increase in disease severity, indicating that B7-H3 is exploited by tumors as an immune evasion pathway. Human B7-H3 is known to be expressed on a variety of other cancer cells (e.g., gastric cancer, ovarian cancer, and non-small cell lung cancer) in addition to neuroblastoma cells. By blocking the binding of B7-H3 to its receptors, or developing a corresponding targeted therapeutic product using B7-H3 as a tumor overexpressed antigen, is a potential means of treating a variety of different tumors.

[0008] Currently, there are many deficiencies in the prior art tumor targeted therapeutic products developed for B7-H3, and there is a need in the art to develop new therapeutic antibody related products. SUMMARY

[0009] The present application aims to provide an anti-B7-H3 antibody and preparation and application thereof.

[0010] In a first aspect of the present application, a heavy chain variable region of an antibody is provided, which comprises the following three complementarity determining regions CDRs:

[0011] a VH-CDR1 as represented by SEQ ID NO: 3n,

[0012] a VH-CDR2 as represented by SEQ ID NO: 3n+1, and

[0013] a VH-CDR3 as represented by SEQ ID NO: 3n+2;

[0014] wherein each n is independently 11, 12, 13, 14, 15, 16, or 17;

[0015] Alternatively, the heavy chain variable region comprises the following three complementarity determining regions CDRs:

[0016] a VH-CDR1 as represented by SEQ ID NO: 97,

[0017] a VH-CDR2 as represented by SEQ ID NO: 54, and

[0018] a VH-CDR3 as represented by SEQ ID NO: 35;

[0019] or

[0020] a VH-CDR1 as represented by SEQ ID NO: 33,

[0021] a VH-CDR2 as represented by SEQ ID NO: 54, and

[0022] a VH-CDR3 as represented by SEQ ID NO: 35;

[0023] or

[0024] a VH-CDR1 as represented by SEQ ID NO: 36,

[0025] a VH-CDR2 as represented by SEQ ID NO: 55, and

[0026] a VH-CDR3 as represented by SEQ ID NO: 56;

[0027] or

[0028] a VH-CDR1 as represented by SEQ ID NO: 57,

[0029] a VH-CDR2 as represented by SEQ ID NO: 40, and

[0030] VH-CDR3 as set forth in SEQ ID NO: 41;

[0031] or

[0032] VH-CDR1 as set forth in SEQ ID NO: 51,

[0033] VH-CDR2 as set forth in SEQ ID NO: 52, and

[0034] VH-CDR3 as set forth in SEQ ID NO: 58;

[0035] or

[0036] VH-CDR1 as set forth in SEQ ID NO: 51,

[0037] VH-CDR2 as set forth in SEQ ID NO: 59, and

[0038] VH-CDR3 as set forth in SEQ ID NO: 60;

[0039] wherein any of the above amino acid sequences further includes a derivative sequence that optionally has at least one amino acid added to, deleted from, modified in, and / or substituted for, and which is capable of retaining B7-H3 binding affinity.

[0040] In another preferred embodiment, the heavy chain variable region comprises the following three complementarity determining regions (CDRs):

[0041]

[0042] In another preferred embodiment, the heavy chain variable region has an amino acid sequence as set forth in any one of SEQ ID NOs: 79-96.

[0043] In a second aspect of the application, there is provided a heavy chain of an antibody, the heavy chain having a heavy chain variable region as described in the first aspect of the application.

[0044] In another preferred embodiment, the heavy chain further comprises a heavy chain constant region.

[0045] In another preferred embodiment, the heavy chain constant region is of human origin.

[0046] In another preferred embodiment, the heavy chain constant region is a human antibody heavy chain IgGl or IgG4 constant region.

[0047] In a third aspect of the application, there is provided a light chain variable region of an antibody, the light chain variable region comprising the following three complementarity determining regions (CDRs):

[0048] VL-CDR1 as shown in SEQ ID NO: 3m+1,

[0049] VL-CDR2 as shown in SEQ ID NO: 3m+2, and

[0050] VL-CDR3 as shown in SEQ ID NO: 3m+3;

[0051] wherein each m is independently 0, 1, 2, 3, 4, 5, 6, or 7;

[0052] Alternatively, the light chain variable region comprises the following three complementarity determining regions CDRs:

[0053] VL-CDR1 as shown in SEQ ID NO: 1,

[0054] VL-CDR2 as shown in SEQ ID NO: 25, and

[0055] VL-CDR3 as shown in SEQ ID NO: 3;

[0056] or

[0057] VL-CDR1 as shown in SEQ ID NO: 1,

[0058] VL-CDR2 as shown in SEQ ID NO: 26, and

[0059] VL-CDR3 as shown in SEQ ID NO: 3;

[0060] or

[0061] VL-CDR1 as shown in SEQ ID NO: 27,

[0062] VL-CDR2 as shown in SEQ ID NO: 5, and

[0063] VL-CDR3 as shown in SEQ ID NO: 6;

[0064] or

[0065] VL-CDR1 as shown in SEQ ID NO: 27,

[0066] VL-CDR2 as shown in SEQ ID NO: 28, and

[0067] VL-CDR3 as shown in SEQ ID NO: 6;

[0068] or

[0069] VL-CDR1 as shown in SEQ ID NO: 29,

[0070] VL-CDR2 as set forth in SEQ ID NO: 11, and

[0071] VL-CDR3 as set forth in SEQ ID NO: 12;

[0072] or

[0073] VL-CDR1 as set forth in SEQ ID NO: 30,

[0074] VL-CDR2 as set forth in SEQ ID NO: 23, and

[0075] VL-CDR3 as set forth in SEQ ID NO: 31;

[0076] or

[0077] VL-CDR1 as set forth in SEQ ID NO: 30,

[0078] VL-CDR2 as set forth in SEQ ID NO: 23, and

[0079] VL-CDR3 as set forth in SEQ ID NO: 32;

[0080] wherein any of the above amino acid sequences further includes a derivative sequence that optionally has at least one amino acid added to, deleted from, modified in, and / or substituted for, and is capable of retaining B7-H3 binding affinity.

[0081] In another preferred embodiment, the light chain variable region comprises the following three complementarity determining regions (CDRs):

[0082]

[0083]

[0084] In another preferred embodiment, the light chain variable region has an amino acid sequence as set forth in any one of SEQ ID NOs: 61-78.

[0085] In a fourth aspect of the application, there is provided a light chain of an antibody, the light chain having a light chain variable region as defined in the third aspect of the application.

[0086] In another preferred embodiment, the light chain further comprises a light chain constant region.

[0087] In another preferred embodiment, the light chain constant region is of human origin.

[0088] In another preferred embodiment, the light chain constant region is of human antibody light chain kappa constant region.

[0089] In a fifth aspect of the application, there is provided an antibody, the antibody having:

[0090] (1) a heavy chain variable region as described in the first aspect of the present application; and / or

[0091] (2) a light chain variable region as described in the third aspect of the present application;

[0092] Alternatively, the antibody has: a heavy chain as described in the second aspect of the present application; and / or a light chain as described in the fourth aspect of the present application,

[0093] wherein any one of the above amino acid sequences further comprises a derivative sequence that optionally has at least one amino acid added to, deleted from, modified in, and / or substituted for, and that is capable of retaining the B7-H3 binding affinity.

[0094] In another preferred embodiment, any one of the above CDR sequences further comprises a derivative CDR sequence that has 1, 2, or 3 amino acids added to, deleted from, modified in, and / or substituted for, and that is capable of retaining the B7-H3 binding affinity of the VH and VL comprising the derivative CDR sequence.

[0095] In another preferred embodiment, the ratio of the B7-H3 binding affinity of the derivative antibody F1 to the B7-H3 binding affinity of the corresponding non-derivative antibody F0 (F1 / F0) is between 0.5 and 2, preferably between 0.7 and 1.5, and more preferably between 0.8 and 1.2.

[0096] In another preferred embodiment, the number of amino acids added to, deleted from, modified in, and / or substituted for is between 1 and 5 (e.g., between 1 and 3, preferably between 1 and 2, and more preferably 1).

[0097] In another preferred embodiment, the derivative sequence that has at least one amino acid added to, deleted from, modified in, and / or substituted for, and that is capable of retaining the B7-H3 binding affinity is an amino acid sequence that has at least 96% homology or sequence identity.

[0098] In another preferred embodiment, the antibody further comprises a heavy chain constant region and / or a light chain constant region.

[0099] In another preferred embodiment, the heavy chain constant region is of human origin, and / or the light chain constant region is of human origin.

[0100] In another preferred embodiment, the heavy chain constant region is a human antibody heavy chain IgGl or IgG4 constant region, and the light chain constant region is a human antibody light chain kappa constant region.

[0101] In another preferred embodiment, the antibody is selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or a combination thereof.

[0102] In another preferred embodiment, the ratio of the immunogenicity of the chimeric antibody in humans (Z1) to the immunogenicity of a non-chimeric antibody (e.g., a murine antibody) in humans (Z0) (Z1 / Z0) is between 0 and 0.5, preferably between 0 and 0.2, more preferably between 0 and 0.05 (e.g., between 0.001 and 0.05).

[0103] In another preferred embodiment, the antibody is a partially or fully humanized, or fully human monoclonal antibody.

[0104] In another preferred embodiment, the antibody is a diabody, or a single chain antibody.

[0105] In another preferred embodiment, the antibody is an antibody full-length protein, or an antigen binding fragment.

[0106] In another preferred embodiment, the antibody is a bispecific antibody, or a multispecific antibody.

[0107] In another preferred embodiment, the antibody has one or more properties selected from the group consisting of:

[0108] (a) inhibiting tumor cell migration or metastasis;

[0109] (b) inhibiting tumor growth; and

[0110] (c) relieving tumor immune suppression of T cells.

[0111] In another preferred embodiment, the antibody has a heavy chain variable region as described in the first aspect of the application and a light chain variable region as described in the third aspect of the application;

[0112] wherein the heavy chain variable region and the light chain variable region are as follows:

[0113]

[0114] wherein any of the above amino acid sequences further includes a derivative sequence that optionally has at least one amino acid added, deleted, modified and / or substituted, and that retains B7-H3 binding affinity.

[0115] In another preferred embodiment, the antibody has a heavy chain variable region as described in the first aspect of the application and a light chain variable region as described in the third aspect of the application; wherein,

[0116] the heavy chain variable region includes the following three complementarity determining regions (CDRs):

[0117] VH-CDR1 as set forth in SEQ ID NO: 97,

[0118] VH-CDR2 as set forth in SEQ ID NO: 54, and

[0119] VH-CDR3 as set forth in SEQ ID NO: 35;

[0120] the light chain variable region comprises the following three complementarity determining regions CDRs:

[0121] VL-CDR1 as set forth in SEQ ID NO: 1,

[0122] VL-CDR2 as set forth in SEQ ID NO: 26, and

[0123] VL-CDR3 as set forth in SEQ ID NO: 3;

[0124] or

[0125] the heavy chain variable region comprises the following three complementarity determining regions CDRs:

[0126] VH-CDR1 as set forth in SEQ ID NO: 33,

[0127] VH-CDR2 as set forth in SEQ ID NO: 54, and

[0128] VH-CDR3 as set forth in SEQ ID NO: 35;

[0129] the light chain variable region comprises the following three complementarity determining regions CDRs:

[0130] VL-CDR1 as set forth in SEQ ID NO: 1,

[0131] VL-CDR2 as set forth in SEQ ID NO: 26, and

[0132] VL-CDR3 as set forth in SEQ ID NO: 3;

[0133] or

[0134] the heavy chain variable region comprises the following three complementarity determining regions CDRs:

[0135] VH-CDR1 as set forth in SEQ ID NO: 33,

[0136] VH-CDR2 as set forth in SEQ ID NO: 54, and

[0137] VH-CDR3 as set forth in SEQ ID NO: 35;

[0138] the light chain variable region comprises the following three complementarity determining regions CDRs:

[0139] VL-CDR1 as set forth in SEQ ID NO: 1,

[0140] VL-CDR2 as set forth in SEQ ID NO: 2, and

[0141] VL-CDR3 as set forth in SEQ ID NO: 3.

[0142] or

[0143] the heavy chain variable region comprises the following three complementarity determining regions (CDRs):

[0144] VH-CDR1 as set forth in SEQ ID NO: 33,

[0145] VH-CDR2 as set forth in SEQ ID NO: 34, and

[0146] VH-CDR3 as set forth in SEQ ID NO: 35.

[0147] the light chain variable region comprises the following three complementarity determining regions (CDRs):

[0148] VL-CDR1 as set forth in SEQ ID NO: 1,

[0149] VL-CDR2 as set forth in SEQ ID NO: 25, and

[0150] VL-CDR3 as set forth in SEQ ID NO: 3.

[0151] In another preferred embodiment, the heavy chain variable region of the antibody comprises an amino acid sequence set forth in any one of SEQ ID NOs: 79-96; and / or the light chain variable region of the antibody comprises an amino acid sequence set forth in any one of SEQ ID NOs: 61-78.

[0152] In another preferred embodiment, the antibody is a humanized antibody, and the heavy chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 83; and the light chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 64.

[0153] Alternatively, the heavy chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 83; and the light chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 62.

[0154] Alternatively, the heavy chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 81; and the light chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 63.

[0155] In another preferred embodiment, the amino acid sequence of the heavy chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 79-96 of the Sequence Listing.

[0156] In another preferred embodiment, the amino acid sequence of the heavy chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 79-96 of the Sequence Listing.

[0157] A recombinant protein, the recombinant protein comprising:

[0158] (i) a heavy chain variable region as described in the first aspect of the application, a heavy chain as described in the second aspect of the application, a light chain variable region as described in the third aspect of the application, a light chain as described in the fourth aspect of the application, or an antibody as described in the fifth aspect of the application; and

[0159] (ii) an optional tag sequence to assist expression and / or purification.

[0160] In another preferred embodiment, the tag sequence comprises a 6His tag.

[0161] In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

[0162] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0163] In another aspect of the application, there is provided a chimeric antigen receptor (CAR) targeting B7-H3, the antigen binding domain of the CAR comprising a heavy chain variable region as described in the first aspect of the application, or a light chain variable region as described in the third aspect of the application.

[0164] In another preferred embodiment, the CAR has the following structure as shown in Formula I:

[0165] L-scFv-H-TM-C-CD3ζ (I)

[0166] wherein,

[0167] each “-” is independently a linking peptide or a peptide bond;

[0168] L is nothing or a signal peptide sequence;

[0169] H is nothing or a hinge region;

[0170] TM represents a transmembrane domain;

[0171] C is a co-stimulatory signaling molecule;

[0172] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ.

[0173] In a seventh aspect of the invention, a polynucleotide is provided, the polynucleotide encoding a polypeptide selected from the group consisting of:

[0174] (1) The heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention; and

[0175] (2) The recombinant protein as described in the sixth aspect of the present invention.

[0176] In an eighth aspect of the invention, a carrier is provided, the carrier containing the polynucleotide described in the seventh aspect of the invention.

[0177] In another preferred embodiment, the vector includes: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0178] In a ninth aspect of the invention, a genetically engineered host cell is provided, the host cell containing the vector or genome of the eighth aspect of the invention in which the polynucleotides of the seventh aspect of the invention are integrated.

[0179] In a tenth aspect of the present invention, an antibody conjugate is provided, the antibody conjugate comprising:

[0180] (a) An antibody portion, said antibody portion being selected from the group consisting of: the heavy chain variable region as described in the first aspect of the invention, the heavy chain as described in the second aspect of the invention, the light chain variable region as described in the third aspect of the invention, the light chain as described in the fourth aspect of the invention, or an antibody as described in the fifth aspect of the invention, or a combination thereof; and

[0181] (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0182] In another preferred embodiment, the antibody portion is coupled to the coupling portion via a chemical bond or a linker.

[0183] In another preferred embodiment, the antibody-drug conjugate (ADC) has the following molecular formula:

[0184]

[0185] wherein:

[0186] Ab is an antibody against B7-H3,

[0187] LU is a linker (also known as a linker);

[0188] D is a drug;

[0189] and subscript p is a value selected from 1-10, preferably 1-8.

[0190] In another preferred embodiment, the drug is selected from the group consisting of a chemotherapeutic drug, a radiotherapy drug, a hormone therapy drug, or an immunotherapy drug.

[0191] In another preferred embodiment, the toxin is selected from the group consisting of a taxane, a maytansinoid, an auristatin, a calicheamicin, an anthracycline, docetaxel, a cathepsin, ricin, gelonin, Pseudomonas exotoxin, diphtheria toxin, RNase, and a radioisotope.

[0192] In an eleventh aspect of the present application, there is provided an immune cell expressing or having exposed on the cell membrane an antibody according to the fifth aspect of the present application.

[0193] In another preferred embodiment, the immune cell comprises an NK cell, a T cell.

[0194] In another preferred embodiment, the immune cell is from a human or a non-human mammal (e.g., a murine).

[0195] In a twelfth aspect of the present application, there is provided a pharmaceutical composition comprising:

[0196] (i) an active ingredient selected from the group consisting of a heavy chain variable region according to the first aspect of the present application, a heavy chain according to the second aspect of the present application, a light chain variable region according to the third aspect of the present application, a light chain according to the fourth aspect of the present application, or an antibody according to the fifth aspect of the present application, a recombinant protein according to the sixth aspect of the present application, an antibody conjugate according to the tenth aspect of the present application, an immune cell according to the eleventh aspect of the present application, or a combination thereof; and

[0197] (ii) a pharmaceutically acceptable carrier.

[0198] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0199] In another preferred embodiment, the pharmaceutical composition is an injection.

[0200] In another preferred embodiment, the pharmaceutical composition comprises 0.01-99.99% of the antibody of the fifth aspect of the present application, the recombinant protein of the sixth aspect of the present application, the antibody conjugate of the tenth aspect of the present application, the immune cell of the eleventh aspect of the present application, or a combination thereof, and 0.01-99.99% of a pharmaceutically acceptable carrier, the percentages being percentages by mass of the pharmaceutical composition.

[0201] In another preferred embodiment, the pharmaceutical composition further comprises a second active ingredient, which comprises a second antibody, or a chemotherapeutic agent.

[0202] In another preferred embodiment, the second antibody is selected from the group consisting of a CTLA4 antibody, a PD-1 antibody, a PD-L1 antibody, a VEGF antibody, a B7-H4 antibody, a HER2 antibody, a LAG3 antibody, a TIM-3 antibody, a 4-1BB antibody, a CD3 antibody, a TIGIT antibody.

[0203] In another preferred embodiment, the chemotherapeutic agent is selected from the group consisting of docetaxel, carboplatin, or a combination thereof.

[0204] In the thirteenth aspect of the present application, there is provided a use of an active ingredient selected from the group consisting of the heavy chain variable region of the first aspect of the present application, the heavy chain of the second aspect of the present application, the light chain variable region of the third aspect of the present application, the light chain of the fourth aspect of the present application, or the antibody of the fifth aspect of the present application, the recombinant protein of the sixth aspect of the present application, the antibody conjugate of the tenth aspect of the present application, the immune cell of the eleventh aspect of the present application, or a combination thereof, wherein the active ingredient is used for (a) preparing a medicament for preventing and / or treating a disease associated with abnormal expression or function of B7-H3; and / or (b) preparing a diagnostic reagent or kit.

[0205] In another preferred embodiment, the diagnostic reagent is a test sheet or a test plate.

[0206] In another preferred embodiment, the disease associated with abnormal expression or function of B7-H3 is selected from the group consisting of a tumor and an autoimmune disease.

[0207] In another preferred embodiment, the tumor is selected from the group consisting of melanoma, mesothelioma, non-small cell lung cancer, breast cancer, liver cancer, gastroesophageal cancer, adenocarcinoma, head and neck cancer, synovial sarcoma, colorectal cancer, renal cancer, bladder cancer, prostate cancer, ovarian cancer, chronic hepatitis C virus infection, advanced solid cancer, gastrointestinal malignancy, endometrial cancer, recurrent melanoma, head and neck squamous cell carcinoma, cutaneous T-cell lymphoma, fallopian tube cancer, peritoneal tumor, muscle-invasive bladder cancer, extensive stage small cell lung cancer, adult acute myeloid leukemia, atypical chronic myeloid leukemia, ovarian epithelial cancer, B-cell chronic lymphocytic leukemia, cutaneous B-cell non-Hodgkin lymphoma, intraocular lymphoma, testicular choriocarcinoma, neuroblastoma, esophageal cancer.

[0208] In another preferred embodiment, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis, ankylosing spondylitis, scleroderma, Takayasu's arteritis, Wegener's granulomatosis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, psoriasis, dermatitis herpetiformis, graft rejection.

[0209] In another preferred embodiment, the diagnostic agent or kit is used for detecting B7-H3 protein in a sample.

[0210] In another preferred embodiment, the diagnostic agent or kit is used for diagnosing a B7-H3 related disease.

[0211] In a fourteenth aspect of the present application, there is provided a method of detecting (including diagnosing) B7-H3 protein in a sample in vitro, the method comprising the steps of:

[0212] (1) contacting, in vitro, the sample with an antibody according to the fifth aspect of the present application;

[0213] (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of B7-H3 protein in the sample.

[0214] In a fifteenth aspect of the present application, there is provided a composition for detecting B7-H3 protein in a sample in vitro, comprising an antibody according to the fifth aspect of the present application, a recombinant protein according to the sixth aspect of the present application, an antibody conjugate according to the tenth aspect of the present application, an immune cell according to the eleventh aspect of the present application, or a combination thereof as an active ingredient.

[0215] In a sixteenth aspect of the present application, a detection plate is provided, which comprises: a substrate (support plate) and a test strip, wherein the test strip comprises the antibody of the fifth aspect of the present application, the recombinant protein of the sixth aspect of the present application, the antibody conjugate of the tenth aspect of the present application, the immune cell of the eleventh aspect of the present application, or a combination thereof.

[0216] In a seventeenth aspect of the present application, a kit is provided, which comprises:

[0217] (1) a first container, wherein the first container comprises the antibody of the present application; and / or

[0218] (2) a second container, wherein the second container comprises a secondary antibody against the antibody of the present application.

[0219] Alternatively,

[0220] The kit comprises the detection plate of the sixteenth aspect of the present application.

[0221] In an eighteenth aspect of the present application, a method for preparing a recombinant polypeptide is provided, which comprises:

[0222] (a) culturing the host cell of the ninth aspect of the present application under conditions suitable for expression;

[0223] (b) isolating the recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody of the fifth aspect of the present application or the recombinant protein of the sixth aspect of the present application.

[0224] In a nineteenth aspect of the present application, a method for treating a disease associated with abnormal expression or function of B7-H3 is provided, which comprises administering to a subject in need thereof an effective amount of the antibody of the fifth aspect of the present application, or the recombinant protein of the sixth aspect of the present application, or the antibody conjugate of the tenth aspect of the present application, or the immune cell of the eleventh aspect of the present application, or the pharmaceutical composition of the twelfth aspect of the present application, or a combination thereof.

[0225] In another preferred embodiment, the disease associated with abnormal expression or function of B7-H3 is cancer.

[0226] It should be understood that, within the scope of the present application, each of the technical features described above and each of the technical features specifically described below (e.g., in the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0227] Figure 1 Relative tumor volume (mean ± standard error) of NCI-H1975 subcutaneous xenograft tumor is shown.

[0228] Figure 2 The relative animal weight (mean ± standard error) of NCI-H1975 subcutaneous xenograft tumor-bearing mice is shown. DETAILED DESCRIPTION

[0229] The present inventors have made extensive and in-depth studies and found an anti-B7-H3 antibody. The present application also provides a preparation method and related applications of the antibody. The antibody of the present application has high affinity and high selectivity for the target protein B7-H3, can be effectively endocytosed, and is easy to express and purify. The ADC prepared by using the antibody of the present application shows obvious tumor inhibition effect in an animal model. On this basis, the present application is completed.

[0230] Specifically, the present application uses B7-H3-his protein and CHO-K cells overexpressing B7-H3 to immunize healthy Balb / c mice by subcutaneous and intraperitoneal multiple injections. After 3-4 times of immunization, the mouse with the highest serum titer is selected, and the spleen is taken after euthanasia to construct a phage display library. After three rounds of liquid phase screening, the enzyme-coated plates are coated with B7-H3-His, and the anti-B7-H3 Fab supernatant is added. The positive clones specifically binding to B7-H3 are obtained by ELISA screening. The positive clones are sequenced, and cell level binding verification and monkey B7-H3 cross-binding verification are performed. The candidate antibodies that can bind to both cell level B7-H3 and monkey B7-H3 are used for full-length antibody construction.

[0231] Meanwhile, more than 4000 human PBMC samples are used for RNA extraction, and different germline primers are designed according to the IMGT database. According to the proportion of human genes reported in the literature, the library construction scheme is set, and four different germline proportion hundred billion libraries are constructed, which are combined into a trillion-level antibody library. After three rounds of liquid phase screening, the enzyme-coated plates are coated with Anti-Fd, and then the anti-B7-H3 Fab supernatant is added. The positive clones specifically binding to B7-H3 are obtained by sandwich ELISA screening. The obtained positive clones are sequenced, and the monoclonal obtained is subjected to cell level binding and monkey B7-H3 binding verification. The candidate antibodies that can bind to both cell level B7-H3 and monkey B7-H3 are used for full-length antibody construction.

[0232] The plasmids carrying the antibody light chains and heavy chains screened from the above mouse immune library and human source library are co-transfected into ExpiCHO-S cells, and the cell expression supernatant is collected after 7 days of expression. Protein A affinity chromatography column is used for purification, and the purity of the antibody is identified by SDS-PAGE and SEC after purification.

[0233] The purified antibodies were subjected to protein level binding, species cross detection, cell level binding, and endocytosis or activity detection. Based on the comprehensive results of all experiments, 8 candidate antibodies from the mouse immune library and 3 candidate antibodies from the human library were obtained. Further, among the candidate antibodies from the mouse immune library, two antibodies were selected for humanization. The CDRs of the murine antibodies were grafted onto the corresponding human antibody scaffolds by CDR grafting, and then a number of light-heavy chain mutants were designed by 3D structure simulation. The activity of the antibodies was verified after transient expression.

[0234] B7-H3

[0235] In the cellular immune response, the proliferation and activation of T cells not only require the first signal of T cell receptor TCR recognizing the MHC presented by APC or tumor cells on the surface, but also require the second signal provided by the costimulatory molecule. The B7-CD28 superfamily is one of the costimulatory molecule families currently discovered, which belongs to the immunoglobulin superfamily. B7 family molecules can provide stimulation signals to enhance and maintain T cell immune response, and can also produce inhibitory signals to limit and weaken T cell immune response. Therefore, the family plays an important role in tumor diseases, organ transplantation and autoimmune diseases.

[0236] According to the function, the B7 family can be divided into three categories.

[0237] The first group: B7-1 (CD80) and B7-2 (CD86).

[0238] The second group: B7-H1 (PD-L1) and B7-DC (PD-L2)

[0239] The third group: B7-H3 (CD276) and B7-H4 (B7x) are composed. Their receptors have not been determined, but are believed to be involved in the co-stimulation and co-inhibition pathways.

[0240] B7-H3 (B7 homolog 3 protein), also known as CD276, is an important immune checkpoint molecule of the B7-CD28 family. It is a member of the B7 family identified by Chapoval et al. in 2001 in a human dendritic cell cDNA library. B7-H3 exists mainly in the form of membrane protein and soluble form. Soluble B7-H3 (sB7-H3) is derived from membrane protein by metalloproteinase cleavage. In addition, B7-H3 protein has also been found in exosomes and other extracellular vesicles.

[0241] B7-H3 is a T cell co-inhibitory molecule with partial co-stimulatory function. B7-H3 can effectively inhibit T cell and NK cell function, and also has an effect on bone development.

[0242] B7-H3 is expressed in a variety of malignancies and is closely related to the growth, metastasis, recurrence and poor prognosis of malignancies. B7-H3 can down-regulate T helper 1 -mediated immune response, inhibit CD4+ T cell activation and suppress cytokine production, thus may play a role in promoting cancer immune escape.

[0243] B7-H3 is a type I transmembrane glycoprotein consisting of 316 amino acids with a molecular weight of 45-66 kDa, which has a similar molecular structure to B7-H1 (PD-L1). It contains a putative 28 AA signal peptide, a 217 AA extracellular region consisting of immunoglobulin constant (IgC) and variable (IgV) structures, a transmembrane region and a 45 amino acid cytoplasmic domain. The gene consists of 10 exons, of which 4-7 exons encode the extracellular IgV-IgC domain.

[0244] Currently, two forms of B7-H3 have been found: 2Ig-B7-H3 and 4Ig-B7-H3. 2Ig-B7-H3 is expressed in murine and human cells, which has extracellular IgV-IgC structure; 4Ig-B7-H3 is only expressed in human cells, which consists of tandem repeats of IgV-IgC-IgV-IgC structure. The main form of human B7-H3 is 4IgB7-H3, which is located on chromosome 15. The mouse B7-H3 gene is located on chromosome 9. B7-H3 is one of the most evolutionarily conserved members of the B7 family, as it is expressed in various species from teleosts to mammals.

[0245] B7-H3 transcripts are widely expressed in heart, liver, placenta, prostate, testis, uterus, pancreas, small intestine and colon, etc. The expression of B7-H3 protein is more limited to the cell surface, such as activated dendritic cells, monocytes, T cells, B cells and NK cells.

[0246] B7-H3 is abnormally highly expressed in a variety of cancer cells or tissues, including gastric cancer, lung cancer, prostate cancer, kidney cancer, pancreatic cancer, ovarian cancer, breast cancer, endometrial cancer, liver cancer, colorectal cancer, oral cancer, bladder cancer, osteosarcoma and hematological malignancies.

[0247] The molecular mechanism regulating B7-H3 expression is not clear, but the expression of B7-H3 protein is inversely proportional to the level of miR-29, and the miR-29 binding site on B7-H3 is conserved in evolution.

[0248] Antibodies

[0249] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons having the same structural core features. It is composed of two identical light (L) polypeptides and two identical heavy (H) polypeptides. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. At one end of each heavy chain is a variable region (VH) followed by a number of constant regions. At one end of each light chain is a variable region (VL) followed by a constant region; the constant region of the light chain is aligned with the first constant region of the heavy chain, and the variable region of the light chain is aligned with the variable region of the heavy chain. Particular amino acid residues are located at the interface between the variable regions of the light and heavy chains.

[0250] As used herein, the term "variable" refers to the fact that portions of the antibody, variable region differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable regions of antibodies; it is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable regions. The more highly conserved portions of the variable regions are called the framework regions (FRs). The variable regions of the heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647- 669 (1991)). The constant regions of the antibodies are not involved directly in binding with the antigen, but exhibit various effector functions.

[0251] The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant regions. Depending on the amino acid sequence of the constant region of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chains of different classes of immunoglobulins have different constant regions. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

[0252] Generally, the antigen binding properties of an antibody can be described by three specific regions on the variable region of the heavy and light chains, called the complementarity determining regions (CDRs), which are interspaced by four framework regions (FRs), the amino acid sequences of which are relatively conserved and not directly involved in binding interactions. The CDRs form loops or complementarity determining regions that are brought together through the FRs in a β-sheet arrangement to create a structure that is complementary to the antigen. The CDRs on the heavy chain and the CDRs on the light chain make up the antigen binding site of an antibody. The identification of the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same class.

[0253] The present application includes not only intact antibodies, but also fragments of antibodies that are immunologically active or fusions of antibodies with other sequences. Thus, the present application also includes fragments, derivatives and analogs of the antibodies.

[0254] In the present application, antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, which include both human and non-human portions, can be obtained by standard DNA recombination techniques and are useful antibodies. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having a variable region from a murine monoclonal antibody and a constant region from a human immunoglobulin (see, e.g., U.S. Patent 4,816,567 and U.S. Patent 4,816,397, both of which are incorporated herein by reference in their entirety). Humanized antibodies are antibodies derived from non-human species that have one or more complementarity determining regions (CDRs) derived from a non-human species and framework regions derived from a human immunoglobulin molecule (see U.S. Patent 5,585,089, which is incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.

[0255] In the present application, antibodies can be monospecific, bispecific, trispecific, or more multispecific.

[0256] In the present application, the antibodies of the present application also include conservative variants thereof, which are polypeptides having up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids replaced by similar or identical amino acids as compared to the amino acid sequence of the antibodies of the present application. These conservative variant polypeptides are preferably generated by amino acid replacements according to Table A.

[0257] Table A

[0258] Original residue Representative substitution Preferred substitution Ala (A) Val; Leu; lie Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Lys; Arg Gin Asp (D) Glu Glu Cys (C) Ser Ser Gin (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gin; Lys; Arg Arg lie (I) Leu; Val; Met; Ala; Phe Leu

[0259] Leu (L) lie; Val; Met; Ala; Phe lie Lys (K) Arg; Gin; Asn Arg Met (M) Leu; Phe; lie Leu Phe (F) Leu; Val; lie; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) lie; Leu; Met; Phe; Ala Leu

[0260] Anti-b7-h3 antibodies

[0261] The present application provides an antibody against B7-H3 with high specificity and high affinity, which comprises a heavy chain containing a heavy chain variable region (VH) amino acid sequence and a light chain containing a light chain variable region (VL) amino acid sequence.

[0262] In another preferred embodiment, the antibody comprises a heavy chain variable region and a light chain variable region;

[0263] The heavy chain variable region comprises the following three complementarity determining regions CDRs:

[0264]

[0265] The light chain variable region comprises the following three complementarity determining regions CDRs:

[0266]

[0267]

[0268] In another preferred embodiment, the heavy chain variable region of the antibody contains an amino acid sequence shown in any one of SEQ ID NO: 79-96; and / or the light chain variable region of the antibody contains an amino acid sequence shown in any one of SEQ ID NO: 61-78.

[0269] In another preferred embodiment, the antibody comprises:

[0270] Name Type Heavy chain variable region Light chain variable region IL-P7-C05 Unhumanized murine sequence 79 61 IL-P7-C05-H4L3 Humanized light-heavy chain sequence combination 83 64 IL-P7-C05-H4L1 Humanized light-heavy chain sequence combination 83 62 IL-P7-C05-H2L2 Humanized light-heavy chain sequence combination 81 63 IL-P7-C05-H2L1 Humanized light-heavy chain sequence combination 81 62 IL-P7-C05-H3L3 Humanized light-heavy chain sequence combination 82 64 IL-P7-C05-H1L1 Humanized light-heavy chain sequence combination 80 62 IL-P7-C05-H3L1 Humanized light-heavy chain sequence combination 82 62 IL-P5-C06 Unhumanized murine sequence 84 65 IL-P5-C06-H2L2 Humanized light-heavy chain sequence combination 86 67 IL-P5-C06-H2L4 Humanized light-heavy chain sequence combination 86 69 IL-P5-C06-H2L3 Humanized light-heavy chain sequence combination 86 68 IL-P5-C06-H1L4 Humanized light-heavy chain sequence combination 85 69 IL-P5-C06-H1L1 Humanized light-heavy chain sequence combination 85 66 IL-P5-C06-H1L3 Humanized light-heavy chain sequence combination 85 68 IL-P5-C06-H3L3 Humanized light-heavy chain sequence combination 87 68 IL-P5-C06-H3L4 Humanized light-heavy chain sequence combination 87 69 IL-P6-D06 Unhumanized murine sequence 88 70 IL-P6-C11 Unhumanized murine sequence 89 71 IL-P6-B07 Unhumanized murine sequence 90 72 IL-P6-C08 Unhumanized murine sequence 91 73 IL-P5-B01 Unhumanized murine sequence 92 74 IL-P7-A06 Unhumanized murine sequence 93 75

[0271] NL24-A17 Sequence obtained from human library screening 94 76 NL24-A68 Sequence obtained from human library screening 95 77 NL24-A10 Sequence obtained from human library screening 96 78。

[0272] In a preferred embodiment, the antibody comprises a heavy chain variable region and a light chain variable region; wherein,

[0273] The heavy chain variable region comprises the following three complementarity determining regions CDRs:

[0274] VH-CDR1 shown in SEQ ID NO: 97,

[0275] VH-CDR2 shown in SEQ ID NO: 54, and

[0276] VH-CDR3 shown in SEQ ID NO: 35;

[0277] The light chain variable region comprises the following three complementarity determining regions CDRs:

[0278] VL-CDR1 shown in SEQ ID NO: 1,

[0279] VL-CDR2 as set forth in SEQ ID NO: 26, and

[0280] VL-CDR3 as set forth in SEQ ID NO: 3;

[0281] or

[0282] the heavy chain variable region comprises the following three complementarity determining regions CDRs:

[0283] VH-CDR1 as set forth in SEQ ID NO: 97,

[0284] VH-CDR2 as set forth in SEQ ID NO: 54, and

[0285] VH-CDR3 as set forth in SEQ ID NO: 35;

[0286] the light chain variable region comprises the following three complementarity determining regions CDRs:

[0287] VL-CDR1 as set forth in SEQ ID NO: 1,

[0288] VL-CDR2 as set forth in SEQ ID NO: 2, and

[0289] VL-CDR3 as set forth in SEQ ID NO: 3;

[0290] or

[0291] the heavy chain variable region comprises the following three complementarity determining regions CDRs:

[0292] VH-CDR1 as set forth in SEQ ID NO: 33,

[0293] VH-CDR2 as set forth in SEQ ID NO: 34, and

[0294] VH-CDR3 as set forth in SEQ ID NO: 35;

[0295] the light chain variable region comprises the following three complementarity determining regions CDRs:

[0296] VL-CDR1 as set forth in SEQ ID NO: 1,

[0297] VL-CDR2 as set forth in SEQ ID NO: 25, and

[0298] VL-CDR3 as set forth in SEQ ID NO: 3.

[0299] In another preferred embodiment, the antibody is a humanized antibody, and the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 83; and the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 64.

[0300] Alternatively, the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 83; and the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 62.

[0301] Alternatively, the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 81; and the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 63.

[0302] In another preferred embodiment, any one of the above-mentioned amino acid sequences further comprises a derivative sequence that optionally has at least one amino acid added, deleted, modified and / or substituted, and that retains the B7-H3 binding affinity.

[0303] In another preferred embodiment, the sequence formed by the addition, deletion, modification and / or substitution of at least one amino acid is preferably an amino acid sequence having at least 80% homology, more preferably at least 85% homology, even more preferably at least 90% homology, and most preferably at least 95% homology. More preferably, the number of amino acids added, deleted, modified and / or substituted is 1-7, more preferably 1-5, even more preferably 1-3, and most preferably 1-2.

[0304] The antibody of the present application can be a double-chain or single-chain antibody, and can be selected from an animal-derived antibody, a chimeric antibody, a humanized antibody, more preferably a humanized antibody, a human-animal chimeric antibody, and more preferably a fully humanized antibody.

[0305] The antibody derivative of the present application can be a single-chain antibody, and / or an antibody fragment such as Fab, Fab', (Fab')2, or other known antibody derivatives in the art, and any one or several of IgA, IgD, IgE, IgG, and IgM antibodies or other subtypes of antibodies.

[0306] Preferably, the animal is a mammal, such as a mouse.

[0307] The antibody of the present application can be a chimeric antibody, a humanized antibody, a CDR grafted and / or modified antibody targeting human B7-H3.

[0308] Preparation of the antibody

[0309] The DNA molecule of the antibody or fragment thereof of the present application can be obtained by conventional techniques, such as by PCR amplification or screening of genomic libraries, etc. Furthermore, the coding sequences of the light and heavy chains can be fused together to form a single chain antibody.

[0310] Once the relevant sequences have been obtained, they can be obtained in large quantities by recombinant means. This is typically done by cloning them into vectors, which are then introduced into cells, and then isolating the relevant sequences from the propagated host cells by conventional means.

[0311] Furthermore, the relevant sequences can be synthesized by artificial synthesis, especially when the fragments are relatively short. Typically, longer fragments are obtained by first synthesizing a number of smaller fragments, which are then ligated together.

[0312] At present, it is possible to obtain the DNA sequence encoding the antibody (or fragment thereof, or derivative thereof) of the present application entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present application by chemical synthesis.

[0313] The present application also relates to vectors comprising the appropriate DNA sequences described above, together with appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells, so that they are able to express the protein.

[0314] The host cells can be prokaryotic cells, such as bacterial cells, or lower eukaryotic cells, such as yeast cells, or higher eukaryotic cells, such as mammalian cells. Preferred animal cells include (but are not limited to): CHO-S, HEK-293 cells.

[0315] Typically, the transformed host cells are cultured under conditions appropriate for the expression of the antibody of the present application. The antibody of the present application is then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, size exclusion chromatography, or affinity chromatography, as well as other conventional separation and purification methods known to those skilled in the art.

[0316] The resulting monoclonal antibodies can be identified using conventional means. For example, the binding specificity of the monoclonal antibodies can be determined using immunoprecipitation or an in vitro binding assay, such as a radioimmunoassay (RIA) or an enzyme-linked immunoabsorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, using the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0317] The antibodies of the present application can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic lysis, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

[0318] Antibody-drug conjugate (ADC)

[0319] The present application also provides an antibody-drug conjugate (ADC) based on the antibody of the present application.

[0320] Typically, the antibody-drug conjugate comprises the antibody, and an effector molecule, which is conjugated, and preferably chemically conjugated, to the antibody. The effector molecule is preferably a therapeutically active drug. In addition, the effector molecule can be one or more of a toxin, a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0321] The antibody of the present application can be conjugated to the effector molecule via a conjugating agent. Examples of the conjugating agent can be any one or several of a non-selective conjugating agent, a conjugating agent using a carboxyl group, a peptide chain, and a conjugating agent using a disulfide bond. The non-selective conjugating agent refers to a compound that allows the effector molecule and the antibody to form a covalent bond, such as glutaraldehyde, etc. The conjugating agent using a carboxyl group can be any one or several of a conjugating agent of aconitic anhydride (such as aconitic anhydride), and a conjugating agent of an acylhydrazone (with an acylhydrazone as a conjugating site).

[0322] Certain residues on the antibody (such as Cys or Lys, etc.) are used to link to a variety of functional groups, including imaging agents (such as chromophoric groups and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizing agents (such as ethylene glycol polymers), and therapeutic agents. The antibody can be conjugated to a functional agent to form an antibody-functional agent conjugate. The functional agent (such as a drug, a detection agent, a stabilizing agent) is conjugated (covalently linked) to the antibody. The functional agent can be linked to the antibody directly, or indirectly via a linker.

[0323] Antibodies can be conjugated to drugs to form antibody drug conjugates (ADCs). Typically, an ADC comprises a linker between the drug and the antibody. The linker can be a degradable linker or a non-degradable linker. Degradable linkers are typically susceptible to degradation in the intracellular environment, e.g., the linker is degraded at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptide-based linkers that are degradable by intracellular proteases, e.g., lysosomal proteases or endosomal proteases, or saccharide linkers, e.g., glucuronide-containing linkers that are degradable by glucuronidases. Peptide-based linkers can include, for example, dipeptides, e.g., valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at a pH less than 5.5, such as hydrazone linkers) and linkers that are degradable under reducing conditions (e.g., disulfide linker). Non-degradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by proteases.

[0324] The linker has a reactive functional group capable of reacting with certain amino acid residues prior to attachment to the antibody, and the attachment is achieved through the reactive functional group. Thiol-specific reactive functional groups are preferred and include, for example, maleimides, haloamides (e.g., iodo-, bromo-, or chloro-); haloesters (e.g., iodo-, bromo-, or chloro-); halo-methyl ketones (e.g., iodo-, bromo-, or chloro-), benzyl halides (e.g., iodo-, bromo-, or chloro-); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-bis-(mercurymethyl) dioxane, and counterions are acetate, chloride, or nitrate; and polymethylene dimethylsulfide sulfonate. The linker can include, for example, a maleimide attached to the antibody through a thio-butyrimide.

[0325] The drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, the linker attaches the antibody to the drug, and the drug has a functional group that can bond to the linker. For example, the drug can have an amino, carboxyl, thiol, hydroxyl, or keto group that can bond to the linker. In cases where the drug is directly attached to the linker, the drug has a reactive functional group prior to attachment to the linker.

[0326] In another preferred embodiment, the drug is selected from the group consisting of a chemotherapeutic drug, a radiotherapy drug, a hormone therapy drug, or an immunotherapy drug. In particular, useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemotherapeutic sensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful classes of cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine containing drugs (e.g., pyrrolo[l,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), and vinca alkaloids.

[0327] Preferred toxins of the application are selected from the group consisting of taxanes, maytansinoids, auristatins, calicheamicins, anthracyclines, docetaxel, cathepsins, ricin, gelonin, Pseudomonas exotoxin, diphtheria toxin, RNase, and radioisotopes.

[0328] In the present application, the drug-linker can be used to form the ADC in a single step. In other embodiments, a bifunctional linker compound can be used to form the ADC in a two- or multi-step process. For example, the cysteine residue is reacted with a reactive portion of the linker in a first step, and in a subsequent step, a functional group on the linker is reacted with the drug, thereby forming the ADC.

[0329] In general, the functional groups on the linker are chosen to facilitate specific reaction with a suitable reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkyne group on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkyne. Other useful functional groups include, for example, ketones and aldehydes (for reaction with hydrazides and alkoxylamines), phosphines (for reaction with azides); isocyanates and isothiocyanates (for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimidyl esters (for reaction with amines and alcohols). These and other ligation strategies, such as those described in Bioconjugate Techniques, 2nd Edition (Elsevier), are well known to those skilled in the art. It will be appreciated by those skilled in the art that for selective reaction of the drug moiety and the linker, each member of a complementary pair of reactive functional groups can be used on either the linker or the drug when the complementary pair is chosen.

[0330] The present application also provides methods of making an ADC, which can further comprise: combining an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0331] In certain embodiments, the methods of the present application comprise: combining an antibody with a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the present application further comprise: combining the antibody-linker conjugate with a drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via the linker.

[0332] In some embodiments, the antibody drug conjugate (ADC) is represented by the following formula:

[0333]

[0334] wherein:

[0335] Ab is an antibody,

[0336] LU is a linker;

[0337] D is a drug;

[0338] and subscript p is a value selected from 1 to 8.

[0339] Applications

[0340] The present application also provides uses of the antibodies, antibody conjugates (ADCs), recombinant proteins, and / or immune cells of the present application, for example, for the manufacture of a diagnostic or a medicament.

[0341] Preferably, the medicament is a medicament for preventing and / or treating a disease associated with abnormal expression or function of B7-H3.

[0342] The uses of the antibodies, ADCs, recombinant proteins, and / or immune cells of the present application include, but are not limited to:

[0343] (i) diagnosing, preventing and / or treating tumorigenesis, growth and / or metastasis, especially of a B7-H3 high expressing tumor. The tumor includes, but is not limited to, melanoma, mesothelioma, non-small cell lung cancer, breast cancer, liver cancer, synovial sarcoma, metastatic colon cancer, renal cancer, bladder cancer, prostate cancer, ovarian cancer, chronic hepatitis C virus infection, advanced solid cancer, gastrointestinal malignancy, endometrial cancer, recurrent melanoma, head and neck squamous cell carcinoma, cutaneous T-cell lymphoma, fallopian tube cancer, peritoneal tumor, muscle-invasive bladder cancer, extensive stage small cell lung cancer, adult acute myeloid leukemia, atypical chronic myeloid leukemia, ovarian epithelial cancer, B-cell chronic lymphocytic leukemia, cutaneous B-cell non-Hodgkin lymphoma, intraocular lymphoma, testicular choriocarcinoma, neuroblastoma, esophageal cancer.

[0344] (ii) diagnosing, preventing and / or treating an autoimmune disease, which includes, but is not limited to, systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis, ankylosing spondylitis, scleroderma, Takayasu's arteritis, Wegener's granulomatosis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, psoriasis, parapsoriasis, graft rejection.

[0345] Detection uses and kits

[0346] The antibodies or ADCs thereof of the present application can be used in detection applications, for example, for detecting a sample, thereby providing diagnostic information.

[0347] In the present application, the sample (specimen) used includes cells, tissue samples, and biopsy specimens. The term "biopsy" used in the present application shall include all kinds of biopsies known to those skilled in the art. Thus, the biopsy used in the present application can include, for example, a resected sample of a tumor, a tissue sample prepared by an endoscopic method or a puncture or needle biopsy of an organ.

[0348] The sample used in the present application includes a fixed or preserved cell or tissue sample.

[0349] The present application also provides a kit comprising the antibody (or fragment thereof) of the present application. In a preferred embodiment of the present application, the kit further comprises a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present application can be immobilized on a detection plate.

[0350] Pharmaceutical compositions

[0351] The present application also provides a composition. In preferred embodiments, the composition is a pharmaceutical composition comprising the antibody or active fragment thereof or fusion protein thereof or ADC thereof or corresponding immune cells described above, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably the pH is about 6-8, although the pH value can vary depending on the nature of the substance to be formulated and the disease to be treated.

[0352] The prepared pharmaceutical composition can be administered by conventional routes, including but not limited to intratumoral, intraperitoneal, intravenous, or topical administration. Typically, the administration route of the pharmaceutical composition of the present application is preferably injection or oral administration. The injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection, etc. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid or liquid form, which can be an aqueous solution, a non-aqueous solution or a suspension, more preferably tablets, capsules, granules, injections or infusions, etc.

[0353] The antibody of the present application can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

[0354] The pharmaceutical composition of the present application is a pharmaceutical composition for preventing and / or treating diseases associated with abnormal expression or function of B7-H3.

[0355] The pharmaceutical composition of the present application can be directly used to bind B7-H3 protein molecules, and thus can be used for preventing and treating diseases such as tumors.

[0356] The pharmaceutical composition of the present application contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody (or conjugate thereof) of the present application and a pharmaceutically acceptable carrier or excipient. Such carriers include but are not limited to saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the administration method. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition such as the needle, the solution is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptide of the present application can also be used with other therapeutic agents.

[0357] In the present application, preferably, the pharmaceutical composition of the present application further comprises one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carriers are conventional pharmaceutically acceptable carriers in the art, which can be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant. The pharmaceutical adjuvant is conventional pharmaceutical adjuvant in the art, preferably including pharmaceutically acceptable excipients, fillers or diluents, etc. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the above-mentioned protein and 0.01-99.99% of the pharmaceutically acceptable carrier, and the percentage is the mass percentage of the pharmaceutical composition.

[0358] In the present application, preferably, the administration amount of the pharmaceutical composition is an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, a degenerative or an injury condition. The effective amount can be determined on an individual basis and will be based in part on the condition to be treated and the considerations of the sought result. The effective amount can be determined by a person skilled in the art by using the above-mentioned factors on an individual basis and using no more than conventional experiments.

[0359] When the pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dose should also take into account the route of administration, the patient's health condition, etc., which are within the skill of a skilled physician.

[0360] The present application provides the use of the above-mentioned pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease related to abnormal expression or function of B7-H3. Preferably, the disease related to abnormal expression or function of B7-H3 is cancer and autoimmune disease.

[0361] The main advantages of the present application include:

[0362] (a) The antibody of the present application has high affinity and high selectivity for the target protein B7-H3.

[0363] (b) In cell experiments, the antibody of the present application can be effectively endocytosed after binding to the target protein.

[0364] (c) The antibody of the present application is easy to express and purify.

[0365] (d) After the antibody of the present application is prepared into an ADC, a single administration has a significant tumor inhibition effect in a tumor-bearing mouse animal model.

[0366] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions suggested by the manufacturer. Unless otherwise specified, the percentages and parts are weight percentages and weight parts.

[0367] Example 1 Mouse immunization

[0368] Healthy Balb / c mice are selected and immunized by subcutaneous injection and intraperitoneal injection at multiple sites, and the immunization is carried out once every two weeks. One group of mice is immunized with B7-H3 his protein, and another group of mice is immunized with CHOK cells overexpressing B7-H3. The first immunization uses Freund's complete adjuvant, and the subsequent immunization uses Freund's incomplete adjuvant. After three to four times of immunization, the mouse serum titer is detected by ELISA, and the mouse with the highest titer is sacrificed to take the spleen for phage display library construction.

[0369] Example 2 Mouse immunization library construction and screening

[0370] The spleen of the mouse with qualified titer after immunization is taken, and the total RNA of the mouse spleen is extracted with chloroform, and then a cDNA library is prepared by reverse transcription PCR. The variable region genes of the light chain and the heavy chain of the mouse are amplified by the combined primers of Zhiyou Bio, and the combined Fab fragments are inserted into the phagemid vector to construct the Fab antibody library. The library is prepared into a phage display library, and B7-H3 his biotin is coated on magnetic beads by using the method of liquid phase screening. After three rounds of liquid phase screening, the enzyme-labeled plate is coated with B7-H3 His, and then the anti-B7-H3 Fab supernatant is added, and the specific binding B7-H3 positive clones are obtained by ELISA screening.

[0371] The obtained positive clones are sequenced, and the obtained single clones are verified at the cell level binding and monkey B7-H3 binding. The candidate antibodies capable of binding to cell level B7-H3 and monkey B7-H3 are constructed into full-length antibodies.

[0372] The names of some antibodies constructed in this example and their sequence numbers are shown in Table 1.

[0373] Table 1 Murine antibodies and their sequence numbers

[0374] Antibody name Heavy chain variable region Light chain variable region

[0375] Amino acid sequence number Amino acid sequence number IL-P7-C05 79 61 IL-P5-C06 84 65 IL-P6-D06 88 70 IL-P6-C11 89 71 IL-P6-B07 90 72 IL-P6-C08 91 73 IL-P5-B01 92 74 IL-P7-A06 93 75

[0376] Example 3 Construction and screening of full human natural library

[0377] Construction of Triumphant hundred billion antibody library: using more than 4000 human PBMC samples, RNA was extracted, and primers for different germlines were designed according to the IMGT database, and the library construction scheme was set according to the reported proportion of human genes, a total of four hundred billion libraries with different germline proportions were constructed, and combined into a hundred billion antibody library.

[0378] Liquid phase screening method was used to coat B7-H3 hisbiotin on magnetic beads for three rounds of liquid phase screening. After three rounds of liquid phase screening, the enzyme-labeled plate was coated with AntiFd, then the Fab supernatant of anti-B7-H3 was added, incubated for 1 hour, then 2 μg / mL of B7-H3 Hisbiotin was added and incubated for 1 hour, and finally Neutravidin HRP was used to detect the signal. Specific binding of B7-H3 positive clones was obtained by sandwich ELISA screening.

[0379] The obtained positive clones were sequenced, and the obtained monoclonal cells were verified for binding to monkey B7-H3, and the candidate antibodies capable of binding to cell level B7-H3 and monkey B7-H3 were constructed into full-length antibodies.

[0380] The names and sequence numbers of some antibodies constructed in this example are shown in Table 2.

[0381] Table 2 Full human antibody and its sequence number

[0382]

[0383] Example 4 Expression and purification of full-length antibodies and humanization of mouse-derived antibodies

[0384] The plasmids carrying the antibody light chain and heavy chain were co-transfected into ExpiCHOS cells, and the cell expression supernatant was collected after 7 days of expression. Protein A affinity chromatography column was used for purification, and the specific experimental steps were referred to the AKTA manual. The purified antibodies were replaced into PBS buffer with pH 7.2 by dialysis. A total of 23 mouse immune library and 39 human library antibodies were expressed and purified, and a total of 57 antibodies were successfully expressed and purified, of which 19 were derived from mouse immune library and 38 were derived from human library.

[0385] The results show that the specific antibodies listed in Examples 2 and 3 are successfully expressed in ExpiCHOS cells.

[0386] The CDRs of the mouse antibodies (IL-P7-C05, IL-P5-C06) were grafted onto the corresponding human antibody skeletons by CDR grafting, and then several pairs of light and heavy chain mutants were designed by 3D structure simulation. The activity of the antibodies was verified after transient expression.

[0387] The partial antibody heavy chain and light chain variable regions and their sequence numbers constructed in this example are shown in Table 3.

[0388] Table 3 Humanized heavy chain and light chain variable regions and their sequence numbers

[0389] Heavy chain variable region Amino acid sequence number Light chain variable region Amino acid sequence number ILP7C05huVH1 80 ILP7C05huVL1 62 ILP7C05huVH2 81 ILP7C05huVL2 63 ILP7C05huVH3 82 ILP7C05huVL3 64 ILP7C05huVH4 83 ILP5C06huVL1 66 ILP5C06huVH1 85 ILP5C06huVL2 67 ILP5C06huVH2 86 ILP5C06huVL3 68 ILP5C06huVH3 87 ILP5C06huVL4 69

[0390] The above heavy chain variable region and light chain variable region can be combined in any combination to form a new humanized antibody.

[0391] Example 5 Protein level binding and species cross detection

[0392] Human B7-H3his, mouse B7-H3his and monkey B7-H3his were diluted to 2 μg / mL with PBS, and 30 μL per well was added to the enzyme-labeled plate, and then the enzyme-labeled plate was incubated in the 4-degree refrigerator for 16-20 hours. The next day, the enzyme-labeled plate was washed once with the washing solution (PBS containing 0.05% Tween 20), and then 5% skimmed milk powder was added for blocking for 1 hour. After blocking was completed, the enzyme-labeled plate was washed once, and then gradient-diluted anti-B7-H3 antibody was added, and incubated at room temperature for 1 hour. After incubation was completed, the enzyme-labeled plate was washed three times, and then goat anti-human enzyme-labeled secondary antibody was added, and incubated at room temperature for 1 hour. After incubation was completed, the enzyme-labeled plate was washed three times, and then TMB was added for color development for 5-15 minutes, and finally 2M sulfuric acid was used for termination, and the data was recorded on the enzyme-labeled instrument.

[0393] The results of species cross detection are shown in Table 4, and almost all antibodies have human-monkey cross, and most have human-mouse cross.

[0394] Subsequently, the human B7-H3 protein level binding of the humanized antibodies constructed in Example 4 and part of the mouse antibodies and the full human antibodies was verified again, and the results are shown in Table 5. The listed antibodies have very high affinity to human B7-H3.

[0395] Table 4 B7-H3 protein binding species cross activity

[0396]

[0397] Table 5 Antibody and human B7-H3 protein level binding activity

[0398] Name Protein binding activity EC50 (pg / mL) IL-P7-C05 0.044 IL-P7-C05-H4L3 0.03953 IL-P7-C05-H4L1 0.035 IL-P7-C05-H2L2 0.051 IL-P7-C05-H2L1 0.111 IL-P7-C05-H3L3 0.071 IL-P7-C05-H1L1 0.053 IL-P7-C05-H3L1 0.026 IL-P5-C06 0.302 IL-P5-C06-H2L2 0.147 IL-P5-C06-H2L4 0.223 IL-P5-C06-H2L3 0.151 IL-P5-C06-H1L4 0.237 IL-P5-C06-H1L1 0.206 IL-P5-C06-H1L3 0.164 IL-P5-C06-H3L3 0.151 IL-P5-C06-H3L4 0.156 IL-P6-D06 0.233 IL-P6-C11 0.182 IL-P6-B07 0.194 IL-P6-C08 0.088 IL-P5-B01 0.248 IL-P7-A06 0.172 NL24-A17 0.280 NL24-A68 0.285 NL24-A10 0.355

[0399] Example 6 Cell level binding detection

[0400] CHOK cells overexpressing B7-H3 or NCIH322 cells endogenously expressing B7-H3 were added to round-bottom 96-well plates at 100,000 cells per well, centrifuged to remove the supernatant, and then a gradient-diluted anti-B7-H3 antibody was added and incubated at 4°C for 1 hour. After incubation, the cells were washed twice with FACS buffer (PBS containing 2% FBS), and then a fluorescently labeled goat anti-human secondary antibody (Abeam, ab98596) was added and incubated for 0.5 hours. After incubation, the cells were washed twice with FACS buffer, resuspended in FACS buffer, and then detected on a flow cytometer (Beckman, cytoFlex).

[0401] The results are shown in Table 6, and the listed antibodies had high binding ability to both CHO cells artificially expressing B7-H3 and human non-small cell lung cancer cells (NCI-H322) highly expressing B7-H3 protein.

[0402] Table 6 Cell-level binding activity of antibodies

[0403]

[0404] Example 7 Detection of endocytosis activity of candidate antibodies

[0405] CHOK cells overexpressing B7-H3 or NCIH322 cells endogenously expressing B7-H3 were seeded into 96-well plates and incubated in a 37°C incubator overnight. Anti-B7-H3 antibodies were labeled with a toxin-containing secondary antibody (ATS, IT51), and then the antibodies were added to the cells and incubated in a 37°C incubator for 3 days. When the antibodies are endocytosed, the toxin labeled on the antibodies is endocytosed into the cells along with the antibodies, and the toxin is toxic to the cells, reducing the viability of the cells. Finally, a cell viability detection kit (Promega, G3581) was used to detect the viability of the cells.

[0406] The results are shown in Table 7, and the listed antibodies were effectively endocytosed by CHO cells expressing B7-H3, and some of the selected antibodies also showed high endocytosis activity in the human non-small cell lung cancer cell (NCI-H322) endocytosis experiment.

[0407] Table 7 Endocytosis activity of candidate antibodies

[0408]

[0409] Example 10 In vitro pharmacodynamic evaluation

[0410] The antibody IL-P7-C05-H4L3 prepared in the foregoing was used to construct an antibody drug conjugate IL-P7-C05-H4L3-MMAE with MMAE (Monomethyl auristatin E) as a conjugated effector molecule, and in vitro pharmacodynamic evaluation was performed.

[0411] The CellTiter-Glo method was used to detect the inhibitory activity of IL-P7-C05-H4L3-MMAE on B7-H3-positive human tumor cell lines NCI-H1975 and BxPC-3 and B7-H3-negative human tumor cell line Raji after 72 h of incubation.

[0412] The results, as shown in Table 8, show that IL-P7-C05-H4L3-MMAE has strong killing ability on B7-H3-high tumor cells (NCI-H1975 and BxPC-3), but poor killing activity on B7-H3-negative tumor cells.

[0413] Table 8. IC values of IL-P7-C05-H4L3-MMAE on human tumor cell lines after 72 h of incubation 50 values

[0414] Cell line Category IC 50 (μM) NCI-H1975 Human non-small cell lung cancer 0.0074 BxPC-3 Human pancreatic cancer 0.0077 Raji Human lymphoma 0.0341

[0415] The CellTiter-Glo method was used to detect the inhibitory activity of IL-P7-C05-H4L3-MMAE and Isotype-MMAE on B7-H3-positive human tumor cell lines Calu-6 and BxPC-3 and B7-H3-negative human tumor cell line Raji after 5 days of incubation.

[0416] The results, as shown in Table 9, show that IL-P7-C05-H4L3-MMAE has strong killing ability on B7-H3-high tumor cells (Calu-6 and BxPC-3), but poor killing activity on B7-H3-negative tumor cells. The killing activity of Isotype-MMAE control on cells is much lower than that of IL-P7-C05-H4L3-MMAE.

[0417] Table 9. IC values of IL-P7-C05-H4L3-MMAE and Isotype-MMAE on human tumor cell lines after 5 days of incubation 50 values

[0418]

[0419] Example 11 In vivo pharmacodynamic evaluation

[0420] In the subcutaneous transplantation tumor model of human non-small cell lung cancer NCI-H1975 cells in BALB / c nude mice, the inhibitory effect of IL-P7-C05-H4L3-MMAE and isotype control Isotype-MMAE on tumor growth was detected after single administration.

[0421] The tumor volume and animal body weight were measured as shown in Figure 1 and Figure 2 .

[0422] The results showed that IL-P7-C05-H4L3-MMAE had a significant inhibitory effect on the growth of NCI-H1975 subcutaneous transplanted tumors at a dose of 1 mg / kg and 3 mg / kg after a single tail vein administration (P<0.01), and the tumor growth inhibition rate (TGI) was 78.4% and 101.8% respectively at the end of the experiment (21 days after administration), and the relative tumor proliferation rate (%T / C) of the two dose groups was 26.2% and 4.2% respectively; and the tumors in the 3 mg / kg dose group atrophied and regressed. After a single administration of isotype control Isotype-MMAE at a dose of 3 mg / kg, there was no statistically significant difference in tumor growth compared with the solvent control group (P>0.05). IL-P7-C05-H4L3-MMAE at a dose of 1 mg / kg and 3 mg / kg, and isotype control Isotype-MMAE at a dose of 3 mg / kg, had no significant effect on the change of animal body weight after single administration, and no obvious abnormal clinical manifestations were observed, and the animals tolerated well.

[0423] All the documents mentioned in the present application are incorporated by reference in the present application as if each document was individually incorporated by reference. In addition, it should be understood that various modifications and changes can be made to the present application by those skilled in the art upon reading the above description of the present application, and these equivalent forms also fall within the scope defined by the claims attached hereto.

[0424] The sequences involved in the present application

[0425] Light chain variable region CDR

[0426]

[0427] Heavy chain variable region CDR

[0428]

[0429]

[0430] Light chain variable region

[0431]

[0432]

[0433] Heavy chain variable region

[0434]

[0435]

[0436] SEQUENCE LISTING <110> Minghui Pharmaceutical (Shanghai) Co., Ltd. Minghui Pharmaceutical (Hangzhou) Co., Ltd. <120> Anti-B7-H3 antibodies and preparation and use thereof <130> P2022-2345 <150> CN202010491448.3 <151> 02-JUN-2020 <160> 97 <170> PatentIn version 3.5 <210> 1 <211> 11 <212> PRT <213> Mus musculus <400> 1 Arg Ala Ser Glu Asn lie Tyr Ser Asn Leu Ala 1 5 10 <210> 2 <211> 7 <212> PRT <213> Mus musculus <400> 2 Ala Ala Thr Asn Leu Ala Asp 1 5 <210> 3 <211> 10 <212> PRT <213> Mus musculus <400> 3 Gln His Phe Trp Gly Thr Pro Pro Trp Thr 1 5 10 <210> 4 <211> 12 <212> PRT <213> Mus musculus <400> 4 Ser Ala Ser Ser Ser lie Ser Ser Asn Tyr Leu His 1 5 10 <210> 5 <211> 7 <212> PRT <213> Mus musculus <400> 5 Ser Thr Ser Asn Leu Ala Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Mus musculus <400> 6 Gln Gln Tyr Ser Gly Tyr Pro Leu Thr 1 5 <210> 7 <211> 11 <212> PRT <213> Mus musculus <400> 7 Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala 1 5 10 <210> 8 <211> 7 <212> PRT <213> Mus musculus <400> 8 Ser Ala Ser Tyr Arg Tyr Ser 1 5 <210> 9 <211> 9 <212> PRT <213> Mus musculus <400> 9 Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr 1 5 <210> 10 <211> 10 <212> PRT <213> Mus musculus <400> 10 Ser Ala Ser Ser Ser Ile Ser Tyr Met His 1 5 10 <210> 11 <211> 7 <212> PRT <213> Mus musculus <400> 11 Asp Thr Ser Lys Leu Ala Ser 1 5 <210> 12 <211> 9 <212> PRT <213> Mus musculus <400> 12 Gln Gln Trp Ser Ser Asn Pro Leu Thr 1 5 <210> 13 <211> 11 <212> PRT <213> Mus musculus <400> 13 Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala 1 5 10 <210> 14 <211> 7 <212> PRT <213> Mus musculus <400> 14 Asn Ala Lys Thr Leu Ala Glu 1 5 <210> 15 <211> 10 <212> PRT <213> Mus musculus <400> 15 Gln His His Tyr Gly Thr Pro Pro Tyr Thr 1 5 10 <210> 16 <211> 11 <212> PRT <213> Mus musculus <400> 16 Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala 1 5 10 <210> 17 <211> 7 <212> PRT <213> Mus musculus <400> 17 Ser Ala Ser Tyr Arg Tyr Ser 1 5 <210> 18 <211> 9 <212> PRT <213> Mus musculus <400> 18 Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr 1 5 <210> 19 <211> 11 <212> PRT <213> Mus musculus <400> 19 Lys Ala Ser Gln Asn Val Gly Thr Ala Val Ala 1 5 10 <210> 20 <211> 7 <212> PRT <213> Mus musculus <400> 20 Ser Ala Ser Asn Arg Tyr Thr 1 5 <210> 21 <211> 9 <212> PRT <213> Mus musculus <400> 21 Gln Gln Tyr Ser Ser Tyr Pro Phe Thr 1 5 <210> 22 <211> 11 <212> PRT <213> Mus musculus <400> 22 Arg Ala Ser Gln Ser Val Ser Thr Tyr Leu Ala 1 5 10 <210> 23 <211> 7 <212> PRT <213> Mus musculus <400> 23 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 24 <211> 11 <212> PRT <213> Mus musculus <400> 24 Gln Gln Arg Ser Asn Trp Pro Pro Leu Phe Thr 1 5 10 <210> 25 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL CDR <400> 25 Ala Ala Thr Asn Leu Gin Ser 1 5 <210> 26 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 <400> 26 Ala Ala Ser Asn Leu Gin Ser 1 5 <210> 27 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 <400> 27 Arg Ala Ser Ser Ser lie Ser Ser Asn Tyr Leu His 1 5 10 <210> 28 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 <400> 28 Ser Thr Ser Asn Leu Gin Ser 1 5 <210> 29 <211> 10 <212> PRT <213> Mus musculus <400> 29 Ser Ala Ser Ser Ser Val Ser Tyr Met His 1 5 10 <210> 30 <211> 11 <212> PRT <213> Mus musculus <400> 30 Arg Ala Ser Gin Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 31 <211> 11 <212> PRT <213> Mus musculus <400> 31 Gln Gin Arg Ser Asn Trp Pro Pro Ser Leu Thr 1 5 10 <210> 32 <211> 11 <212> PRT <213> Mus musculus <400> 32 Gln Gin Arg Ser Asn Trp Pro Pro Met Tyr Thr 1 5 10 <210> 33 <211> 10 <212> PRT <213> Mus musculus <400> 33 Gly Phe Thr Phe Ser Ser Tyr Gly Met Ser 1 5 10 <210> 34 <211> 10 <212> PRT <213> Mus musculus <400> 34 Thr lie Asn Ser Asn Gly Gly Thr Thr Tyr 1 5 10 <210> 35 <211> 10 <212> PRT <213> Mus musculus <400> 35 Asn Glu Glu Phe Arg Arg Gly Leu Ala Tyr 1 5 10 <210> 36 <211> 10 <212> PRT <213> Mus musculus <400> 36 Gly Tyr Thr Phe Thr Asp Tyr Ala Ile His 1 5 10 <210> 37 <211> 10 <212> PRT <213> Mus musculus <400> 37 Ile Ile Asn Thr Tyr Asn Gly Asn Ser Asn 1 5 10 <210> 38 <211> 12 <212> PRT <213> Mus musculus <400> 38 Asn Tyr Gly Ser Ser Phe Asn Trp Tyr Phe Asp Val 1 5 10 <210> 39 <211> 10 <212> PRT <213> Mus musculus <400> 39 Gly Tyr Thr Phe Thr Asp Tyr Val Ile His 1 5 10 <210> 40 <211> 10 <212> PRT <213> Mus musculus <400> 40 Gly lie Asn Arg Asp Ser Gly Gly Thr Thr 1 5 10 <210> 41 <211 > 9 <212> PRT <213> Mus musculus <400> 41 Trp Lys Phe Pro Trp Tyr Phe Asp Val 1 5 <210> 42 <211 > 10 <212> PRT <213> Mus musculus <400> 42 Gly Phe Thr Phe Ser Ser Tyr Gly Met Ser 1 5 10 <210> 43 <211 > 10 <212> PRT <213> Mus musculus <400> 43 Thr lie Ser Gly Gly Gly Arg Tyr Thr Tyr 1 5 10 <210> 44 <211 > 8 <212> PRT <213> Mus musculus <400> 44 His Tyr Asp Gly Tyr Leu Asp Tyr 1 5 <210> 45 <211 > 10 <212> PRT <213> Mus musculus <400> 45 Gly Asp Ser lie Thr Ser Gly Ser Trp Asn 1 5 10 <210> 46 <211> 9 <212> PRT <213> Mus musculus <400> 46 Tyr lie Ser Tyr Ser Gly Ser lie Tyr 1 5 <210> 47 <211> 8 <212> PRT <213> Mus musculus <400> 47 Gly Asn Pro Ala Trp Phe Ala Tyr 1 5 <210> 48 <211> 10 <212> PRT <213> Mus musculus <400> 48 Gly Tyr Thr Phe Thr Asp Tyr Ala Met His 1 5 10 <210> 49 <211> 10 <212> PRT <213> Mus musculus <400> 49 Ile lie Asn Thr Tyr Asn Gly Asn Thr Asn 1 5 10 <210> 50 <211> 11 <212> PRT <213> Mus musculus <400> 50 Ser Arg Gly Pro Trp Asp Arg Tyr Phe Asp Val 1 5 10 <210> 51 <211> 10 <212> PRT <213> Mus musculus <400> 51 Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser 1 5 10 <210> 52 <211> 10 <212> PRT <213> Mus musculus <400> 52 Gly Ile Ile Pro Ile Leu Gly Ile Ala Asn 1 5 10 <210> 53 <211> 9 <212> PRT <213> Mus musculus <400> 53 Gly Asp Ser Gly Tyr Asp Ser Asn Tyr 1 5 <210> 54 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region CDR <400> 54 Ala Ile Asn Ser Asn Gly Gly Ser Thr Tyr 1 5 10 <210> 55 <211> 10 <212> PRT <213> Mus musculus <400> 55 Val Ile Asn Thr Tyr Ser Gly Asn Thr Asn 1 5 10 <210> 56 <211> 15 <212> PRT <213> Mus musculus <400> 56 Gly Val Thr Tyr Gly Ser Gly Tyr Pro Tyr Trp Tyr Phe Asp Val 1 5 10 15 <210> 57 <211> 10 <212> PRT <213> Mus musculus <400> 57 Gly Tyr Thr Phe Thr Glu Tyr Val Ile His 1 5 10 <210> 58 <211> 10 <212> PRT <213> Mus musculus <400> 58 Gly Gly Ser Gly Arg Tyr Leu Pro Asp Tyr 1 5 10 <210> 59 <211> 10 <212> PRT <213> Mus musculus <400> 59 Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn 1 5 10 <210> 60 <211> 10 <212> PRT <213> Mus musculus <400> 60 Gly Glu Arg Gly Ser Tyr Leu Ile Asp Tyr 1 5 10 <210> 61 <211> 108 <212> PRT <213> Mus musculus <400> 61 Asp Ile Gln Met Ile Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Pro 85 90 95 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 62 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain variable region <400> 62 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val 35 40 45 Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 63 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain variable region <400> 63 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val 35 40 45 Tyr Ala Ala Thr Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 64 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain variable region <400> 64 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Asn Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gin His Phe Trp Gly Thr Pro Pro 85 90 95 Trp Thr Phe Gly Gin Gly Thr Lys Leu Gin Ile Lys 100 105 <210> 65 <211> 108 <212> PRT <213> Mus musculus <400> 65 Asp Ile Val Leu Thr Gin Ser Pro Thr Thr Met Ala Ala Ser Pro Gly 1 5 10 15 Glu Lys Ile Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Ser Asn 20 25 30 Tyr Leu His Trp Tyr Gin Gin Lys Pro Gly Phe Ser Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu Glu Asp Phe Ala Thr Tyr Tyr Cys Gin His Phe Trp Gly Thr Pro Pro65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro 85 90 95 Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 66 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain variable region <400> 66 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Met Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Ile Thr Ile Thr Cys Arg Ala Ser Ser Ser Ile Ser Ser Asn 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro 85 90 95 Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 67 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain variable region <400> 67 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Ile Thr Ile Thr Cys Arg Ala Ser Ser Ser Ile Ser Ser Asn 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro 85 90 95 Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 68 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain variable region <400> 68 Asp lie Gin Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr lie Thr Cys Arg Ala Ser Ser Ser lie Ser Ser Asn 20 25 30 Tyr Leu His Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 lie Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Tyr Ser Gly Tyr Pro 85 90 95 Leu Thr Phe Gly Gin Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 69 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain variable region <400> 69 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Ile Ser Ser Asn 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro 85 90 95 Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 70 <211> 107 <212> PRT <213> Mouse (Mus musculus) <220> <223> Humanized light chain variable region <400> 70[[ID=4l]] Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly<00l837>1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 71 <211> 106 <212> PRT <213> Mouse (Mus musculus) <220> <223> Humanized light chain variable region <400> 71 Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 72 <211> 106 <212> PRT <213> Mouse (Mus musculus) <220> <223> Humanized light chain variable region <400> 72 Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr ​​​​​​​​65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 73 <211> 108 <212> PRT <213> Mouse (Mus musculus) <220> <223> Humanized light chain variable region <400> 73 Asp Ile Gln Met Ile Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Pro 85 90 95 Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 74 <211> 107 <212> PRT <213> Mouse (Mus musculus) <400> 74 Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 75 <211> 107 <212> PRT <213> Mouse (Mus musculus) <400> 75 Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Met Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 76 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Human light chain variable region <400> 76 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Tyr 20 25 30 Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu lie 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly lie Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Tyr Ala Val Tyr Tyr Cys Gin Gin Arg Ser Asn Trp Pro Pro 85 90 95 Leu Phe Thr Phe Gly Pro Gly Thr Lys Val Asp lie Lys 100 105 <210> 77 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Human light chain variable region <400> 77 Glu lie Val Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu lie 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Ser Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 78 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Human-derived light chain variable region <400> 78 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Met Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 79 <211> 119 <212> PRT <213> Mouse (Mus musculus) <400> 79 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Leu Val 35 40 45 Ala Thr Ile Asn Ser Asn Gly Gly Thr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys His Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Thr Arg Asn Glu Glu Phe Arg Arg Gly Leu Ala Tyr Trp Gly Gin Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 <210> 80 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain variable region <400> 80 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Leu Val 35 40 45 Ala Thr He Asn Ser Asn Gly Gly Thr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys His Thr Leu Tyr 65 70 75 80 Leu Gin Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asn Glu Glu Phe Arg Arg Gly Leu Ala Tyr Trp Gly Gin Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 81 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain variable region <400> 81 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val 35 40 45 Ala Thr Ile Asn Ser Asn Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asn Glu Glu Phe Arg Arg Gly Leu Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 82 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain variable region <400> 82 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Leu Val 35 40 45 Ala Ala He Asn Ser Asn Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gin Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asn Glu Glu Phe Arg Arg Gly Leu Ala Tyr Trp Gly Gin Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 83 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain variable region <400> 83 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Leu Val 35 40 45 Ala Ala He Asn Ser Asn Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gin Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asn Glu Glu Phe Arg Arg Gly Leu Ala Tyr Trp Gly Gin Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 84 <211> 121 <212> PRT <213> Mus musculus <220> <223> Humanized heavy chain variable region <400> 84 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Val 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Ile His Trp Ala Lys Gln Gly His Ala Lys Ser Leu Glu Trp Ile 35 40 45 Gly Ile Ile Asn Thr Tyr Asn Gly Asn Ser Asn Tyr Asn Gln Asn Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Thr Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Asn Tyr Gly Ser Ser Phe Asn Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 85 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain variable region <400> 85 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Ile His Trp Ala Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ile Ile Asn Thr Tyr Asn Gly Asn Ser Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Tyr Gly Ser Ser Phe Asn Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 86 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain variable region <400> 86 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Ile His Trp Gly Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ile Ile Asn Thr Tyr Asn Gly Asn Ser Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Ala Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Tyr Gly Ser Ser Phe Asn Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 87 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain variable region <400> 87 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Ile His Trp Gly Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Thr Tyr Asn Gly Asn Ser Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Tyr Gly Ser Ser Phe Asn Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 88 <211> 124 <212> PRT <213> Mouse (Mus musculus) <400> 88 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Val 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Ile His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile 35 40 45 Gly Val Ile Asn Thr Tyr Ser Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Phe Cys 85 90 95 Ala Arg Gly Val Thr Tyr Gly Ser Gly Tyr Pro Tyr Trp Tyr Phe Asp 100 105 110 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 89 <211> 118 <212> PRT <213> Mouse (Mus musculus) <400> 89 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 1**0** 15 Ser Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Val Ile His Trp Val Lys Gln Asn His Gly Gly Ser Leu Glu Trp Ile 35 40 45 Note: There seems to be a typo in the original text where "10" in line was misaligned. It's corrected in the translation for better readability.Gly Gly lie Asn Arg Asp Ser Gly Gly Thr Thr Tyr Asn Gin lie Phe 50 55 60 Gln Asp Lys Val Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Lys Trp Lys Phe Pro Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 90 <211> 118 <212> PRT <213> Mus musculus <400> 90 Glu Val Gin Leu Gin Gin Ser Gly Pro Gin Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Arg lie Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30 Val lie His Trp Val Lys Gin Asn His Gly Gly Ser Leu Glu Trp lie 35 40 45 Gly Gly lie Asn Arg Asp Ser Gly Gly Thr Thr Tyr Asn Gin lie Phe 50 55 60 Gln Asp Lys Val Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Lys Trp Lys Phe Pro Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 91 <211> 117 <212> PRT <213> Mouse (Mus musculus) <400> 91 Glu Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Thr Ser Asp Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Val Arg His Tyr Asp Gly Tyr Leu Asp Tyr Trp Gly Leu Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 92 <211> 116 <212> PRT <213> Mouse (Mus musculus) <400> 92 Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly 20 25 30 Ser Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Ser Ile Tyr Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu 65 70 75 80<00023!8>Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Gly Asn Pro Ala Trp Phe Ala Tyr Trp Gly Gin Gly Thr Leu Val 100 105 110 Thr Val Ser Ala 115 <210> 93 <211> 120 <212> PRT <213> Mus musculus <400> 93 Gln Val Gin Leu Gin Gin Ser Gly Pro Gin Val Val Arg Pro Gly Val 1 5 10 15 Ser Val Lys lie Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Lys Gin Ser His Ala Lys Ser Leu Glu Trp lie 35 40 45 Gly lie lie Asn Thr Tyr Asn Gly Asn Thr Asn Tyr Asn Gin Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Phe Ala Arg Leu Thr Ser Glu Asp Ser Ala lie Tyr Tyr Cys 85 90 95 Ala Arg Ser Arg Gly Pro Trp Asp Arg Tyr Phe Asp Val Trp Gly Ala 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 94 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Human heavy chain variable region <400> 94 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Ser Gly Tyr Asp Ser Asn Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 95 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Human heavy chain variable region <400> 95 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Ser Gly Arg Tyr Leu Pro Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 <210> 96 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Human heavy chain variable region <400> 96 Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala lie Ser Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45 Gly Gly lie lie Pro lie Phe Gly Thr Ala Asn Tyr Ala Gin Lys Phe 50 55 60 Gln Gly Arg Val Thr lie Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Glu Arg Gly Ser Tyr Leu lie Asp Tyr Trp Gly Gin Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 <210> 97 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> H chain variable region CDR <400> 97 Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser 1 5 10

Claims

1. An antibody against B7-H3, characterized in that, The antibody has a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region includes the following three complementary determinant regions (CDRs): VH-CDR1, as shown in SEQ ID NO:97, VH-CDR2 shown in SEQ ID NO:54, and VH-CDR3 as shown in SEQ ID NO:35; The light chain variable region includes the following three complementary determinant regions (CDRs): VL-CDR1 shown in SEQ ID NO:1 VL-CDR2 as shown in SEQ ID NO:26 or 2, and VL-CDR3 as shown in SEQ ID NO:3; or The heavy chain variable region includes the following three complementary determinant regions (CDRs): VH-CDR1 shown in SEQ ID NO:33, VH-CDR2 shown in SEQ ID NO:34, and VH-CDR3 as shown in SEQ ID NO:35; The light chain variable region includes the following three complementary determinant regions (CDRs): VL-CDR1 shown in SEQ ID NO:1 VL-CDR2 shown in SEQ ID NO:2 or 25, and VL-CDR3 as shown in SEQ ID NO:3; or The heavy chain variable region includes the following three complementary determinant regions (CDRs): VH-CDR1 shown in SEQ ID NO:33, VH-CDR2 shown in SEQ ID NO:54, and VH-CDR3 as shown in SEQ ID NO:35; The light chain variable region includes the following three complementary determinant regions (CDRs): VL-CDR1 shown in SEQ ID NO:1 VL-CDR2 shown in SEQ ID NO:2 or 26, and VL-CDR3 as shown in SEQ ID NO:

3.

2. The antibody as described in claim 1, characterized in that, The heavy chain variable region and light chain variable region of the antibody are selected from the following group: 。 3. A recombinant protein, characterized in that, The recombinant proteins include: (i) the antibody as described in claim 1 or 2; and (ii) Optional tag sequences to assist in expression and / or purification.

4. An antibody conjugate, characterized in that, This antibody conjugate contains: (a) An antibody portion, wherein the antibody portion is the antibody as described in claim 1 or 2; and (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

5. The antibody conjugate as described in claim 4, characterized in that, The coupling part is MMAE.

6. A polynucleotide, characterized in that, The polynucleotides encode polypeptides selected from the following group: (1) The antibody as described in claim 1 or 2; or (2) The recombinant protein as described in claim 3.

7. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 6.

8. A genetically engineered host cell, characterized in that, The host cell contains the vector as described in claim 7 or the genome is integrated with the polynucleotide as described in claim 6.

9. The use of an active ingredient, characterized in that, The active ingredient is selected from the group consisting of: the antibody as described in claim 1 or 2, the recombinant protein as described in claim 3, or the antibody-drug conjugate as described in claim 4 or 5, or combinations thereof, wherein the active ingredient is used to prepare diagnostic reagents or kits for detecting human B7-H3 protein.

10. The use of an active ingredient, characterized in that, The active ingredient is an antibody conjugate, and the antibody conjugate contains: (a) An antibody portion, wherein the antibody portion is the antibody as described in claim 1 or 2; and (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof; The active ingredient is used to prepare drugs for the prevention and / or treatment of human non-small cell lung cancer, human pancreatic cancer, human undifferentiated lung cancer, colorectal cancer, gastric cancer, prostate cancer, or liver cancer that highly express B7-H3.

11. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (i) the antibody conjugate according to claim 4 or 5; and (ii) Pharmaceutically acceptable carriers.

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