Anti-trop-2 antibodies, antigen-binding fragments thereof or mutants thereof, and medical uses thereof

Abstract

The present disclosure relates to anti-TROP-2 antibodies, antigen-binding fragments thereof, or mutants thereof, and medical uses thereof. Further, the present disclosure relates to a mutant of a humanized anti-TROP-2 antibody, and a pharmaceutical composition comprising the mutant of the anti-TROP-2 antibody, and uses thereof in the manufacture of a medicament for treating a TROP-2-mediated disease or disorder, and in tumor detection and diagnosis.

Description

[0001] This application claims priority to patent application filed on October 14, 2020 (application number 202011099107.8). Technical Field

[0002] This disclosure relates to an anti-TROP-2 antibody, its antigen-binding fragment or a mutant thereof; a chimeric antibody, a humanized antibody or a mutant thereof comprising the CDR region of the anti-TROP-2 antibody; and a pharmaceutical composition comprising a human anti-TROP-2 antibody, its antigen-binding fragment or a mutant thereof; and its use as an anticancer drug and for detecting or diagnosing tumors. Background Technology

[0003] With the deepening research into tumor genomics, proteomics, and signal transduction pathways, the interaction between oncogenes and tumor suppressor genes in tumor cells and their impact on the tumor microenvironment have become increasingly clear. This has made it possible to design new anti-tumor therapies targeting specific molecular targets of tumors.

[0004] Molecular targeted therapy for tumors is a novel treatment modality distinct from traditional surgery, radiotherapy, and chemotherapy. Its advantage lies in the fact that drugs typically bind only to specific target sites, directly affecting the function of target molecules or the physical or chemical effect molecules they carry to kill or inhibit target cells. Because the target sites are clearly defined, drugs usually exhibit high selectivity, effectively killing or inhibiting target cells while producing little or no toxic side effects on normal tissue cells. Therefore, the development of molecularly targeted drugs has become a hot topic in clinical tumor research.

[0005] Trophoblast cell surface antigen 2 (TROP-2), also known as tumor-associated calcium signal transducer 2 (TACSTD2), is a transmembrane glycoprotein encoded by the TACSTD2 gene. TROP-2 consists of approximately 323 amino acids, with a 26-amino acid signal peptide, 248 amino acids in the extracellular domain, 23 amino acids in the transmembrane domain, and 26 amino acids in the cytoplasmic domain. The extracellular domain of TROP-2 contains four heterogeneous N-binding glycosylation sites; the addition of glycans increases the apparent molecular weight by 11 to 13 kDa. Within the TACSTD gene family, the extracellular domain possesses a characteristic thyroglobulin (TY) sequence, generally considered to be associated with cancer cell proliferation, invasion, and metastasis.

[0006] To date, no physiological ligand for TROP-2 has been identified, and its molecular function remains unclear. However, because the residue at position 303 of serine in the cell acts as a protein kinase C (PKC, belonging to the Ca2+ family), it is known to function as a protein kinase C. 2+The fact that TROP-2 is phosphorylated by a protein kinase-dependent enzyme and that its intracellular domains contain a PIP2-binding sequence suggests that TROP-2 has a signal transduction function in tumor cells.

[0007] Numerous clinical studies and literature reports have shown that TROP-2 antigen is overexpressed in various epithelial-derived cancers, including gastric cancer, lung cancer, colorectal cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer, and esophageal cancer. TROP-2 antigen is rarely expressed or not expressed in normal adult tissues, with only small amounts expressed in epithelial cells, and the expression level is lower than in cancer cells, indicating that TROP-2 is associated with tumorigenesis. Overexpression of TROP-2 in tumor tissues is closely related to poor prognosis and cancer cell metastasis, and also affects overall survival. Therefore, TROP-2 has become a promising target for molecular targeted therapy of tumors.

[0008] Currently, although studies on the anti-tumor effects of various TROP-2 antibodies have been reported:

[0009] U.S. Patent No. 5,840,854 reports the cytotoxicity of an anti-TROP-2 monoclonal antibody (BR110) conjugated with cytotoxin against human cancer cell lines H3619, H2987, MCF-7, H3396, and H2981.

[0010] US Patent No. 6,653,104 discloses an antibody (RS7) that was tested in an in vivo model using an antibody labeled with a radioactive substance and showed antitumor activity in a nude mouse xenograft model, but no antitumor effect was reported when using the nude antibody alone.

[0011] U.S. Patent No. 7,420,040 also reports that isolated monoclonal antibodies produced from hybridoma cell lines AR47A6.4.2 or AR52A301.5, obtained by immunizing mice with human ovarian cancer tissue, bind to the TROP-2 antigen and exhibit antitumor activity in a nude mouse xenograft model.

[0012] CN102827282A discloses a human anti-TROP-2 genetically engineered antibody IgG and its application. In vitro test results show that the anti-TROP-2 antibody IgG has a significant inhibitory effect on the proliferation of pancreatic cancer cells.

[0013] CN104114580A discloses an antibody (particularly a humanized antibody) that specifically reacts with the TROP-2 antigen and has anti-tumor activity in vivo; as well as a hybridoma that produces the antibody, a complex of the antibody and a pharmaceutical agent, a pharmaceutical composition for the diagnosis or treatment of tumors, a method for the detection of tumors, and a kit for the detection or diagnosis of tumors.

[0014] However, finding monoclonal antibodies with high affinity, high specificity, and potent cytotoxicity or tumor-killing / inhibitory / regression activity remains challenging. Therefore, there is still an urgent need to develop TROP-2 antibodies and other immunotherapeutic agents with good efficacy, high safety profiles, and suitability for human subjects. Summary of the Invention

[0015] This disclosure provides an anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, comprising a heavy chain variable region and a light chain variable region.

[0016] In a preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, wherein:

[0017] The heavy chain variable region contains at least one HCDR selected from the following sequences:

[0018] SEQ ID NO: 4, SEQ ID NO: 43 and SEQ ID NO: 6;

[0019] and

[0020] The light chain variable region comprises at least one LCDR selected from the following sequence:

[0021] SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9;

[0022] Wherein, SEQ ID NO: 43 is RIDPXDSETHYNQKFKD, and X is selected from amino acid residues of R, Y, Q, L, T, I, F, E or A.

[0023] In a preferred embodiment of this disclosure, the heavy chain variable region HCDR2 is selected from SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 42.

[0024] In a preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, wherein the antibody heavy chain variable region comprises:

[0025] HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 26, and HCDR3 shown in SEQ ID NO: 6; or

[0026] HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 27, and HCDR3 shown in SEQ ID NO: 6; or

[0027] HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 28, and HCDR3 shown in SEQ ID NO: 6; or

[0028] HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 29, and HCDR3 shown in SEQ ID NO: 6; or

[0029] HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 30, and HCDR3 shown in SEQ ID NO: 6; or

[0030] HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 39, and HCDR3 shown in SEQ ID NO: 6; or

[0031] HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 40, and HCDR3 shown in SEQ ID NO: 6; or

[0032] HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 41, and HCDR3 shown in SEQ ID NO: 6; or

[0033] HCDR1 shown in SEQ ID NO:4, HCDR2 shown in SEQ ID NO:42, and HCDR3 shown in SEQ ID NO:6.

[0034] In a preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, wherein the light chain variable region comprises:

[0035] LCDR1 shown in SEQ ID NO: 7, LCDR2 shown in SEQ ID NO: 8, and LCDR3 shown in SEQ ID NO: 9.

[0036] In a preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, wherein:

[0037] The heavy chain variable region includes: HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 26, and HCDR3 shown in SEQ ID NO: 6; and

[0038] The light chain variable region includes: LCDR1 shown in SEQ ID NO: 7, LCDR2 shown in SEQ ID NO: 8, and LCDR3 shown in SEQ ID NO: 9.

[0039] In a preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, wherein:

[0040] The heavy chain variable region includes: HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 27, and HCDR3 shown in SEQ ID NO: 6; and

[0041] The light chain variable region includes: LCDR1 shown in SEQ ID NO: 7, LCDR2 shown in SEQ ID NO: 8, and LCDR3 shown in SEQ ID NO: 9.

[0042] In a preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, wherein:

[0043] The heavy chain variable region includes: HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 28, and HCDR3 shown in SEQ ID NO: 6; and

[0044] The light chain variable region includes: LCDR1 shown in SEQ ID NO: 7, LCDR2 shown in SEQ ID NO: 8, and LCDR3 shown in SEQ ID NO: 9.

[0045] In a preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, wherein:

[0046] The heavy chain variable region includes: HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 29, and HCDR3 shown in SEQ ID NO: 6; and

[0047] The light chain variable region includes: LCDR1 shown in SEQ ID NO: 7, LCDR2 shown in SEQ ID NO: 8, and LCDR3 shown in SEQ ID NO: 9.

[0048] In a preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, wherein:

[0049] The heavy chain variable region includes: HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 30, and HCDR3 shown in SEQ ID NO: 6; and

[0050] The light chain variable region includes: LCDR1 shown in SEQ ID NO: 7, LCDR2 shown in SEQ ID NO: 8, and LCDR3 shown in SEQ ID NO: 9.

[0051] In a preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, wherein:

[0052] The heavy chain variable region includes: HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 39, and HCDR3 shown in SEQ ID NO: 6; and

[0053] The light chain variable region includes: LCDR1 shown in SEQ ID NO: 7, LCDR2 shown in SEQ ID NO: 8, and LCDR3 shown in SEQ ID NO: 9.

[0054] In a preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, wherein:

[0055] The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 40, and HCDR3 shown in SEQ ID NO: 6; and

[0056] The light chain variable region includes: LCDR1 shown in SEQ ID NO: 7, LCDR2 shown in SEQ ID NO: 8, and LCDR3 shown in SEQ ID NO: 9.

[0057] In a preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, wherein:

[0058] The heavy chain variable region includes: HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 41, and HCDR3 shown in SEQ ID NO: 6; and

[0059] The light chain variable region includes: LCDR1 shown in SEQ ID NO: 7, LCDR2 shown in SEQ ID NO: 8, and LCDR3 shown in SEQ ID NO: 9.

[0060] In a preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, wherein:

[0061] The heavy chain variable region includes: HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 42, and HCDR3 shown in SEQ ID NO: 6; and

[0062] The light chain variable region includes: LCDR1 shown in SEQ ID NO: 7, LCDR2 shown in SEQ ID NO: 8, and LCDR3 shown in SEQ ID NO: 9.

[0063] In a preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, wherein the antibody is a murine antibody, a chimeric antibody, a human antibody, or a humanized antibody.

[0064] In a preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, wherein the anti-TROP-2 antibody or its antigen-binding fragment further comprises a constant region derived from human IgG1, IgG2, IgG3, or IgG4 or a mutant thereof.

[0065] In a further preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof further comprises a mutant weight chain constant region derived from human IgG1, IgG2, or IgG4.

[0066] In a further preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof further comprises a heavy chain constant region derived from IgG1, which exhibits enhanced ADCC toxicity after amino acid mutation.

[0067] In a further preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof further comprises a constant region of the IgG1 heavy chain introducing 239D and 241L mutations.

[0068] In a further preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof further comprises a heavy chain constant region selected from SEQ ID NO: 12.

[0069] In a further preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof further comprises a light chain constant region derived from the human κ chain, λ chain, or a mutant thereof.

[0070] In a further preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof further comprises a light chain constant region selected from SEQ ID NO: 13.

[0071] In a preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, wherein the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof comprises a heavy chain variable region selected from the following: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with them.

[0072] In a preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, wherein the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof comprises a light chain variable region SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it.

[0073] In a preferred embodiment of this disclosure, the anti-TROP-2 antibody or its mutant comprises a heavy chain selected from the following: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or a full-length heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with them.

[0074] In a preferred embodiment of this disclosure, the anti-TROP-2 antibody or its mutant comprises a light chain SEQ ID NO: 15, or a full-length light chain having at least 80%, 85%, 90%, 95%, or 99% identity with it.

[0075] In a preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, wherein:

[0076] The anti-TROP-2 antibody, its antigen-binding fragment, or its mutants have the heavy chain variable region shown in SEQ ID NO: 16, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it; or,

[0077] The anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof has the heavy chain variable region shown in SEQ ID NO: 17, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it; or,

[0078] The anti-TROP-2 antibody, its antigen-binding fragment, or its mutants have the heavy chain variable region shown in SEQ ID NO: 18, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it; or,

[0079] The anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof has the heavy chain variable region shown in SEQ ID NO: 19, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it; or,

[0080] The anti-TROP-2 antibody, its antigen-binding fragment, or its mutants have the heavy chain variable region shown in SEQ ID NO: 20, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it; or,

[0081] The anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof has the heavy chain variable region shown in SEQ ID NO: 31, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it; or,

[0082] The anti-TROP-2 antibody, its antigen-binding fragment, or its mutants have the heavy chain variable region shown in SEQ ID NO: 32, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it; or,

[0083] The anti-TROP-2 antibody, its antigen-binding fragment, or its mutants have the heavy chain variable region shown in SEQ ID NO: 33, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it; or,

[0084] The anti-TROP-2 antibody, its antigen-binding fragment, or its mutants have the heavy chain variable region shown in SEQ ID NO: 34, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it.

[0085] In a preferred embodiment of this disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, wherein:

[0086] The anti-TROP-2 antibody or its mutant comprises the heavy chain shown in SEQ ID NO: 21, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity with it; or,

[0087] The anti-TROP-2 antibody or its mutant comprises the heavy chain shown in SEQ ID NO: 22, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity with it; or,

[0088] The anti-TROP-2 antibody or its mutant comprises the heavy chain shown in SEQ ID NO: 23, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity with it; or,

[0089] The anti-TROP-2 antibody or its mutant comprises the heavy chain shown in SEQ ID NO: 24, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity with it; or,

[0090] The anti-TROP-2 antibody or its mutant comprises the heavy chain shown in SEQ ID NO: 25, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity with it; or,

[0091] The anti-TROP-2 antibody or its mutant comprises the heavy chain shown in SEQ ID NO: 35, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity with it; or,

[0092] The anti-TROP-2 antibody or its mutant comprises the heavy chain shown in SEQ ID NO: 36, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity with it; or,

[0093] The anti-TROP-2 antibody or its mutant comprises the heavy chain shown in SEQ ID NO: 37, or the heavy chain having at least 80%, 85%, 90%, 95% or 99% identity with it, and the light chain shown in SEQ ID NO: 15, or the light chain having at least 80%, 85%, 90%, 95% or 99% identity with it;

[0094] The anti-TROP-2 antibody or its mutant comprises the heavy chain shown in SEQ ID NO: 38, or the heavy chain having at least 80%, 85%, 90%, 95% or 99% identity with it, and the light chain shown in SEQ ID NO: 15, or the light chain having at least 80%, 85%, 90%, 95% or 99% identity with it.

[0095] This disclosure also relates to a polynucleotide encoding the aforementioned anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof.

[0096] This disclosure also relates to an expression vector containing the aforementioned polynucleotides.

[0097] This disclosure also relates to a host cell that transforms the above-described expression vector.

[0098] This disclosure also relates to a preferred embodiment in which the host cell is selected from bacteria, yeast or mammalian cells;

[0099] This disclosure further includes a preferred embodiment in which the host cell is selected from Escherichia coli, Pichia pastoris, CHO cells, or HEK293 cells.

[0100] This disclosure also relates to a method for producing the above-described anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, comprising the steps of: culturing the above-described host cells, preferably HEK293 cells; isolating the antibody from the culture, preferably from the cell culture medium; and purifying the antibody, preferably by chromatography.

[0101] This disclosure also relates to a pharmaceutical composition comprising: the above-described anti-TROP-2 antibody, an antigen-binding fragment or a mutant thereof, and a pharmaceutically acceptable excipient, diluent or carrier.

[0102] This disclosure also relates to a detection or diagnostic kit containing: the above-described anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof.

[0103] This disclosure also relates to the use of the above-mentioned anti-TROP-2 antibody, its antigen-binding fragment or a mutant thereof in the preparation of a medicament for the treatment or prevention of TROP-2 mediated diseases or conditions.

[0104] This disclosure also relates to the use of the described anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof in the preparation of reagents, wherein the reagents are used to detect, diagnose, and prognose TROP-2-mediated diseases or conditions.

[0105] This disclosure also relates to a preferred embodiment of the above-described use, wherein: the TROP-2-mediated disease or condition is cancer; preferably, the TROP-2-mediated disease or condition is a cancer expressing TROP-2; preferably, the cancer is selected from: breast cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma, and melanoma.

[0106] This disclosure also relates to a preferred embodiment of the above-described anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, for the treatment or prevention of TROP-2-mediated diseases; said diseases are selected from: breast cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma, and melanoma.

[0107] This disclosure also relates to a preferred embodiment of the above-described anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof, for the detection, diagnosis, and prognosis of TROP-2-mediated diseases; said diseases are selected from: breast cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma, and melanoma.

[0108] This disclosure also relates to a method of treating or preventing TROP-2-mediated diseases, comprising the steps of: providing a subject in need with a therapeutically effective or preventatively effective amount of any of the above-described anti-TROP-2 antibody, its antigen-binding fragment, or a mutant thereof; or providing a subject in need with a therapeutically effective or preventatively effective amount of a pharmaceutical composition according to this disclosure; wherein the TROP-2-mediated diseases are selected from breast cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma, and melanoma. Detailed Implementation

[0109] the term

[0110] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0111] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

[0112] The term "antibody" as used in this disclosure refers to immunoglobulin, which is a tetrapeptide chain structure composed of two heavy chains and two light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in its heavy chain, it can be further divided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ chains or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ chain or a λ chain.

[0113] In this disclosure, the variable region of the antibody light chain may further include a constant region of the light chain, which includes a human or mouse κ, λ chain or a variant thereof.

[0114] In this disclosure, the variable region of the antibody heavy chain may further include a constant region of the heavy chain, which includes human or mouse IgG1, IgG2, IgG3, IgG4 or variants thereof.

[0115] The sequence of approximately 110 amino acids near the N-terminus of the antibody heavy and light chains varies considerably, forming the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable, forming the constant region (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). The three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The number and position of the CDR amino acid residues in the VL and VH regions of the antibody or antigen-binding fragment described in this disclosure conform to the known Kabat numbering rules and Kabat or ABM definition rules.

[0116] The term "TROP-2" includes any variant or isotype of naturally expressed TROP-2. The antibodies disclosed herein are cross-reactive with TROP-2 derived from non-human species. Alternatively, the antibody may be human TROP-2 specific and may not exhibit cross-reactivity with other species. TROP-2 or any variant or isotype thereof may be isolated from cells or tissues that naturally express them, or generated via recombinant techniques using those techniques common in the art and those described herein. Preferably, the anti-TROP-2 antibody targets human TROP-2 having a normal glycosylation pattern.

[0117] The term "recombinant human antibody" includes human antibodies prepared, expressed, created, or isolated by recombinant methods, the techniques and methods of which are well known in the art, such as:

[0118] 1. Antibodies isolated from transgenic, transchromosomal animals (e.g., mice) of the human immunoglobulin gene or hybridomas prepared therefrom;

[0119] 2. Antibodies isolated from host cells transformed to express antibodies, such as transfected tumors;

[0120] 3. Antibodies isolated from recombinant human antibody libraries; and

[0121] 4. Antibodies prepared, expressed, created, or isolated by methods such as splicing human immunoglobulin gene sequences into other DNA sequences.

[0122] These recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations that occur during antibody maturation.

[0123] The term "mouse antibody" in this disclosure refers to a monoclonal antibody against human TROP-2 prepared in accordance with the knowledge and skills in the art. Preparation involves injecting a test subject with TROP-2 antigen, followed by isolation of a hybridoma expressing an antibody having the desired sequence or functional characteristics. In one exemplary embodiment of this disclosure, the mouse TROP-2 antibody or its antigen-binding fragment may further comprise a light chain constant region of a mouse κ, λ chain, or a variant thereof, or further comprise a heavy chain constant region of a mouse IgG1, IgG2, IgG3, or IgG4, or a variant thereof.

[0124] The term "human antibody" includes antibodies having variable and constant regions of human immunoglobulin sequences. Human antibodies disclosed herein may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, the term "human antibody" does not include humanized antibodies.

[0125] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody created by grafting a non-human species' CDR sequence into the variable region framework of a human antibody. Humanized antibodies overcome the drawback of chimeric antibodies, which often induce a strong immune response due to carrying a large number of non-human protein components. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse mutations can be performed on the variable region of the human antibody to maintain its activity.

[0126] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of an antibody from a first species with the constant region of an antibody from a second species. This chimeric antibody can mitigate the immune response induced by the first species antibody. As an example, the development of a chimeric antibody involves creating a hybridoma that secretes a murine-specific monoclonal antibody, cloning the variable region gene from mouse hybridoma cells, and then cloning the constant region gene of a human antibody as needed. The mouse variable region gene and the human constant region gene are then linked to form a chimeric gene, which is inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system. The constant region of the human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or their variants, preferably containing the heavy chain constant region of human IgG1, IgG2, or IgG4, or using the IgG1 heavy chain constant region that enhances ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity through amino acid mutations.

[0127] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. The antibody fragment retains at least some of the binding specificity of the parent antibody. Typically, when activity is expressed on a molar basis, the antibody fragment retains at least 10% of the parent antibody's binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity to the target. Examples of antigen-binding fragments include, but are not limited to: Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. A review of engineered antibody variants is available in Holliger and Hudson, 2005, Nat. Biotechnol. 23: 1126-1136.

[0128] A "Fab fragment" consists of a light chain, a heavy chain (CH1), and a variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0129] The “Fc” region contains two heavy chain segments, including the CH1 and CH2 domains of the antibody. The two heavy chain segments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain.

[0130] The “Fab’ fragment” contains a light chain and a heavy chain portion containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, thereby enabling the formation of interchain disulfide bonds between the two heavy chains of two Fab’ fragments to form the F(ab’)2 molecule.

[0131] The “F(ab')2 segment” contains two light chains and two heavy chains containing portions of a constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab')2 segment consists of two Fab' segments held together by disulfide bonds between the two heavy chains.

[0132] The “Fv region” contains variable regions from both the heavy and light chains, but lacks constant regions.

[0133] The term "multispecific antibody," used in its broadest sense, encompasses antibodies that exhibit multi-epitope specificity. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit exhibits multi-epitope specificity; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies, triabodies, and antibody fragments covalently or non-covalently linked together, etc.

[0134] The term "single-chain antibody" refers to a single-chain recombinant protein composed of the heavy chain variable region (VH) and light chain variable region (VL) of an antibody linked by a linker peptide. It is the smallest antibody fragment with a complete antigen-binding site.

[0135] The term "domain antibody fragment" refers to an immunoglobulin fragment with immunological function containing only the heavy chain variable region or the light chain variable region. In some cases, two or more VH regions are covalently linked to peptide linkers to form a bivalent domain antibody fragment. The two VH regions of a bivalent domain antibody fragment can target the same or different antigens.

[0136] The term "binding to TROP-2" refers to the ability to interact with TROP-2 (or its epitopes).

[0137] The term "antigen binding site" refers to the three-dimensional spatial site recognized by the antibody or antigen binding fragment disclosed herein.

[0138] The term "epitope" refers to a site on an antigen that binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or from non-adjacent amino acids through the ternary folding of a protein. Epitopes formed from adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost after treatment with denaturing solvents. Epitopes typically comprise at least 3-15 amino acids in a unique spatial conformation. Methods for determining which epitopes bind to a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of epitopes include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0139] The terms "specific binding" and "selective binding" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, when human TROP-2 is used as the analyte and an antibody as the ligand, the antibody binds at approximately less than 10^66 ppm when measured in an instrument using surface plasmon resonance (SPR) technology. -7 M or even smaller equilibrium dissociation constant (K) D An antibody binds to a predetermined antigen (or epitope); and the affinity of the antibody for the predetermined antigen (or epitope) is at least twice that for a nonspecific antigen (such as BSA) other than the predetermined antigen (or closely related antigens). The term "antibody that recognizes an antigen" may be used interchangeably with the term "antibody that specifically binds" herein.

[0140] The term "cross-reactivity" refers to the ability of the antibodies of this disclosure to bind to TROP-2 from different species. For example, an antibody of this disclosure that binds to human TROP-2 may also bind to TROP-2 from another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays (e.g., SPR and ELISA), or binding or functional interaction with cells physiologically expressing TROP-2. Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.

[0141] The terms “inhibition” and “blocking” are used interchangeably and encompass both partial and complete inhibition / blocking. Preferably, inhibition or blocking of a ligand reduces or alters the level or type of activity when the ligand binds, compared to the normal level or type of activity when the ligand binds without inhibition or blocking. Inhibition and blocking are also intended to include any measurable reduction in ligand binding affinity upon contact with an anti-TROP-2 antibody compared to a ligand not contacted with an anti-TROP-2 antibody.

[0142] The term “growth inhibition” (e.g., involving cells) is intended to include any measurable reduction in cell growth.

[0143] The terms “inducing an immune response” and “enhancing an immune response” are used interchangeably and refer to the stimulation of an immune response by a specific antigen (i.e., passive or adaptive). The term “inducing” in relation to induced CDC or ADCC refers to the stimulation of a specific direct cell-killing mechanism.

[0144] The term "ADCC," or antibody-dependent cell-mediated cytotoxicity, refers to the direct killing of target cells by antibodies by cells expressing Fc receptors, which recognize the Fc fragment of the antibody. The ADCC effector function of antibodies can be enhanced, reduced, or eliminated by modifying the Fc fragment of IgG. This modification involves mutations in the constant region of the antibody's heavy chain.

[0145] Methods for producing and purifying antibodies and antigen-binding fragments are well-known and readily available in the prior art, such as in Cold Spring Harbor's Guide to Antibody Experimentation, Chapters 5-8 and 15. As an example, mice can be immunized with human TROP-2 or fragments thereof; the resulting antibody can be refolded, purified, and sequenced amino acids using conventional methods. Similarly, antigen-binding fragments can be prepared using conventional methods. The antibodies or antigen-binding fragments of this disclosure are obtained by adding one or more human FR regions to a non-human CDR region using genetic engineering methods. Human FR germline sequences are available from the ImMunoGeneTics (IMGT) website or from the journal Immunoglobulins, 2001 ISBN012441351.

[0146] The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. The corresponding antibody cDNA sequence can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminus of the FC region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. The culture medium secreting antibodies can be purified and collected using conventional techniques. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

[0147] The antibodies disclosed herein refer to monoclonal antibodies. The monoclonal antibodies (mAbs) described herein refer to antibodies obtained from a single clonal cell line, and the cell line is not limited to eukaryotic, prokaryotic, or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using techniques such as hybridoma technology, recombinant technology, phage display technology, synthetic technology (such as CDR-grafting), or other existing technologies.

[0148] "Administration," "dosing," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "dosing," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Administration," "dosing," and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. When applied to humans, veterinary, or research subjects, "administration," "dosing," and "treatment" refer to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0149] "Treatment" means providing a subject with an oral or topical therapeutic agent (such as an antibody or fragment thereof comprising any of the present disclosure) that has symptoms of one or more diseases, and that the therapeutic agent has a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered in the treated subject (or subject population) in an amount that effectively relieves one or more disease symptoms, either by inducing the regression of such symptoms or by inhibiting their development to any clinically measured extent. The amount of therapeutic agent that effectively relieves any specific disease symptom (also referred to as the "therapeuticly effective amount") can vary depending on a variety of factors, such as the subject's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may be ineffective in alleviating the symptoms of the target disease in each subject, they should significantly reduce the symptoms of the target disease in a statistically valid number of subjects, as determined by any statistical test known in the art, such as the Studentt test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0150] An "effective dose" includes an amount sufficient to improve or prevent a medical condition or its symptoms. An effective dose also means an amount sufficient to allow or facilitate diagnosis. The effective dose for a specific subject or veterinary subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective dose may be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0151] "Exogenous" refers to substances that are produced outside of an organism, cell, or human body, depending on the context.

[0152] "Endogenous" refers to substances that are produced in an organism, cell, or human body in accordance with their context.

[0153] "Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same bases or amino acid residues—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100%. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of homology is obtained by aligning the two sequences.

[0154] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include their progeny. Therefore, the terms “transformation” and “transformed cell” include primary cells and cultures derived from them, regardless of passage number. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in DNA content. This includes mutant progeny with the same function or biological activity as those selected from primary cells. Where different names are intended, the context will be clear.

[0155] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur. For example, "optionally contains 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may, but do not have to, be present.

[0156] "Pharmaceutical composition" means containing one or more antibodies or antigen-binding fragments thereof described herein, as well as other components such as physiological / pharmaceutical-grade carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0157] In the context, unless explicitly stated otherwise, the singular forms “a,” “one,” or “the” also include their corresponding plural forms.

[0158] The following embodiments are used to further describe this disclosure, but these embodiments are not intended to limit the scope of this disclosure. Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents that do not specify a specific source are commercially available, conventional reagents.

[0159] Example 1: Antigen Preparation

[0160] The human TROP-2 (TROP-2-His) protein encoding the His tag was synthesized by SinoBiologics (10428-H08H).

[0161] TROP-2-His sequence:

[0162] MARGPGLAPPPLRLPLLLLVLAAVTGHTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHILIDLRHRPTAG AFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSMKRLTAGLIAVIVVVVALVAGMAVLVITNRRKSGKYKKVEIKELGELRKEPSLHHHHHHHH

[0163] SEQ ID NO: 1.

[0164] Example 2: Obtaining mouse hybridoma and antibody sequences

[0165] Animals were immunized with human antigen TROP-2-His. Five Balb / c mice and five A / J mice, female and 10 weeks old, were used. The immunogen and immune adjuvant were thoroughly mixed and emulsified in a 1:1 ratio to prepare a stable "water-in-oil" liquid. The injection dose was 25 μg / 200 μL / mouse.

[0166] Table 1. Immunization Regimens

[0167] Day 1 First immunization, with Freund's adjuvant. Day 21 Second immunization, incomplete Freund's adjuvant. Day 35 The third immunization was administered with an incomplete Freund's adjuvant. Day 42 Blood collection and serum titer testing (3-immunoassay) Day 49 Fourth immunization, with incomplete Freund's adjuvant. Day 56 Blood collection and serum titer testing (4 immune blood tests)

[0168] Serum titers and binding capacity to cell surface antigens were assessed using the indirect ELISA method described in Example 3 on immunized mouse serum. Cell fusion was initiated based on control titer (greater than 100,000-fold dilution). Mice with high serum titers, affinity, and FACS binding were selected for a single final immunization, followed by sacrifice. Spleen cells were fused with SP2 / 0 myeloma cells and plated to obtain hybridomas. Target hybridomas were screened using indirect ELISA, and monoclonal cell lines were established using limiting dilution. Positive antibody lines were further screened using indirect ELISA to select hybridomas binding recombinant proteins. Hybridoma cells in logarithmic growth phase were collected, and RNA was extracted using Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript). TM Reverse Transcriptase (Takara#2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-primers (Novagen, TB326 Rev.B 0503) and then sequenced to obtain the sequence of the mouse antibody.

[0169] The heavy and light chain variable region sequences of mouse monoclonal antibody M1 are as follows:

[0170] M1 HCVR

[0171] QVQLQQPGAELVRPGASVKLSCRASGYTFT NYWMN WVKQRPEQGLEWIG RIDPNDSETHYNQKFKD RAILTVDKASNTAYMQLSGLTSEDSAVHYCAR SGFGSTYWFFDV WGAGTTVTVSS

[0172] SEQ ID NO: 2

[0173] M1 LCVR

[0174] DIVMTQSHKFMSTSVGDRVSITC KASQDVSTAVA WYQQKPGQSPKLLIY SASYR YT GVPDRFAGSGYGTDFTFTISSVQTEDLTVYHC QQHYSTPLT FGPGTRLELK

[0175] SEQ ID NO: 3.

[0176] Table 2. CDR sequences of the heavy and light chain variable regions of mouse monoclonal antibody M1

[0177] name sequence serial number HCDR1 NYWMN SEQ ID NO: 4 HCDR2 RIDPNDSETHYNQKFKD SEQ ID NO: 5 HCDR3 SGFGSTYWFFDV SEQ ID NO: 6 LCDR1 KASQDVSTAVA SEQ ID NO: 7 LCDR2 SASYRYT SEQ ID NO: 8 LCDR3 QQHYSTPLT SEQ ID NO: 9

[0178] Example 3: Method for detecting the in vitro binding activity of antibodies

[0179] In vitro indirect ELISA binding assay:

[0180] TROP-2His protein (Sino Biological Inc., cat#10428-H08H) was diluted to 1 μg / mL with PBS at pH 7.4, and 100 μL was added to each well of a 96-well high-affinity microplate. The plate was incubated overnight (16-20 hours) at 4°C. After washing four times with PBST (pH 7.4 PBS containing 0.05% Tween-20), 150 μL of 3% bovine serum albumin (BSA) blocking buffer diluted with PBST was added to each well, and the plate was incubated at room temperature for 1 hour. After blocking, the blocking buffer was discarded, and the plate was washed four times with PBST buffer.

[0181] The antibody to be tested was diluted with PBST containing 3% BSA, starting at 10 μM, with a 5-fold gradient of 9 doses, and added to the microplate at 100 μL / well. The plate was incubated at room temperature for 1 hour. After incubation, the plate was washed 4 times with PBST, and 100 μL / well of HRP-labeled goat anti-human secondary antibody (Abcam, cat#ab97225) diluted with PBST containing 3% BSA was added. The plate was incubated at room temperature for 1 hour. After washing 4 times with PBST, 100 μL / well of TMB chromogenic substrate (Cell Signaling Technology, cat#7004S) was added, and the plate was incubated at room temperature in the dark for 1 minute. The reaction was terminated by adding 100 μL / well of stop solution (Cell Signaling Technology, cat#7002S). The absorbance was read at 450 nm using a BioTek Synergy H1 microplate reader, and the data were analyzed. The results of concentration signal curve analysis are shown in Table 3 below.

[0182] Table 3. Affinity of mouse antibodies to human TROP-2 antigen (ECG) 50 value)

[0183] mouse antibodies <![CDATA[EC binding to human TROP-2His antigen 50 (nM)]]> M1 0.56

[0184] Example 4: Mouse Antibody Humanization Experiment

[0185] Humanization of murine anti-human TROP-2 monoclonal antibodies is performed using methods disclosed in many publications in the art. In short, by replacing the parental (murine antibody) constant domain with a human constant domain and selecting a human antibody sequence based on the homology between the murine and human antibodies, this disclosure humanizes murine antibody M1.

[0186] Based on the typical structure of the mouse antibody VH / VL CDR obtained, the heavy and light chain variable region sequences were compared with the human antibody germline database to obtain a human germline template with high homology.

[0187] The CDR region of the murine antibody M1 was transplanted into a selected humanized template. Then, based on the three-dimensional structure of the murine antibody, reverse mutations were performed on the embedded residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of VL and VH. After expression testing and comparison of the number of reverse mutations, an antibody composed of the humanized heavy chain variable region (HCVR) and light chain variable region (LCVR) sequences was selected and designed. The sequence is as follows:

[0188] HU6 HCVR

[0189] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDPNDSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQGTTVTVSS

[0190] SEQ ID NO: 10

[0191] HU6 LCVRDVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIK

[0192] SEQ ID NO: 11.

[0193] The designed heavy and light chain variable region sequences were ligated to the IgG1 heavy chain constant region and light chain constant region sequences, respectively. The ligated human IgG1 heavy chain constant region sequence is as follows:

[0194] IgG1 C1

[0195] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0196] SEQ ID NO: 12;

[0197] The designed heavy and light chain variable region sequences were ligated to the IgG1 heavy and light chain constant region sequences, respectively. The ligated human kappa chain constant region sequence is as follows:

[0198] Ig kappa C

[0199] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0200] SEQ ID NO: 13;

[0201] After concatenation, the following are exemplary heavy and light chain sequences:

[0202] HU6DL HCEVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDPNDSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0203] SEQ ID NO: 14

[0204] HU6DL LC

[0205] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0206] SEQ ID NO: 15.

[0207] Example 5: Experiment on the Design of HU6DL Variants

[0208] The humanized antibody HU6DL has N in HCDR2 54 D 55 S 56The motif is prone to glycosylation. Using computer-aided techniques, site-directed mutagenesis of N54 was performed to reduce the potential risk of glycosylation without affecting its binding to the antigen or its thermal stability, resulting in mutants of the antibody HU6DL: HU6DL.R54, HU6DL.Y54, HU6DL.Q54, HU6DL.L54, HU6DL.T54, HU6DL.I54, HU6DL.F54, HU6DL.E54, and HU6DL.A54. The corresponding heavy and light chain variable regions are as follows:

[0209] HU6DL.R54 HCVR

[0210] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDP R DSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQGTTVTVSS

[0211] SEQ ID NO: 16

[0212] HU6DL.R54 LCVR

[0213] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIK

[0214] SEQ ID NO: 11

[0215] HU6DL.Y54 HCVR

[0216] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDP Y DSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQGTTVTVSS

[0217] SEQ ID NO: 17

[0218] HU6DL.Y54 LCVR

[0219] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIK

[0220] SEQ ID NO:11

[0221] HU6DL.Q54 HCVR

[0222] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDP Q DSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQGTTVTVSS

[0223] SEQ ID NO:18

[0224] HU6DL.Q54 LCVR

[0225] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIK

[0226] SEQ ID NO:11

[0227] HU6DL.L54 HCVR

[0228] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDP L DSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQGTTVTVSS

[0229] SEQ ID NO:19

[0230] HU6DL.L54 LCVR

[0231] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIK

[0232] SEQ ID NO:11

[0233] HU6DL.T54 HCVR

[0234] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDP T DSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQGTTVTVSS

[0235] SEQ ID NO:20

[0236] HU6DL.T54 LCVR

[0237] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIK

[0238] SEQ ID NO:11

[0239] HU6DL.I54 HCVR

[0240] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDP I DSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQGTTVTVSS

[0241] SEQ ID NO:31

[0242] HU6DL.I54 LCVR

[0243] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIK

[0244] SEQ ID NO:11

[0245] HU6DL.F54 HCVR

[0246] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDP F DSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQGTTVTVSS

[0247] SEQ ID NO:32

[0248] HU6DL.F54 LCVR

[0249] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIK

[0250] SEQ ID NO:11

[0251] HU6DL.E54 HCVR

[0252] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDP E DSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQGTTVTVSS

[0253] SEQ ID NO:33

[0254] HU6DL.E54 LCVRDVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIK

[0255] SEQ ID NO: 11

[0256] HU6DL.A54 HCVR

[0257] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDP A DSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQGTTVTVSS

[0258] SEQ ID NO: 34

[0259] HU6DL.A54 LCVR

[0260] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIK

[0261] SEQ ID NO: 11.

[0262] The designed heavy and light chain variable region sequences were linked to the IgG1 heavy chain constant region and light chain constant region sequences, respectively. The resulting exemplary heavy and light chain sequences are as follows (wherein, the heavy chains HU6DL.R54, HU6DL.Y54, HU6DL.Q54, HU6DL.L54, HU6DL.T54, HU6DL.I54, HU6DL.F54, HU6DL.E54, and HU6DL.A54 are derived from sequences SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34 are linked to sequence SEQ ID NO: 12, respectively):

[0263] HU6DL.R54 HC

[0264] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDPRDSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0265] SEQ ID NO:21

[0266] HU6DL.R54 LC

[0267] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIKRTV

[0268] AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT

[0269] EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0270] SEQ ID NO:15

[0271] HU6DL.Y54 HC

[0272] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRID

[0273] PYDSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYW

[0274] FFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV

[0275] SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV

[0276] DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0277] SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE

[0278] YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY

[0279] PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

[0280] MHEALHNHYTQKSLSLSPGK

[0281] SEQ ID NO:22

[0282] HU6DL.Y54 LC

[0283] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYR

[0284] YTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIKRTV

[0285] AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT

[0286] EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0287] SEQ ID NO:15

[0288] HU6DL.Q54 HC

[0289] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRID

[0290] PQDSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYW

[0291] FFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV

[0292] SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV

[0293] DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0294] SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE

[0295] YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY

[0296] PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

[0297] MHEALHNHYTQKSLSLSPGK

[0298] SEQ ID NO:23

[0299] HU6DL.Q54 LC

[0300] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYR

[0301] YTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIKRTV

[0302] AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT

[0303] EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0304] SEQ ID NO:15

[0305] HU6DL.L54 HC

[0306] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRID

[0307] PLDSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYW

[0308] FFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV

[0309] SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV

[0310] DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0311] SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE

[0312] YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY

[0313] PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

[0314] MHEALHNHYTQKSLSLSPGK

[0315] SEQ ID NO:24

[0316] HU6DL.L54 LC

[0317] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYR

[0318] YTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIKRTV

[0319] AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT

[0320] EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0321] SEQ ID NO:15

[0322] HU6DL.T54 HC

[0323] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRID

[0324] PTDSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYW

[0325] FFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV

[0326] SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV

[0327] DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0328] SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE

[0329] YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY

[0330] PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

[0331] MHEALHNHYTQKSLSLSPGK

[0332] SEQ ID NO:25

[0333] HU6DL.T54 LC

[0334] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYR

[0335] YTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIKRTV

[0336] AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT

[0337] EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0338] SEQ ID NO:15

[0339] HU6DL.I54 HC

[0340] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRID

[0341] PIDSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYW

[0342] FFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV

[0343] SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV

[0344] DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0345] SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE

[0346] YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY

[0347] PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

[0348] MHEALHNHYTQKSLSLSPGK

[0349] SEQ ID NO:35

[0350] HU6DL.I54 LC

[0351] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYR

[0352] YTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIKRTV

[0353] AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT

[0354] EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0355] SEQ ID NO:15

[0356] HU6DL.F54 HC

[0357] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRID

[0358] PFDSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYW

[0359] FFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV

[0360] SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV

[0361] DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0362] SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE

[0363] YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY

[0364] PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

[0365] MHEALHNHYTQKSLSLSPGK

[0366] SEQ ID NO:36

[0367] HU6DL.F54 LC

[0368] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYR

[0369] YTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIKRTV

[0370] AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT

[0371] EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0372] SEQ ID NO:15

[0373] HU6DL.E54 HC

[0374] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRID

[0375] PEDSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYW

[0376] FFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV

[0377] SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV

[0378] DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0379] SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE

[0380] YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY

[0381] PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

[0382] MHEALHNHYTQKSLSLSPGK

[0383] SEQ ID NO:37

[0384] HU6DL.E54 LC

[0385] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYR

[0386] YTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIKRTV

[0387] AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0388] SEQ ID NO:15

[0389] HU6DL.A54 HC

[0390] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDPADSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0391] SEQ ID NO:38

[0392] HU6DL.A54 LC

[0393] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0394] SEQ ID NO:15。

[0395] Table 4. Heavy chain variable region HCDR2 sequences of HU6DL mutants

[0396] name sequence serial number HU6DL.R54 RIDPRDSETHYNQKFKD SEQ ID NO: 26 HU6DL.Y54 RIDPYDSETHYNQKFKD SEQ ID NO: 27 HU6DL.Q54 RIDPQDSETHYNQKFKD SEQ ID NO: 28 HU6DL.L54 RIDPLDSETHYNQKFKD SEQ ID NO: 29 HU6DL.T54 RIDPTDSETHYNQKFKD SEQ ID NO: 30 HU6DL.I54 RIDPIDSETHYNQKFKD SEQ ID NO: 39 HU6DL.F54 RIDPFDSETHYNQKFKD SEQ ID NO: 40 HU6DL.E54 RIDPEDSETHYNQKFKD SEQ ID NO: 41 HU6DL.A54 RIDPADSETHYNQKFKD SEQ ID NO: 42

[0397] Table 5. HU�DL mutant sequence numbers

[0398]

[0399]

[0400] Based on the amino acid sequences of the light and heavy chains of the above-mentioned humanized antibodies, cDNA fragments were synthesized. Protein expression services were provided by Beijing Yiqiao Shenzhou Technology Co., Ltd. HU6DL protein mutants were expressed by transient transfection of HEK293 cells. The purity of the antibodies was detected by molecular size exclusion chromatography, and their concentration and purity are shown in Table 6 below.

[0401] Table 6. Concentration and purity of HU6DL mutant

[0402] name Concentration (mg / mL) purity(%) HU6DL.R54 1.99 95.8 HU6DL.Y54 1.34 96.4 HU6DL.Q54 1.73 93.1 HU6DL.L54 1.36 94.9 HU6DL.T54 2.73 96.1 HU6DL.I54 2.00 95.5 HU6DL.F54 1.68 93.5 HU6DL.E54 1.56 95.6 HU6DL.A54 1.00 95.1

[0403] Example 6: Affinity test of HU6DL mutant to TROP-2 antigen

[0404] 1. Experimental objective:

[0405] The differences in affinity levels of different anti-TROP-2 mutants for the TROP-2 antigen were evaluated using the ELISA sandwich method, namely "antigen-antibody-HRP-labeled secondary antibody".

[0406] 2. Experimental steps:

[0407] TROP-2, his-tag protein (SinoBiologics, Cat: 10428-H08H), was diluted to 1 μg / mL using DPBS at pH 7.4. 100 μL / well was added to each well of a high-affinity 96-well plate (Corning, Cat: 3590), and incubated overnight at 4°C. The next day, the TROP-2 antigen solution was removed, and 200 μL / well was added to each well containing 0.05% Tween 20 PBS at pH 7.4 (PBST). After washing three times, 200 μL / well of 2% BSA (dissolved in PBST) was added, and the plate was blocked at 37°C for 1 h. After washing three times with PBST, 100 μL / well was added to each well, following a 10-fold, 8-concentration gradient (starting concentration 10 nM - 1 × 10⁻⁶). -6 Candidate antibodies diluted (nM), 0.5% BSA as negative control, blocked at 37℃ for 1 h, washed 3 times with PBST; goat anti-human IgG, Fc-HRP (abcam, cat: ab97225) secondary antibody solution diluted 1:10000 was added to 100 μL / well, blocked at 37℃ for 1 h, washed 3 times with PBST.

[0408] Add 100 μL of TMB (CST, Cat: 7004P6) substrate to each well and react at room temperature for 3 min until the color of the solution at the highest antibody concentration turns dark blue. Add 50 μL of stop solution (CST, Cat: 7002P6) to each well to terminate the reaction and read the absorbance value (OD) at 450 nm.

[0409] 3. Data Processing:

[0410] Using the logarithm of candidate antibody concentration as the X-axis and OD450 absorbance as the Y-axis, the EC50 of each HU6DL mutant to antigen affinity was calculated using the GraphPadPRISM 8.0 log (agonist) vs. response-viable slope (four-parameter) formula. 50 The affinity (EC) of the HU6DL mutant for the human TROP-2 antigen. 50 As shown in Table 7 below:

[0411] Table 7. Affinity of HU6DL mutant to human TROP-2 antigen (EC50) 50 )

[0412] name <![CDATA[TOP OD 450 ]]> <![CDATA[Affinity EC 50 (nM)]]> HU6DL.R54 2.05 0.052 HU6DL.Y54 1.93 0.051 HU6DL.Q54 1.91 0.086 HU6DL.L54 1.96 0.048 HU6DL.T54 1.90 0.052 HU6DL.I54 1.96 0.063 HU6DL.F54 1.93 0.067 HU6DL.E54 1.96 0.064 HU6DL.A54 2.07 0.062

[0413] 4. Experimental conclusions:

[0414] The data above show that the HU6DL mutants disclosed in this paper have good affinity for human TROP-2 antigen.

[0415] Example 7: Affinity experiment of HU6DL mutant to tumor cells

[0416] 1. Experimental objective:

[0417] Flow cytometry was used to evaluate the differences in affinity levels of HU6DL mutants for tumor cell lines expressing TROP-2 antigen.

[0418] 2. Experimental reagents:

[0419] Gastric cancer cells NCI-N87 (purchased from the Chinese Academy of Sciences Cell Bank, TCHU130);

[0420] Non-small cell lung cancer cells HCC827 (purchased from the Chinese Academy of Sciences Cell Bank, TCHU153);

[0421] Bladder cancer cells SW780 (purchased from the Chinese Academy of Sciences Cell Bank, TCHU219);

[0422] Bladder cancer cells RT4 (purchased from the Chinese Academy of Sciences Cell Bank, TCHU226);

[0423] 3. Experimental steps:

[0424] Tumor cells in good growth condition were treated with Accutase (Sigma, cat: A6964) digestion solution, and single-cell suspensions were prepared with 2% FBS (DBPS, diluted at pH 7.4) solution to adjust the cell density to 1×10⁻⁶. 6 Cells / mL. Dispense 100 μL / well into 96-well V-type plates, centrifuge at 300 g × 5 min, 4 °C, discard the supernatant, and add 100 μL / well of the supernatant at a 10-fold, 10-fold concentration gradient (1000 nM⁻¹ × 10⁻¹). -6 The candidate antibody solution diluted (nM) was incubated at 4°C for 1 hour.

[0425] Centrifuge at 300g for 5 min at 4℃, wash twice, and add 5μL / 1000μL of the solution to each well. 6 Cells were proportionally diluted with mouse anti-human IgG Fc and PE-labeled secondary antibody (Biolegend, cat: 409304) solution and incubated at 4°C for 1 h; centrifuged at 300 g × 5 min at 4°C, washed twice, and resuspended in 70 μL of 2% FBS solution. The mean fluorescence intensity (MFI) of the PE channel was detected by ZE5 flow cytometer (Bio-Rad, ZE5).

[0426] 4. Data Processing:

[0427] Using the logarithm of mutant antibody concentration as the X-axis and MFI as the y-axis, the EC50 of each candidate antibody's affinity for tumor cells was calculated using the GraphPad PRISM 8.0 log (agonist) vs. response-viable slope (four-parameter) formula. 50 As shown in Table 8 below:

[0428] Table 8. Affinity of HU6DL mutant to NCI-N87, HCC827, SW780 and RT4 tumor cells (EC50) 50 )

[0429]

[0430] 5. Experimental Conclusion:

[0431] The data above show that each mutant of HU6DL disclosed in this paper has good affinity for NCI-N87, HCC827, SW780 and RT4 tumor cells.

[0432] Example 8: HU6DL mutant-mediated TROP2 endocytosis experiment

[0433] 1. Experimental objective:

[0434] The antibody endocytosis activity of the HU6DL mutant in TROP-2 antigen-expressing tumor cell lines was evaluated by flow cytometry.

[0435] 2. Experimental steps:

[0436] Gastric cancer cells NCI-N87 (purchased from the Chinese Academy of Sciences Cell Bank, TCHU130) in good growth condition were treated with Accutase (Sigma, cat: A6964) digestion solution. Single-cell suspensions were prepared with 2% FBS (DBPS, diluted at pH 7.4) solution, and the cell density was adjusted to 1×10⁻⁶ cells / cells. 7 Cells / mL. 100 μL / well was evenly distributed into a 96-well V-type plate, and candidate antibody solution was added to a final concentration of 20 μg / mL. The mixture was then incubated at 4°C for 1 h.

[0437] Centrifuge at 300g for 5 min at 4℃, and add 5μL / 1000μL of the solution to each well. 6 The mouse anti-human IgG Fc, PE-labeled secondary antibody (Biolegend, cat: 409304) solution, diluted to the specified cell ratio, was incubated at 4°C for 1 h.

[0438] Centrifuge at 300g for 5 min at 4℃, wash twice, resuspend the cell pellet in 1 mL of pre-warmed complete culture medium, and divide into four equal parts, designated as 0 min group, blank group, 30 min group, and 120 min group. Take out the 0 min and blank control and place them on ice, and place the rest in a 37℃ incubator for endocytosis for 30 min and 120 min respectively. Take out the corresponding group at the corresponding time point and pre-cool on ice for 5 min. Centrifuge all treatment groups and discard the supernatant (4℃, 1500 rpm × 5 min), wash once with FACS buffer and discard the supernatant. Add 250 μL of strip buffer to all treatment groups except the 0 min group, incubate at room temperature for 8 min, centrifuge and discard the supernatant (4℃, 1500 rpm × 5 min), wash twice with FACS buffer and discard the supernatant. Add 80 μL of 2% FBS to each sample to resuspend the cells, and detect the fluorescence signal of the sample under test using a ZE5 flow cytometer (Bio-Rad, ZE5).

[0439] 3. Data Processing:

[0440] The endocytosis efficiency of each candidate antibody was calculated using the following formula: Antibody endocytosis percentage (%) = (MFI of treated group - MFI of blank control group) / (MFI of 0 min group - MFI of blank control group) * 100%. The endocytosis rate of the HU6DL mutant detected using the above method is shown in Table 9 below.

[0441] Table 9. Endocytosis efficiency of HU6DL mutant in NCI-N87 cells

[0442]

[0443] 4. Experimental conclusions:

[0444] The above data show that the TROP-2 protein mediated by the HU6DL mutant of this disclosure has good endocytosis activity in gastric cancer cells NCI-N87.

[0445] Example 9: Detection of impurity components in the HU6DL mutant

[0446] 1. Experimental objective: To detect and compare the content and change level of impurity peaks in mutated antibodies using capillary electrophoresis based on the Maurice-nrCE-SDS method.

[0447] 2. Experimental Procedure

[0448] 2.1. Sample preparation:

[0449] (1) Sample replacement and concentration: If the protein concentration of the sample is lower than 5 mg / ml, or the salt concentration of the sample buffer is high, the sample needs to be replaced and concentrated to ensure that the protein concentration is around 5 mg / ml and the salt concentration in the sample is less than 50 mM.

[0450] (2) Non-reduced CE sample preparation: Add the sample to the EP tube. The protein amount for each sample is 50 μg. Add 1 μl of 10 kD internal standard (Protein Simple, 046-144), 2.5 μl of 250 mM IAM (Sigma, I1149-5G), and 1× sample buffer (Protein Simple, 046-567) to a final volume of 50 μl.

[0451] (3) After shaking and mixing, heat and incubate at 70℃ for 10 min, then remove and incubate on ice for 5 min. After cooling, centrifuge at 12000 rpm for 5 min. After centrifugation, take 35 μl of supernatant and transfer it to the instrument-matched 96-well plate. Then centrifuge at 1000 rpm for 5 min. Place the 96-well sample plate into a Maurice (Protein Simple) container and prepare for sample loading and analysis.

[0452] 2.2. On-machine testing

[0453] Turn on the instrument and software, perform a self-test according to the instrument operating procedures, install the capillary cartridge, and prepare the necessary reagents by placing them in the appropriate positions on the instrument. Set the corresponding parameters according to the instrument operating procedures to perform non-reductive CE analysis. Set the sample sequences by editing the corresponding sequences according to the sample names; the number of samples per sequence should not exceed 48. After editing the sequences, click "start" to begin sequence detection.

[0454] The contents of the main peak and impurity peaks in the sample are calculated using the following formula.

[0455]

[0456] Note:

[0457] In the formula, non-reduced purity is the percentage of the corrected peak area of ​​the main peak;

[0458] CA 主峰 The corrected peak area of ​​the main peak;

[0459] CA 总 It is the sum of the areas of the main peak and the corrected peaks of impurities.

[0460] 3. Experimental Results:

[0461] Table 10. Detection of impurity components

[0462] sample Main peak content (%) Impurity content (%) HU6DL.T54 93.62 N / A HU6DL 84.78 4.72

[0463] The above data shows that the main peak content of sample HU6DL.T54 reached 93.62%, and no impurities were detected. Experimental results indicate that the N content in HCDR2 of HU6DL... 54 D 55 S 56 Motifs are prone to glycosylation. By performing site-directed mutagenesis on N54, glycosylation impurities can be effectively reduced and better purity can be obtained.

[0464] Example 10: Competitive binding of humanized antibodies to antigens

[0465] 1. Experimental objective: To study the binding modes and binding sites of different antibodies and antigens, using a competitive binding experiment.

[0466] 2. Experimental Procedure

[0467] Dilute the coated antibodies hRS7 and HU6DL mutants to a concentration of 1 μg / ml with PBS (pH 7.4), and add 100 μl / well to each well of a 96-well high-affinity microplate. Incubate overnight (16-20 hours) at 4°C. Wash the plate four times with PBST (pH 7.4 PBS containing 0.05% Tween-20), then add 200 μl / well of 2% bovine serum albumin (BSA) blocking buffer diluted with PBST and incubate at room temperature for 1 hour to block. After blocking, discard the blocking buffer and wash the plate four times with PBST buffer.

[0468] Dilute the competitive antibodies hRS7 and HU6DL to 100 μg / ml with PBST containing 0.5% BSA, and add 50 μl / well to each well of the ELISA plate. Dilute TROP-2His protein (Acro biosystems, cat#TR2-H5223) with PBST containing 0.5% BSA to concentrations corresponding to 2xEC80 or 2xEC90 affinity for the coating antibody, and add 50 μl / well to each well of the ELISA plate. Incubate the ELISA plate at room temperature for 1 hour. After incubation, wash the plate four times with PBST, add 100 μl / well of anti-His HRP-labeled secondary antibody (Sino Biological, cat#105327-MM02T-H) diluted 1:5000 with PBST containing 0.5% BSA, and incubate at room temperature for 1 hour. After washing the plate four times with PBST, add 100 μl / well of TMB chromogenic substrate (Cell Signaling Technology, cat#7004S), incubate at room temperature in the dark for 1 minute, and then add 50 μl / well of stop buffer (Cell Signaling Technology, cat#7002S) to terminate the reaction.

[0469] 3. Data Processing

[0470] The absorbance was read at 450 nm using a Thermo (Varioskan LUX) microplate reader, and the data were analyzed as shown in Table 11 below.

[0471] Table 11. Competitive binding of humanized antibodies to hRS7 antigen.

[0472] Humanized antibodies Inhibition rate hRS7 94.67％ HU6DL.T54 23.30％

[0473] 4. Experimental Results

[0474] The mutant humanized antibody of the present invention has a very low inhibition rate of binding between hRS7 antibody and TROP2 protein, suggesting that the mutant humanized antibody of the present invention does not compete with hRS7 antibody for binding to the same epitope. sequence list <110> Shanghai Hansoh Biomedical Technology Co., Ltd. Jiangsu Hansoh Pharmaceutical Group Co., Ltd. <120> Anti-TROP-2 antibodies, their antigen-binding fragments or mutants thereof, and their pharmaceutical uses <130> 721120CPCT <140> PCT / CN2021 / <141> 2021-10-14 <150> 202011099107.8 <151> 2020‑10‑14 <160> 43 <170> SIPOSequenceListing 1.0 <210> 1 <211> 331 <212> PRT <213> Homo sapiens <400> 1 Met Ala Arg Gly Pro Gly Leu Ala Pro Pro Pro Leu Arg Leu Pro Leu 1 5 10 15 Leu Leu Leu Val Leu Ala Ala Val Thr Gly His Thr Ala Ala Gln Asp 20 25 30 Asn Cys Thr Cys Pro Thr Asn Lys Met Thr Val Cys Ser Pro Asp Gly 35 40 45 Pro Gly Gly Arg Cys Gln Cys Arg Ala Leu Gly Ser Gly Met Ala Val 50 55 60 Asp Cys Ser Thr Leu Thr Ser Lys Cys Leu Leu Leu Lys Ala Arg Met 65 70 75 80 Ser Ala Pro Lys Asn Ala Arg Thr Leu Val Arg Pro Ser Glu His Ala 85 90 95 Leu Val Asp Asn Asp Gly Leu Tyr Asp Pro Asp Cys Asp Pro Glu Gly 100 105 110 Arg Phe Lys Ala Arg Gln Cys Asn Gln Thr Ser Val Cys Trp Cys Val 115 120 125 Asn Ser Val Gly Val Arg Arg Thr Asp Lys Gly Asp Leu Ser Leu Arg 130 135 140 Cys Asp Glu Leu Val Arg Thr His His Ile Leu Ile Asp Leu Arg His 145 150 155 160 Arg Pro Thr Ala Gly Ala Phe Asn His Ser Asp Leu Asp Ala Glu Leu 165 170 175 Arg Arg Leu Phe Arg Glu Arg Tyr Arg Leu His Pro Lys Phe Val Ala 180 185 190 Ala Val His Tyr Glu Gln Pro Thr Ile Gln Ile Glu Leu Arg Gln Asn 195 200 205 Thr Ser Gln Lys Ala Ala Gly Asp Val Asp Ile Gly Asp Ala Ala Tyr 210 215 220 Tyr Phe Glu Arg Asp Ile Lys Gly Glu Ser Leu Phe Gln Gly Arg Gly 225 230 235 240 Gly Leu Asp Leu Arg Val Arg Gly Glu Pro Leu Gln Val Glu Arg Thr 245 250 255 Leu Ile Tyr Tyr Leu Asp Glu Ile Pro Pro Lys Phe Ser Met Lys Arg 260 265 270 Leu Thr Ala Gly Leu Ile Ala Val Ile Val Val Val Val Val Ala Leu 275 280 285 Val Ala Gly Met Ala Val Leu Val Ile Thr Asn Arg Arg Lys Ser Gly 290 295 300 Lys Tyr Lys Lys Val Glu Ile Lys Glu Leu Gly Glu Leu Arg Lys Glu 305 310 315 320 Pro Ser Leu His His His His His His His His 325 330 <210> 2 <211> 121 <212> PRT <213> Mus musculus <400> 2 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Arg Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Ala Ile Leu Thr Val Asp Lys Ala Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val His Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 3 <211> 107 <212> PRT <213> Mus musculus <400> 3 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser 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 Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ala Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Thr 65 70 75 80 Glu Asp Leu Thr Val Tyr His Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Pro Gly Thr Arg Leu Glu Leu Lys 100 105 <210> 4 <211> 5 <212> PRT <213> Mus musculus <400> 4 Asn Tyr Trp Met Asn 1 5 <210> 5 <211> 17 <212> PRT <213> Mus musculus <400> 5 Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 6 <211> 12 <212> PRT <213> Mus musculus <400> 6 Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val 1 5 10 <210> 7 <211> 11 <212> PRT <213> Mus musculus <400> 7 Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala 1 5 10 <210> 8 <211> 7 <212> PRT <213> Mus musculus <400> 8 Ser Ala Ser Tyr Arg Tyr Thr 1 5 <210> 9 <211> 9 <212> PRT <213> Mus musculus <400> 9 Gln Gln His Tyr Ser Thr Pro Leu Thr 1 5 <210> 10 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> <223> Variable region <400> 10 Glu Val Gln Leu Leu 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 Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 11 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> <223> Variable Region <400> 11 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Phe Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 。 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 12 <211> 330 <212> PRT <213> Homo sapiens <400> 12 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 13 <211> 107 <212> PRT <213> Homo sapiens <400> 13 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 14 <211> 451 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> <223> Heavy chain <400> 14 Glu Val Gln Leu Leu 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 Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 15 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> <223> Light chain <400> 15 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Phe Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 16 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> <223> Variable Region <400> 16 Glu Val Gln Leu Leu 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 Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Arg Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 17 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> <223> Variable Region <400> 17 Glu Val Gln Leu Leu 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 Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 18 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> <223> Variable Region <400> 18 Glu Val Gln Leu Leu 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 Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Gln Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 19 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> <223> Variable Region <400> 19 Glu Val Gln Leu Leu 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 Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 20 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> <223> Variable Region <400> 20 Glu Val Gln Leu Leu 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 Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Thr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 21 <211> 451 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> <223> Heavy chain <400> 21 Glu Val Gln Leu Leu 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 Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Arg Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 22 <211> 451 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> <223> Heavy chain <400> 22 Glu Val Gln Leu Leu 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 Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 23 <211> 451 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> <223> Heavy chain <400> 23 Glu Val Gln Leu Leu 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 Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Gln Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 24 <211> 451 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> <223> Heavy chain <400> 24 Glu Val Gln Leu Leu 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 Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Leu Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 25 <211> 451 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> <223> Heavy chain <400> 25 Glu Val Gln Leu Leu 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 Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Thr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 26 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> <223> CDR <400> 26 Arg Ile Asp Pro Arg Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 27 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> <223> CDR <400> 27 Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 28 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> <223> CDR <400> 28 Arg Ile Asp Pro Gln Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 29 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> <223> CDR <400> 29 Arg Ile Asp Pro Leu Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 30 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> [[ID=,41]]<223> CDR <400> 30 Arg Ile Asp Pro Thr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 31 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> <223> Variable Region <400> 31 Glu Val Gln Leu Leu 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 Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ile Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 32 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> <223> Variable Region <400> 32 Glu Val Gln Leu Leu 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 Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Phe Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 33 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> <223> Variable region <400> 33 Glu Val Gln Leu Leu 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 Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Glu Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 34 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> <223> Variable region <400> 34 Glu Val Gln Leu Leu 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 Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 35 <211> 451 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> <223> Heavy chain <400> 35 Glu Val Gln Leu Leu 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 Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ile Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val[[ID=?]] 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 36 <211> 451 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> <223> Heavy chain <400> 36 (注:原文中“[[ID=?]]”处的“?”可能是排版错误,按照要求保留原文形式。)Glu Val Gln Leu Leu 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 Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Phe Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 37 <211> 451 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> <223> Heavy chain <400> 37 Glu Val Gln Leu Leu 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 Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Glu Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 38 <211> 451 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> <223> Heavy chain <400> 38 Glu Val Gln Leu Leu 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 Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 39 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> <223> CDR <400> 39 Arg Ile Asp Pro Ile Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 40 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> <223> CDR <400> 40 Arg Ile Asp Pro Phe Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 41 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> <223> CDR <400> 41 Arg Ile Asp Pro Glu Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 42 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> <223> CDR <400> 42 Arg Ile Asp Pro Ala Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 43 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (5)..(5) <223> X is selected from R, Y, Q, L, T, I, F, E or A <220> <221> DOMAIN <222> <223> CDR <400> 43 Arg Ile Asp Pro Xaa Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp

Claims

1. An anti-TROP-2 antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 43, and HCDR3 shown in SEQ ID NO:

6. The light chain variable region includes LCDR1 shown in SEQ ID NO: 7, LCDR2 shown in SEQ ID NO: 8, and LCDR3 shown in SEQ ID NO:

9.

2. The anti-TROP-2 antibody or its antigen-binding fragment according to claim 1, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region CDR sequence selected from: The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 30, and HCDR3 shown in SEQ ID NO: 6; the light chain variable region includes LCDR1 shown in SEQ ID NO: 7, LCDR2 shown in SEQ ID NO: 8, and LCDR3 shown in SEQ ID NO: 9; or The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 26, and HCDR3 shown in SEQ ID NO: 6; the light chain variable region includes LCDR1 shown in SEQ ID NO: 7, LCDR2 shown in SEQ ID NO: 8, and LCDR3 shown in SEQ ID NO: 9; or, The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 27, and HCDR3 shown in SEQ ID NO: 6; the light chain variable region includes LCDR1 shown in SEQ ID NO: 7, LCDR2 shown in SEQ ID NO: 8, and LCDR3 shown in SEQ ID NO: 9; or, The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 28, and HCDR3 shown in SEQ ID NO: 6; the light chain variable region includes LCDR1 shown in SEQ ID NO: 7, LCDR2 shown in SEQ ID NO: 8, and LCDR3 shown in SEQ ID NO: 9; or, The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 29, and HCDR3 shown in SEQ ID NO: 6; the light chain variable region includes LCDR1 shown in SEQ ID NO: 7, LCDR2 shown in SEQ ID NO: 8, and LCDR3 shown in SEQ ID NO: 9; or, The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 39, and HCDR3 shown in SEQ ID NO: 6; the light chain variable region includes LCDR1 shown in SEQ ID NO: 7, LCDR2 shown in SEQ ID NO: 8, and LCDR3 shown in SEQ ID NO: 9; or, The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 40, and HCDR3 shown in SEQ ID NO: 6; the light chain variable region includes LCDR1 shown in SEQ ID NO: 7, LCDR2 shown in SEQ ID NO: 8, and LCDR3 shown in SEQ ID NO: 9; or, The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 41, and HCDR3 shown in SEQ ID NO: 6; the light chain variable region includes LCDR1 shown in SEQ ID NO: 7, LCDR2 shown in SEQ ID NO: 8, and LCDR3 shown in SEQ ID NO: 9; or, The heavy chain variable region includes HCDR1 shown in SEQ ID NO:4, HCDR2 shown in SEQ ID NO:42, and HCDR3 shown in SEQ ID NO:6, while the light chain variable region includes LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:8, and LCDR3 shown in SEQ ID NO:

9.

3. The anti-TROP-2 antibody or its antigen-binding fragment according to claim 1, wherein the antibody is a murine antibody, a chimeric antibody, a human antibody, or a humanized antibody.

4. The anti-TROP-2 antibody or its antigen-binding fragment according to claim 1, wherein: The antibody or its antigen-binding fragment further comprises a constant region derived from human IgG1, IgG2, IgG3 or IgG4 or mutants thereof.

5. The anti-TROP-2 antibody or its antigen-binding fragment according to claim 4, wherein the antibody or its antigen-binding fragment further comprises a heavy chain constant region derived from a human IgG1, IgG2 or IgG4 mutant.

6. The anti-TROP-2 antibody or its antigen-binding fragment according to claim 4, wherein the antibody or its antigen-binding fragment further comprises an IgG1 heavy chain constant region incorporating 239D and 241L mutations.

7. The anti-TROP-2 antibody or its antigen-binding fragment according to claim 4, wherein the antibody or its antigen-binding fragment further comprises a heavy chain constant region selected from SEQ ID NO:

12.

8. The anti-TROP-2 antibody or its antigen-binding fragment according to claim 1, wherein the antibody or its antigen-binding fragment further comprises a light chain constant region derived from the human κ chain, λ chain or mutant thereof.

9. The anti-TROP-2 antibody or its antigen-binding fragment according to claim 8, wherein the antibody or its antigen-binding fragment further comprises a light chain constant region selected from SEQ ID NO:

13.

10. The anti-TROP-2 antibody or antigen-binding fragment thereof according to any one of claims 1-9, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the following: SEQ ID NO: 20, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the thereof.

11. The anti-TROP-2 antibody or antigen-binding fragment thereof according to any one of claims 1-9, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it.

12. The anti-TROP-2 antibody or its antigen-binding fragment according to any one of claims 1-9, wherein the antibody or its antigen-binding fragment comprises a heavy chain selected from the group consisting of: SEQ ID NO: 25, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with the same.

13. The anti-TROP-2 antibody or its antigen-binding fragment according to any one of claims 1-9, wherein the antibody or its antigen-binding fragment comprises a light chain SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity with it.

14. The anti-TROP-2 antibody or its antigen-binding fragment according to any one of claims 1-9, wherein: The antibody or its antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO: 20, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it; or, The antibody or its antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO: 16, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it; or, The antibody or its antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO: 17, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it; or, The antibody or its antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO: 18, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it; or, The antibody or its antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO: 19, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it; or, The antibody or its antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO: 31, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it; or, The antibody or its antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO: 32, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it; or, The antibody or its antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO: 33, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it; or, The antibody or its antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO: 34, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with it.

15. The anti-TROP-2 antibody or its antigen-binding fragment according to any one of claims 1-9, wherein: The antibody or its antigen-binding fragment comprises the heavy chain shown in SEQ ID NO: 25, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity with it; or, The antibody or its antigen-binding fragment comprises the heavy chain shown in SEQ ID NO: 21, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity with it; or, The antibody or its antigen-binding fragment comprises the heavy chain shown in SEQ ID NO: 22, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity with it; or, The antibody or its antigen-binding fragment comprises the heavy chain shown in SEQ ID NO: 23, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity with it; or, The antibody or its antigen-binding fragment comprises the heavy chain shown in SEQ ID NO: 24, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity with it; or, The antibody or its antigen-binding fragment comprises the heavy chain shown in SEQ ID NO: 35, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity with it; or, The antibody or its antigen-binding fragment comprises the heavy chain shown in SEQ ID NO: 36, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity with it, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity with it; or, The antibody or its antigen-binding fragment comprises the heavy chain shown in SEQ ID NO: 37, or the heavy chain having at least 80%, 85%, 90%, 95% or 99% identity with it, and the light chain shown in SEQ ID NO: 15, or the light chain having at least 80%, 85%, 90%, 95% or 99% identity with it; The antibody or its antigen-binding fragment comprises the heavy chain shown in SEQ ID NO: 38, or the heavy chain having at least 80%, 85%, 90%, 95% or 99% identity with it, and the light chain shown in SEQ ID NO: 15, or the light chain having at least 80%, 85%, 90%, 95% or 99% identity with it.

16. A polynucleotide encoding an anti-TROP-2 antibody or an antigen-binding fragment thereof as described in any one of claims 1-15.

17. An expression vector comprising the polynucleotide of claim 16.

18. A host cell transformed with the expression vector of claim 17, wherein the host cell is selected from bacteria, yeast or mammalian cells.

19. The host cell according to claim 18, wherein the host cell is selected from Escherichia coli, Pichia pastoris, CHO cells or HEK293 cells.

20. A method for producing the anti-TROP-2 antibody or its antigen-binding fragment as described in any one of claims 1-15, comprising the steps of: Culture the host cells as described in claim 18 or 19; Isolating antibodies from cultures; and The antibody was purified.

21. The method according to claim 20, wherein the host cell is HEK293 cell, the culture medium is cell culture medium, and the purification method is chromatography.

22. A pharmaceutical composition comprising the anti-TROP-2 antibody or its antigen-binding fragment as described in any one of claims 1-15, and a pharmaceutically acceptable excipient, diluent, or carrier.

23. A detection or diagnostic reagent comprising the anti-TROP-2 antibody or its antigen-binding fragment as described in any one of claims 1-15.

24. Use of the anti-TROP-2 antibody or antigen-binding fragment thereof according to any one of claims 1-15 or the pharmaceutical composition according to claim 22 in the preparation of a medicament for treating or preventing cancers expressing TROP-2, wherein the cancers expressing TROP-2 are selected from gastric cancer, lung cancer, bladder cancer, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, liver cancer, colon cancer, esophageal cancer, or cervical cancer.

25. The use according to claim 24, wherein the lung cancer is non-small cell lung cancer.

26. Use of the anti-TROP-2 antibody or its antigen-binding fragment according to any one of claims 1-15 or the detection or diagnostic reagent according to claim 23 in the preparation of a kit, wherein the kit is used to detect, diagnose, and prognose TROP-2-mediated diseases or conditions.

27. The use according to claim 26, wherein: The TROP-2-mediated disease or condition is cancer.

28. The use according to claim 26, wherein, The TROP-2-mediated disease or condition is a cancer that expresses TROP-2; the cancer is selected from breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma, or melanoma.

29. The use according to claim 28, wherein the lung cancer is non-small cell lung cancer.

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