Anti-TROP-2 antibodies, their antigen-binding fragments or variants, and their medical use

Abstract

The present invention relates to anti-TROP-2 antibodies, antigen-binding fragments thereof or variants thereof, and their medical uses. Furthermore, the present application relates to humanized anti-TROP-2 antibody variants, pharmaceutical compositions comprising the anti-TROP-2 antibody variants, their use in the preparation of medicaments for treating TROP-2-mediated diseases or conditions, and their use in tumor detection and diagnosis.

Description

[Technical Field]

[0001] This application claims priority to Patent Application No. 202011099107.8, filed on 14 October 2020.

[0002] This disclosure relates to anti-TROP-2 antibodies, their antigen-binding fragments or variants thereof, to chimeric antibodies or humanized antibodies comprising the CDR region of anti-TROP-2 antibodies or their variants, to pharmaceutical compositions comprising human anti-TROP-2 antibodies, their antigen-binding fragments or variants thereof, and to their use as anticancer drugs and for the detection or diagnosis of tumors. [Background technology]

[0003] As research into tumor genomics, proteomics, and signaling pathways deepens, the interactions between oncogenes and tumor suppressor genes in tumor cells, as well as their effects on the tumor microenvironment, are becoming clearer. This is making it possible to design novel antitumor therapeutic schemes targeting specific molecular targets of tumors.

[0004] Molecular targeted therapy for tumors is a new treatment approach that differs from conventional surgery, radiotherapy, and chemotherapy. Its advantage lies in the fact that drugs typically bind only to their corresponding targets, directly affecting the function of the target molecule, or killing or inhibiting target cells through the physical or chemical effector molecules they carry. For specific targets, drugs are usually highly selective and can effectively kill or inhibit target cells with little to no toxic side effects on normal tissue cells. Therefore, the development of molecularly targeted drugs has become a hot spot in oncology clinical research.

[0005] Trophoblastic cell surface antigen 2 (TROP-2), also known as tumor-associated calcium signaling molecule 2 (TACSTD2), is a transmembrane glycoprotein encoded by the TACSTD2 gene. TROP-2 consists of approximately 323 amino acids, of which 26 are in the signal peptide, 248 in the extracellular domain, 23 in the transmembrane domain, and 26 in the cytosolic domain. The extracellular domain of TROP-2 contains four heterologous N-linked glycosylation sites, and after glycosylation, its apparent molecular weight increases to 11–13 kD. Within the TACSTD gene family, the extracellular domain possesses a characteristic thyroglobulin (TY) sequence, which is typically thought to be associated with cancer cell proliferation, invasion, and metastasis.

[0006] To date, no physiological ligands for TROP-2 have been identified, and the molecular function of TROP-2 remains unclear. However, protein kinase C (PKC, Ca) 2+ Due to phosphorylation of the intracellular serine 303 residue by a protein-dependent kinase (belonging to the group of protein-dependent kinases), and the presence of a PIP2 binding sequence in its intracellular domain, TROP-2 has been shown to have a signaling function in tumor cells.

[0007] Numerous clinical studies and literature reports have shown that the TROP-2 antigen is overexpressed in various epithelial cancers, including gastric cancer, lung cancer, colorectal cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer, and esophageal cancer. The TROP-2 antigen is rarely expressed or absent in normal adult tissues, and is expressed very rarely in epithelial cells. Furthermore, its expression level is lower than that in cancer cells, indicating that TROP-2 is associated with tumorigenesis. Overexpression of TROP-2 in tumor tissue is closely associated with poor prognosis and cancer cell metastasis, and further affects overall survival. As a result, TROP-2 has become a promising target in molecular targeted therapies for tumors.

[0008] Currently, research has reported the antitumor effects of various TROP-2 antibodies.

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

[0010] U.S. Patent No. 6,653,104 discloses an antibody (RS7) labeled with a radioactive substance and tested in an in vivo model, which showed antitumor activity in a nude mouse xenograft model; however, the antitumor effect of a naked antibody has not been reported.

[0011] Furthermore, U.S. Patent No. 7420040 reports that isolated monoclonal antibodies produced by 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 nude mouse xenograft models.

[0012] Chinese Patent No. 102827282A discloses a genetically modified human anti-TROP-2 antibody IgG and its use. In vitro studies have shown that this anti-TROP-2 antibody IgG has a significant inhibitory effect on the proliferation of pancreatic cancer cells.

[0013] Chinese Patent No. 104114580A discloses an antibody (particularly a humanized antibody) that specifically reacts with the TROP-2 antigen and has antitumor activity in vivo, as well as a hybridoma that produces the antibody, an antibody-drug conjugate, a pharmaceutical composition for the diagnosis or treatment of tumors, a method for detecting tumors, and a kit for the detection or diagnosis of tumors. [Prior art documents] [Patent Documents]

[0014] [Patent Document 1] U.S. Patent No. 5,840,854 [Patent Document 2] U.S. Patent No. 6,653,104 [Patent Document 3] U.S. Patent No. 7,420,040 [Patent Document 4] Chinese Patent No. 102827282A<000,0080>[Patent Document 5] Chinese Patent No. 104114580A [Non-Patent Document]

[0015] [Non-Patent Document 1] J. Biol. Chem., 243, 3558 (1968) [Non-Patent Document 2] Holliger and Hudson, 2005, Nat. Biotechnol. 23: 1126-1136 [Non-Patent Document 3] Antibodies: A Laboratory Manual, Cold Spring Harbor, Chapters 5-8 and 15 [Summary of the Invention] [Problems to be Solved by the Invention]

[0016] However, it is difficult to find monoclonal antibodies having high affinity, high specificity, and strong cytotoxic or tumor killing / inhibiting / modifying activity. Therefore, there still exists an urgent need to develop TROP-2 antibodies and other immunotherapeutic agents having good efficacy and high safety and suitable for human subjects. [Means for Solving the Problems]

[0017] The present disclosure provides an anti-TROP-2 antibody, an antigen-binding fragment thereof, or a variant thereof, comprising a heavy-chain variable region and a light-chain variable region.

[0018] In a preferred embodiment of the anti-TROP-2 antibody, antigen-binding fragment thereof, or variant thereof of the present disclosure, The heavy chain variable region includes at least one HCDR represented by a sequence selected from the group consisting of SEQ ID NOs: 4, 43, and 6. The light chain variable region includes at least one LCDR represented by a sequence selected from the group consisting of SEQ ID NOs: 7, SEQ ID NOs: 8, and SEQ ID NOs: 9. Sequence ID 43 is as shown in RIDPXDSETHYNQKFKD, where X is selected from the group consisting of amino acid residues R, Y, Q, L, T, I, F, E, and A.

[0019] In preferred embodiments of this disclosure, the heavy chain variable region HCDR2 is selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, 30, 39, 40, 41, and 42.

[0020] In preferred embodiments of the anti-TROP-2 antibody, its antigen-binding fragment, or its variants, the antibody heavy chain variable region is: HCDR1 shown in Sequence ID No. 4, HCDR2 shown in Sequence ID No. 26, and HCDR3 shown in Sequence ID No. 6, or HCDR1 shown in Sequence ID No. 4, HCDR2 shown in Sequence ID No. 27, and HCDR3 shown in Sequence ID No. 6, or HCDR1 shown in Sequence ID No. 4, HCDR2 shown in Sequence ID No. 28, and HCDR3 shown in Sequence ID No. 6, or HCDR1 shown in Sequence ID No. 4, HCDR2 shown in Sequence ID No. 29, and HCDR3 shown in Sequence ID No. 6, or HCDR1 shown in Sequence ID No. 4, HCDR2 shown in Sequence ID No. 30, and HCDR3 shown in Sequence ID No. 6, or HCDR1 shown in Sequence ID No. 4, HCDR2 shown in Sequence ID No. 39, and HCDR3 shown in Sequence ID No. 6, or HCDR1 shown in Sequence ID No. 4, HCDR2 shown in Sequence ID No. 40, and HCDR3 shown in Sequence ID No. 6, or HCDR1 shown in Sequence ID No. 4, HCDR2 shown in Sequence ID No. 41, and HCDR3 shown in Sequence ID No. 6, or HCDR1 shown in Sequence ID 4, HCDR2 shown in Sequence ID 42, and HCDR3 shown in Sequence ID 6 Includes.

[0021] In preferred embodiments of the anti-TROP-2 antibody, its antigen-binding fragment, or its variant, the light chain variable region is: LCDR1 shown in Sequence ID 7, LCDR2 shown in Sequence ID 8, and LCDR3 shown in Sequence ID 9 Includes.

[0022] In preferred embodiments of the anti-TROP-2 antibody, its antigen-binding fragment, or its variants, 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.

[0023] In preferred embodiments of the anti-TROP-2 antibody, its antigen-binding fragment, or its variants, 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.

[0024] In preferred embodiments of the anti-TROP-2 antibody, its antigen-binding fragment, or its variants, 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.

[0025] In preferred embodiments of the anti-TROP-2 antibody, its antigen-binding fragment, or its variants, 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.

[0026] In preferred embodiments of the anti-TROP-2 antibody, its antigen-binding fragment, or its variants, 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.

[0027] In preferred embodiments of the anti-TROP-2 antibody, its antigen-binding fragment, or its variants, 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.

[0028] In preferred embodiments of the anti-TROP-2 antibody, its antigen-binding fragment, or its variants, 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.

[0029] In preferred embodiments of the anti-TROP-2 antibody, its antigen-binding fragment, or its variants, 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.

[0030] In preferred embodiments of the anti-TROP-2 antibody, its antigen-binding fragment, or its variants, 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. 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.

[0031] In preferred embodiments of the anti-TROP-2 antibody, its antigen-binding fragment, or its variants, the antibody is a mouse antibody, a chimeric antibody, a human antibody, or a humanized antibody.

[0032] In preferred embodiments of the anti-TROP-2 antibody, its antigen-binding fragment, or its variants according to the present disclosure, the anti-TROP-2 antibody or its antigen-binding fragment further comprises a constant region derived from human IgG1, IgG2, IgG3, or IgG4, or their variants.

[0033] In further preferred embodiments of the present disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or its variant further comprises a heavy chain constant region derived from human IgG1, IgG2, or IgG4 or their variants.

[0034] In further preferred embodiments of the present disclosure, an anti-TROP-2 antibody, its antigen-binding fragment, or its variant further comprises a heavy chain constant region derived from IgG1 having enhanced ADCC toxicity after amino acid mutation.

[0035] In further preferred embodiments of the present disclosure, an anti-TROP-2 antibody, its antigen-binding fragment, or its variant further comprises the heavy chain constant region of IgG1 into which the 239D and 241L mutations have been introduced.

[0036] In further preferred embodiments of the present disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or its variant further comprises a heavy chain constant region selected from SEQ ID NO: 12.

[0037] In further preferred embodiments of the present disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or its variant further comprises a light chain constant region derived from a human κ chain, λ chain, or a variant thereof.

[0038] In further preferred embodiments of the present disclosure, the anti-TROP-2 antibody, its antigen-binding fragment, or its variant further comprises a light chain constant region selected from SEQ ID NO: 13.

[0039] In preferred embodiments of the anti-TROP-2 antibody, its antigen-binding fragment, or its variants, the anti-TROP-2 antibody, its antigen-binding fragment, or its variants include a heavy chain variable region selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 31, 32, 33, and 34, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0040] In preferred embodiments of the anti-TROP-2 antibody, its antigen-binding fragment, or its variants, the anti-TROP-2 antibody, its antigen-binding fragment, or its variants include the light chain variable region of SEQ ID NO: 11, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity therewith.

[0041] In preferred embodiments of the present disclosure, the anti-TROP-2 antibody or its variant comprises a heavy chain selected from the group consisting of SEQ ID NOs: 21, 22, 23, 24, 25, 35, 36, 37, and 38, or a full-length heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto.

[0042] In preferred embodiments of the present disclosure, the anti-TROP-2 antibody or its variant comprises the light chain of SEQ ID NO: 15, or a full-length light chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto.

[0043] In preferred embodiments of the anti-TROP-2 antibody, its antigen-binding fragment, or its variants, The anti-TROP-2 antibody, its antigen-binding fragment, or its variants include 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 thereto, 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 thereto, or The anti-TROP-2 antibody, its antigen-binding fragment, or its variants include 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 thereto, 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 thereto, or The anti-TROP-2 antibody, its antigen-binding fragment, or its variants include 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 thereto, 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 thereto, or The anti-TROP-2 antibody, its antigen-binding fragment, or its variants include 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 thereto, 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 thereto, or The anti-TROP-2 antibody, its antigen-binding fragment, or its variants include 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 thereto, 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 thereto, or The anti-TROP-2 antibody, its antigen-binding fragment, or its variants include 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 thereto, 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 thereto, or The anti-TROP-2 antibody, its antigen-binding fragment, or its variants include 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 thereto, 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 thereto, or The anti-TROP-2 antibody, its antigen-binding fragment, or its variants include 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 thereto, 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 thereto, or The anti-TROP-2 antibody, its antigen-binding fragment, or its variants include 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 thereto, 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 thereto.

[0044] In preferred embodiments of the anti-TROP-2 antibody, its antigen-binding fragment, or its variants, The anti-TROP-2 antibody or its variant contains the heavy chain shown in SEQ ID NO: 21, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody or its variant contains the heavy chain shown in SEQ ID NO: 22, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody or its variant contains the heavy chain shown in SEQ ID NO: 23, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody or its variant contains the heavy chain shown in SEQ ID NO: 24, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody or its variant contains the heavy chain shown in SEQ ID NO: 25, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody or its variant contains the heavy chain shown in SEQ ID NO: 35, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody or its variant contains the heavy chain shown in SEQ ID NO: 36, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody or its variant contains the heavy chain shown in SEQ ID NO: 37, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody or its variant includes the heavy chain shown in SEQ ID NO: 38, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity thereto.

[0045] This disclosure also relates to polynucleotides encoding the aforementioned anti-TROP-2 antibody, its antigen-binding fragment, or its variants.

[0046] This disclosure also relates to an expression vector comprising the aforementioned polynucleotides.

[0047] This disclosure also relates to host cells transformed with the aforementioned expression vector.

[0048] This disclosure also relates to preferred embodiments in which the host cells are selected from the group consisting of bacteria, yeast, and mammalian cells.

[0049] The disclosure also relates to further preferred embodiments in which the host cells are selected from the group consisting of Escherichia coli, Pichia pastoris, CHO cells, or HEK293 cells.

[0050] The disclosure also relates to a method for producing the aforementioned anti-TROP-2 antibody, its antigen-binding fragment, or its variant, comprising the steps of: culturing the aforementioned host cells, preferably HEK293 cells; isolating the antibody from the culture, preferably from the cell culture medium; and purifying the antibody, preferably by chromatography.

[0051] This disclosure also relates to a pharmaceutical composition comprising the aforementioned anti-TROP-2 antibody, its antigen-binding fragment or variant thereof, and a pharmaceutically acceptable excipient, diluent or carrier.

[0052] This disclosure also relates to a detection or diagnostic kit comprising the aforementioned anti-TROP-2 antibody, its antigen-binding fragment, or its variant.

[0053] This disclosure also relates to the use of the above-mentioned anti-TROP-2 antibody, its antigen-binding fragment, or its variant in the preparation of a pharmaceutical product, wherein the pharmaceutical product is intended to treat or prevent a TROP-2-mediated disease or condition.

[0054] This disclosure also relates to the use of the above-mentioned anti-TROP-2 antibody, its antigen-binding fragment, or its variant in the preparation of reagents, wherein the reagent is for the detection, diagnosis, and prognosis of a TROP-2-mediated disease or condition.

[0055] This disclosure also relates to preferred embodiments of the above-mentioned use, wherein the TROP-2-mediated disease or condition is cancer, preferably, the TROP-2-mediated disease or condition is a cancer that expresses TROP-2, preferably, the cancer is selected from the group consisting of 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.

[0056] The disclosure also relates to preferred embodiments of the above-described anti-TROP-2 antibodies, their antigen-binding fragments, or their variants used to treat or prevent TROP-2-mediated diseases, wherein the disease is selected from the group consisting of 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.

[0057] The disclosure also relates to preferred embodiments of the above-described anti-TROP-2 antibodies, their antigen-binding fragments, or their variants for use in the detection, diagnosis, and prognosis of TROP-2-mediated diseases, wherein the disease is selected from the group consisting of 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.

[0058] The Disclosure also relates to a method for treating or preventing a TROP-2-mediated disease, comprising the steps of: providing a therapeutic or preventive amount of the above-mentioned anti-TROP-2 antibody, its antigen-binding fragment, or its variant to a subject in need of such a subject; or providing a therapeutic or preventive amount of the pharmaceutical composition according to the Disclosure to a subject in need of such a subject, wherein the TROP-2-mediated disease is selected from the group consisting of 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. [Modes for carrying out the invention]

[0059] term To facilitate understanding of this disclosure, certain technical and scientific terms are defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings generally understood by those skilled in the art.

[0060] The three-letter and one-letter amino acid codes used in this disclosure are as described in J. Biol. Chem, 243, pp. 3558 (1968).

[0061] As used in this disclosure, the term “antibody” refers to immunoglobulins, which have a tetrapeptide chain structure consisting of two heavy chains and two light chains linked by interchain disulfide bonds. Because the amino acid composition and sequence order of the constant region of the heavy chains of immunoglobulins differ, their antigenicity also differs. This allows immunoglobulins to be classified into five types, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, γ, α, and ε chains, respectively. Ig of the same type can be further classified into different subclasses depending on the differences in the amino acid composition of the hinge region, as well as the number and position of the heavy chain disulfide bonds. For example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4. The light chains are classified into κ or λ chains depending on the differences in the constant region. Each of the five types of Ig may have either a κ or λ chain.

[0062] In this disclosure, the antibody light chain may further comprise a light chain constant region containing a human or mouse κ chain, λ chain, or variants thereof.

[0063] In this disclosure, the antibody heavy chain may further comprise a heavy chain constant region containing human or mouse IgG1, IgG2, IgG3, and IgG4 or variants thereof.

[0064] The sequence of approximately 110 amino acids near the N-terminus of the antibody's heavy and light chains is highly variable and is named the variable region (V region), while the remaining amino acid sequence near the C-terminus is relatively stable and is named the constant region (C region). The variable region includes three hypervariable regions (HVRs) and four framework regions (FRs) with relatively conserved sequences. The three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each of the light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3, while the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The number and position of CDR amino acid residues in the VL and VH regions of the antibody or antigen-binding fragments in this disclosure conform to known Kabat numbering and Kabat or ABM standards.

[0065] The term "TROP-2" includes any naturally expressed variant or isoform of TROP-2. The antibodies of this disclosure may cross-react with TROP-2 derived from non-human species. Alternatively, the antibodies may also be specific to human TROP-2 and may not exhibit cross-reactivity with other species. TROP-2 or any variant or isoform thereof may be isolated from cells or tissues that naturally express them, or may be produced by recombinant technologies using techniques commonly used in the art and techniques described herein. Preferably, the anti-TROP-2 antibody targets human TROP-2 having a normal glycosylation pattern.

[0066] The term "recombinant human antibody" includes human antibodies prepared, expressed, produced, or isolated by recombinant methods, and related techniques and methods are well known in the art, including, for example, the following: 1. Antibodies isolated from transgenic and transchromosomal animals (e.g., mice) of human immunoglobulin genes, or from hybridomas prepared therefrom. 2. Antibodies isolated from host cells transformed to express antibodies such as transfectionomas. 3. Antibodies isolated from recombinant combinatorial human antibody libraries, and 4. Antibodies prepared, expressed, produced, or isolated by techniques such as splicing human immunoglobulin gene sequences onto other DNA sequences.

[0067] Such recombinant human antibodies include 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.

[0068] In this disclosure, the term “mouse antibody” refers to a monoclonal antibody against human TROP-2 obtained by the knowledge and skills of the art. During preparation, a test subject is injected with the TROP-2 antigen, and then a hybridoma expressing an antibody having the desired sequence or functional properties is isolated. In exemplary embodiments of this disclosure, the mouse TROP-2 antibody or its antigen-binding fragment may further comprise the light chain constant region of the mouse κ chain, λ chain, or variants thereof, or the heavy chain constant region of the mouse IgG1, IgG2, IgG3, or IgG4, or variants thereof.

[0069] The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies in this disclosure may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or somatic mutation in vivo). However, the term "human antibody" does not include humanized antibodies.

[0070] The term "humanized antibody," also known as CDR-transplanted antibody, refers to antibodies produced by transplanting non-human CDR sequences into the framework of a human antibody variable region. Humanized antibodies can overcome the drawbacks of chimeric antibodies, such as the strong immune response induced by carrying a large amount of non-human protein components. To avoid the decrease in activity caused by reduced immunogenicity, the human antibody variable region can be subjected to minimal reverse mutations to maintain its activity.

[0071] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of an antibody of a first species with the constant region of an antibody of a second species, which can mitigate the immune response induced by the antibody of the first species. For example, establishing a chimeric antibody requires first establishing a hybridoma that secretes a mouse-specific monoclonal antibody, then cloning the variable region gene from the mouse hybridoma cells, then cloning the constant region gene of a human antibody as needed, ligating the mouse variable region gene with the human constant region gene to form a chimeric gene, inserting this into a human expression vector, and finally expressing the chimeric antibody molecule in a eukaryotic or prokaryotic industrial system. The constant region of the human antibody may preferably include the human IgG1, IgG2, or IgG4 heavy chain constant region, or it may be selected from the heavy chain constant regions of human IgG1, IgG2, IgG3, and IgG4 and their variants, using an IgG1 heavy chain constant region with enhanced ADCC (antibody-dependent cell-mediated cytotoxicity) after amino acid mutation.

[0072] The term “antigen-binding fragment” refers to an antigen-binding fragment of an antibody and an antibody analog that typically contains 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 a portion of the binding specificity of the parent antibody. Typically, when activity is expressed in molar terms, the antibody fragment retains at least 10% of the binding activity of the parent antibody. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity of the parent antibody 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. Manipulated antibody variants are outlined in Holliger and Hudson, 2005, Nat. Biotechnol. 23: pp. 1126-1136.

[0073] A "Fab fragment" consists of a variable region comprising one light chain and CH1, as well as one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with other heavy chain molecules.

[0074] The "Fc" region contains two heavy chain fragments, each containing the CH1 and CH2 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domain.

[0075] A "Fab' fragment" contains one light chain and a portion of one heavy chain including a VH domain, a CH1 domain, and a region between the CH1 and CH2 domains. Therefore, an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments, thereby forming an F(ab')2 molecule.

[0076] The "F(ab')2 fragment" contains two light chains, as well as two heavy chains that include a portion of the constant region between the CH1 domain and the CH2 domain, thereby forming an interchain disulfide bond between the two heavy chains. Thus, the F(ab')2 fragment is composed of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0077] The "Fv region" includes variable regions from both the heavy and light chains, but lacks a steady-state region.

[0078] The term "multispecific antibody" is used in its broadest sense to encompass antibodies that are specific to multiple epitopes. These multispecific antibodies include, but are not limited to, antibodies containing a heavy chain variable region (VH) and a light chain variable region (VL) (where the VH-VL unit is specific to multiple epitopes); antibodies having two or more VL and VH regions (each VH-VL unit binds to a different target or a different epitope of the same target); antibodies having two or more single variable regions (each single variable region binds to a different target or a different epitope of the same target); full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, covalently or non-covalently bonded antibody fragments, etc.

[0079] The term "single-chain antibody" refers to a single-chain recombinant protein formed by linking the heavy chain variable region (VH) and light chain variable region (VL) of an antibody via a linker peptide. It is the smallest antibody fragment with a complete antigen-binding site.

[0080] The term "domain antibody fragment" refers to an immunoglobulin fragment that contains only the heavy chain variable region or the light chain variable region and possesses immunological function. In certain cases, two or more VH regions are covalently linked by a peptide linker to form a bivalent domain antibody fragment. The two VH regions of a bivalent domain antibody fragment can target the same antigen or different antigens.

[0081] The term "binding to TROP-2" refers to the ability to interact with TROP-2 or its epitope.

[0082] The term "antigen-binding site" refers to a three-dimensional site recognized by the antibody or antigen-binding fragment of this disclosure.

[0083] The term "epitope" refers to an antigenic site that specifically binds to an immunoglobulin or antibody. Epitopes can be formed by adjacent or non-adjacent amino acids through tertiary folding of a protein. Epitopes formed by adjacent amino acids are usually retained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with a denaturing solvent. Epitopes typically contain at least 3 to 15 amino acids in their unique spatial conformation. Methods for determining which epitopes bind to a given antibody are well known in the art and include immunoblotting, immunoprecipitation detection analysis, etc. Methods for determining the spatial conformation of an epitope include the art in the art and the art described herein, such as X-ray crystallography, two-dimensional nuclear magnetic resonance, etc.

[0084] The terms "specifically bind" and "selectively bind" refer to the binding of an antibody to a specific epitope on an antigen. Typically, when recombinant human TROP-2 is used as the analyte and the antibody is used as the ligand, when measured using the instrument's surface plasmon resonance (SPR) technique, the antibody will bind to approximately 10 -7 Equilibrium dissociation constant (K) less than or equal to M D ) binds to a predetermined antigen (or epitope), and its binding affinity to the predetermined antigen (or epitope) is at least twice as high as its binding affinity to nonspecific antigens other than the predetermined antigen or closely related antigen, such as BSA.

[0085] 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 binding assays (e.g., SPR and ELISA) by detecting specific reactivity with purified antigens or binding or functional interaction with cells that physiologically express TROP-2. Methods for measuring cross-reactivity include standard binding assays described herein, such as surface plasmon resonance (SPR) analysis or flow cytometry.

[0086] The terms “inhibition” and “blockage” can be used interchangeably and encompass both partial and complete inhibition / blockage. Preferably, the inhibition or blockage of a ligand reduces or alters the level or type of activity compared to the normal level or type of activity in the absence of inhibition or blockage. Inhibition and blockage are also intended to include a measurable decrease in either the binding affinity to the ligand when in contact with the anti-TROP-2 antibody compared to the ligand not in contact with the anti-TROP-2 antibody.

[0087] The term “inhibition of proliferation” (for example, with respect to cells) is intended to include any measurable reduction in cell proliferation.

[0088] The terms “induced immune response” and “enhanced immune response” can be used interchangeably and refer to an immune response stimulated (i.e., passive or adaptive) by a particular antigen. The term “inducing” in relation to inducing CDC or ADCC refers to stimulating a particular direct cell-killing mechanism.

[0089] The term "ADCC," or antibody-dependent cell-mediated cytotoxicity, refers to the direct killing of target cells by an antibody by cells expressing the Fc receptor upon recognition of the antibody's Fc segment. The ADCC effector function of an antibody can be enhanced, reduced, or eliminated by modifying the Fc segment of IgG. Modification refers to mutations in the constant region of the antibody's heavy chain.

[0090] Methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in prior art such as Antibodies: A Laboratory Manual, Cold Spring Harbor, Chapters 5-8 and 15. For example, human TROP-2 or its fragments can be used to immunize mice, and the resulting antibodies can be regenerated, purified, and subjected to amino acid sequencing by conventional methods. Similarly, antigen-binding fragments can be prepared by conventional methods. To obtain the antibody or antigen-binding fragment of the present invention, one or more human FR regions are added to a non-human CDR region by genetic engineering. Human FR germline sequences can be obtained from ImMunoGeneTics (IMGT) or The Immunoglobulin FactsBook, 2001 ISBN 012441351.

[0091] The manipulated antibodies or antigen-binding fragments of this disclosure can be prepared and purified by conventional methods. The cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems can induce 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 grown in serum-free medium in a bioreactor to produce antibodies. The culture medium from which the antibodies are secreted can be purified and collected by conventional techniques. The antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, e.g., molecular sieving and ion exchange. The resulting product needs to be immediately frozen or lyophilized, for example, at -70°C.

[0092] The antibodies in this disclosure refer to monoclonal antibodies. The monoclonal antibodies (mAbs) described in this disclosure refer to antibodies obtained from monoclonal cell lines, which are eukaryotes, prokaryotes, or phage clone cell lines, but are not limited to these. Monoclonal antibodies or antigen-binding fragments can be obtained, for example, by recombination using hybridoma technology, recombinant technology, phage display technology, synthesis technology (such as CDR transplantation), or other existing technologies.

[0093] When applied to animals, humans, experimental subjects, cells, tissues, organs, or bodily fluids, “administration,” “dosing,” and “treatment” refer to the contact of an exogenous pharmaceutical, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or bodily fluid. “Administration,” “dosing,” and “treatment” can also refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods. Treatment of cells includes contacting a reagent with cells and contacting a reagent with a liquid, where the liquid is in contact with the cells. “Administration,” “dosing,” and “treatment” also refer to, for example, treating cells in vitro and ex vivo with a reagent, diagnostic agent, conjugate composition, or another cell. When applied to humans, veterinary subjects, or research subjects, “administration,” “dosing,” and “treatment” refer to therapeutic treatment, inhibitory or prophylactic measures, research, and diagnostic uses.

[0094] "Treatment" means providing an internal or external therapeutic agent (e.g., a drug containing either one of the antibodies or fragments thereof of this disclosure) to a subject having one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in the subject or population being treated, inducing regression of such symptoms or inhibiting the progression of such symptoms to either a clinically measurable degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also called the "therapeutic dose") may vary depending on a variety of factors, such as the subject's disease state, age, and weight, as well as the drug's ability to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been alleviated can be assessed by any clinical trial method commonly used by a physician or other healthcare professional to assess the severity or progression of symptoms. Embodiments of the present disclosure (e.g., treatment methods or products) may not be effective in alleviating the symptoms of the target disease in any subject, but they should significantly reduce the symptoms of the target disease in a statistically valid number of subjects, as determined by any statistical test method known in the art, such as the Student t-test, chi-square test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0095] An "effective dose" includes an amount sufficient to improve or prevent a medical condition or its symptoms. An effective dose also refers to an amount sufficient to enable or facilitate a diagnosis. The effective dose for a particular subject or veterinary subject may vary depending on factors such as the condition being treated, the patient's overall health, the method, route, and dosage of administration, and the severity of side effects. An effective dose may be the maximum dose or dosage regimen that avoids serious side effects or toxic effects.

[0096] "Exogenous" refers to substances produced outside of an organism, cell, or human body, depending on the context.

[0097] "Endogenous" refers to substances produced within cells, organisms, or the human body, depending on the context.

[0098] "Homologousity" or "identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. Two aligned sequences are homologous at a given position if the positions of the same base or amino acid residues are occupied by the same base or amino acid residue. For example, if each position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The homology percentage between two sequences is a function of the number of identical or homologous positions shared by the two sequences divided by the number of aligned positions, multiplied by 100%. For example, in optimal sequence alignment, two sequences are 60% homologous if 6 out of 10 positions of the two sequences are identical or homologous. Generally, alignment is performed when two sequences are aligned to obtain the maximum homology percentage.

[0099] The terms “cell,” “cell line,” and “cell culture” used herein are interchangeable, and all such names include their offspring. Therefore, the terms “transformed organism” and “transformed cell” include primary cells and cultures derived therefrom, regardless of the number of passages. It should also be understood that, due to intentional or unintentional mutations, not all offspring will be exactly the same in terms of DNA content. This includes mutant offspring that have the same function or biological activity as those screened in the primary cells. Where different names are referred to, this should be clearly understood from the context.

[0100] "Optional" or "optionally" means that the event or situation described thereafter may occur, but does not necessarily occur, and that description includes cases where the event or situation occurs or does not occur. For example, "optionally containing 1 to 3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a specific sequence may be present, but are not required to be present.

[0101] "Pharmaceutical composition" means containing one or more antibody or antigen-binding fragments described herein, and other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate drug administration to a living organism and to aid in the absorption of the active ingredient in order to impart biological activity.

[0102] Unless otherwise explicitly stated in the context, the singular forms "a / an," "one," or "the" also include their corresponding plural forms.

[0103] The present disclosure will be further illustrated with reference to the following examples, but these examples should not be considered to limit the scope of the present disclosure. Experimental methods without specific conditions in the examples of the present disclosure typically follow conventional conditions, such as those described in Antibodies: A Laboratory Manual and Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, or those recommended by the manufacturer of the raw materials or products. Reagents without specific sources are conventional reagents purchased from the market. [Examples]

[0104] Antigen preparation The protein encoding His-tagged human TROP-2 (TROP-2-His) was synthesized by SinoBiologics (10428-H08H). TROP-2-His sequence:

[0105] [ka] [Examples]

[0106] Acquisition of mouse hybridoma and antibody sequences A total of five 10-week-old female Balb / c and five A / J mice were immunized with the human antigen TROP-2-His. Sigma's complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) were used. The immunogen and immunoadjuvant were completely mixed in a 1:1 ratio and emulsified to prepare a stable "water in oil" solution. The injection dose was 25 μg / 200 μL / mouse.

[0107] [Table 1]

[0108] Serum from immunized mice was evaluated for serum titer and ability to bind to cell surface antigens using the indirect ELISA described in Example 3. The initiation of cell fusion was measured according to the titer detection results (dilution greater than 100,000-fold). Immunized mice with high serum titer, affinity, and FACS binding were selected for a single final immunization and then sacrificed. Splenocytes were collected, fused with SP2 / 0 myeloma cells, and seeded to obtain hybridomas. Target hybridomas were screened by indirect ELISA and established as monoclonal cell lines by limiting dilution. To select hybridomas bound to recombinant proteins, the resulting antibody-positive lines were further screened by indirect ELISA. Hybridoma cells in the logarithmic growth phase were collected. RNA was extracted with Trizol (Invitrogen, 15596-018) and subjected to reverse transcription (PrimeScript® reverse transcriptase, Takara, #2680A). The cDNA obtained by reverse transcription was amplified by PCR using a mouse Ig-primer set (Novagen, TB326 Rev.B0503), then sequenced, and finally the sequence of the mouse antibody was obtained.

[0109] The sequences of the heavy chain and light chain variable regions of the mouse monoclonal antibody M1 are as follows:

[0110] [ka]

[0111] [Table 2] [Examples]

[0112] Method for detecting the in vitro binding activity of antibodies In vitro indirect ELISA coupling experiment: The TROP-2 His protein (Sino Biological Inc., cat# 10428-H08H) was diluted to a concentration of 1 μg / ml in pH 7.4 PBS and added to a 96-well high-affinity ELISA plate at a rate of 100 μl / well. The plate was incubated overnight (16-20 hours) in a refrigerator at 4°C. After washing the plate four times with PBST (pH 7.4 PBS containing 0.05% Tween-20), 150 μl / well of 3% bovine serum albumin (BSA) blockade solution diluted with PBST was added, and the plate was incubated at room temperature for 1 hour for blockade. After blockade was complete, the blockade solution was discarded, and the plate was washed four times with PBST buffer.

[0113] The test antibody was diluted in PBST containing 3% BSA to obtain a 5-fold dilution series (starting at 10 μM and totaling 9 doses), and added to an ELISA plate at 100 μl / well, and incubated at room temperature for 1 hour. After incubation, the plate was washed four times with PBST. HRP-labeled goat anti-human secondary antibody (Abcam, cat#ab97225), diluted in PBST containing 3% BSA, was added at 100 μl / well and incubated at room temperature for 1 hour. After washing the plate four times with PBST, TMB chromogenic substrate (Cell Signaling Technology, cat#7004S) was added at 100 μl / well and incubated at room temperature in the dark for 1 minute. The reaction was stopped by adding 100 μl / well of stop solution (Cell Signaling Technology, cat#7002S). Absorbance at 450 nm was read using a microplate reader (BioTek, Model Synergy H1), and the data was analyzed. As shown in Table 3 below, the concentration-signal curves were plotted and the results were analyzed.

[0114] [Table 3] [Examples]

[0115] Humanization experiments of mouse antibodies Humanization of a mouse anti-human TROP-2 monoclonal antibody was performed using methods disclosed in numerous publications in the relevant art. Briefly, the constant domain of the parent (mouse antibody) was replaced with the constant domain of a human antibody. A human germline antibody sequence was selected according to the homology between the mouse antibody and the human antibody. In this disclosure, mouse antibody M1 was humanized.

[0116] Based on the typical structure of the VH / VL CDR obtained from the mouse antibody, the sequences of the heavy chain and light chain variable regions were aligned in a human antibody germline database to obtain a human germline template with high homology.

[0117] The CDR region of mouse antibody M1 was transplanted into a selected corresponding humanized template. Next, based on the three-dimensional structure of the mouse antibody, embedded residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of VL and VH were subjected to reverse mutations. After expression tests and comparison of the number of reverse mutations, antibodies designed by combining the humanized heavy chain variable region (HCVR) and light chain variable region (LCVR) sequences were selected, and the sequences are as follows.

[0118] [ka]

[0119] The designed heavy chain and light chain variable region sequences were concatenated to the IgG1 heavy chain and light chain constant region sequences, respectively. The human IgG1 heavy chain constant region sequence is as follows:

[0120] [ka]

[0121] The designed heavy chain and light chain variable region sequences were concatenated to the IgG1 heavy chain and light chain constant region sequences, respectively. The concatenated human kappa chain constant region sequences are as follows:

[0122] [ka]

[0123] The following is an example of the heavy and light chain arrangement obtained after linking.

[0124] [ka] [Examples]

[0125] HU6DL Variant Design Experiment The humanized antibody HU6DL is N to HCDR2. 54 D55 S 56 It possesses a motif and is susceptible to glycosylation. N54 was subjected to site-directed mutation using computer-aided technology to reduce the potential risk of glycosylation without affecting antigen binding or its unique thermal stability. Mutants of the antibody HU6DL, namely HU6DL.R54, HU6DL.Y54, HU6DL.Q54, HU6DL.L54, HU6DL.T54, HU6DL.I54, HU6DL.F54, HU6DL.E54, and HU6DL.A54, were obtained, with the corresponding heavy and light chain variable regions being as follows.

[0126] [ka]

[0127] [ka]

[0128] [ka]

[0129] The designed heavy chain and light chain variable region sequences were concatenated to the IgG1 heavy chain constant region and light chain constant region sequences, respectively. The resulting exemplary heavy chain and light chain sequences are as follows (wherein the HU6DL.R54, HU6DL.Y54, HU6DL.Q54, HU6DL.L54, HU6DL.T54, HU6DL.I54, HU6DL.F54, HU6DL.E54, and HU6DL.A54 heavy chains are produced by concatenating the sequences of SEQ ID NOs. 16, 17, 18, 19, 20, 31, 32, 33, and 34 to SEQ ID NO. 12, respectively).

[0130] [ka]

[0131] [ka]

[0132] [ka]

[0133] [ka]

[0134] [ka]

[0135] [Table 4]

[0136] [Table 5]

[0137] cDNA fragments were synthesized according to the amino acid sequences of the light and heavy chains of the humanized antibody described above. Protein expression services were provided by Beijing Sinobiological Co., Ltd. HEK293 cells were transiently transfected with the HU6DL protein variant for expression. Antibodies were detected against their purity using molecular exclusion chromatography, and their concentrations and purities are shown in Table 6 below.

[0138] [Table 6] [Examples]

[0139] Affinity experiments of HU6DL mutants against TROP-2 antigen 1. Objective of the experiment: The differences in affinity levels of different anti-TROP-2 variants against the TROP-2 antigen were evaluated using sandwich ELISA, i.e., "antigen-antibody-HRP-labeled secondary antibody".

[0140] 2. Experimental procedure: The his-tagged TROP-2 protein (SinoBiologics, Cat:10428-H08H) was diluted to 1 μg / mL using DPBS pH 7.4 and added to a high-affinity 96-well plate (Corning, Cat:3590) at 100 μL / well, and incubated overnight at 4°C. The TROP-2 antigen solution was discarded the following day. PBS pH 7.4 containing 0.05% Tween 20 (PBST) solution was added at 200 μL / well for three washes. 2% BSA (dissolved in PBST) was added at 200 μL / well for 1 hour blockage at 37°C, and the plate was washed three times with PBST. Serial dilutions of candidate antibodies (from 10 nM to 1 × 10⁶) were prepared. -6 100 μL / well of a 10-fold dilution (giving eight concentration levels up to nM) was added, and 0.5% BSA was used as a negative control. The plates were incubated at 37°C for 1 hour for blockade. The plates were washed three times with PBST. 100 μL / well of a 1:10000 diluted goat anti-human IgG, Fc-HRP (Abcam, cat:ab97225) secondary antibody solution was added, and the plates were incubated at 37°C for 1 hour for blockade. The plates were washed three times with PBST.

[0141] TMB (CST, Cat: 7004P6) substrate was added at a rate of 100 μL / well for a 3-minute reaction at room temperature until the solution in the well with the highest antibody concentration turned dark blue. The reaction was stopped by adding 50 μL / well of stop solution (CST, Cat: 7002P6). Absorbance (OD) was read at a wavelength of 450 nm.

[0142] 3. Data processing: EC 50(Affinity of each HU6DL variant for the antigen) was calculated by plotting the logarithm of the concentration of the candidate antibody on the X-axis and the OD450 absorbance on the Y-axis, and fitting the log(agonist) vs. response-executable slope (4-parameter) equation in GraphPad PRISM 8.0. The affinity (EC 50 ) of the HU6DL variant for the human TROP-2 antigen was as shown in Table 7 below.

[0143]

Table 7

[0144] 4. Conclusions of the experiment: The above data indicate that each HU6DL variant of the present disclosure has good affinity for the human TROP-2 antigen.

Example

[0145] Affinity experiment of HU6DL variant for tumor cells 1. Purpose of the experiment: The difference in the affinity levels of the HU6DL variant for tumor cell lines expressing the TROP-2 antigen was evaluated by flow cytometry.

[0146] 2. Experimental reagents: Gastric cancer cell NCI-N87 (purchased from the Chinese Academy of Sciences Cell Bank, TCHu130), Non-small cell lung cancer cell HCC827 (purchased from the Chinese Academy of Sciences Cell Bank, TCHu153), Bladder cancer cell SW780 (purchased from the Chinese Academy of Sciences Cell Bank, TCHu219), Bladder cancer cell RT4 (purchased from the Chinese Academy of Sciences Cell Bank, TCHu226).

[0147] 3. Experimental procedure: Well-proliferating tumor cells were treated with Accutase (Sigma, cat: A6964) digestion solution. Single-cell suspensions were prepared in 2% FBS (diluted in DBPS, pH 7.4) solution, and the cell concentration was increased to 1 × 10⁻⁶. 6 The concentration was adjusted to cells / mL. The suspension was aliquoted at 100 μL / well into a 96-well V-bottom plate and centrifuged at 4°C at 300 g for 5 minutes. The supernatant was discarded, and serial dilutions of the candidate antibody solution (from 1000 nM to 1 × 10⁶) were prepared. -6 A 10-fold dilution (giving 10 concentration levels up to nM) was added at 100 μL / well and incubated at 4°C for 1 hour.

[0148] The plate was centrifuged at 4°C at 300g for 5 minutes and washed twice. 5μL / 10 6 A 100 μL / well of mouse anti-human IgG Fc, PE-labeled secondary antibody (Biolegend, cat:409304), diluted to the cell ratio, was added and incubated at 4°C for 1 hour. The plate was centrifuged at 4°C at 300 g for 5 minutes and washed twice. Cells were resuspended by adding 70 μL of 2% FBS solution. The mean fluorescence intensity (MFI) of PE channels was detected by ZE5 flow cytometry (Bio-Rad, ZE5).

[0149] 4. Data processing: EC 50 The affinity of each candidate antibody against tumor cells was calculated by plotting the logarithmic concentration of the mutant antibody as the X-coordinate and the MFI as the Y-coordinate, and fitting the log(agonist) vs. response-feasible slope (4 parameters) equation in GraphPad PRISM 8.0, as shown in Table 8 below.

[0150] [Table 8]

[0151] 5. Conclusion of the experiment: The data above demonstrates that each HU6DL variant in this disclosure exhibits good affinity for NCI-N87, HCC827, SW780, and RT4 tumor cells. [Examples]

[0152] HU6DL mutant-mediated TROP2 endocytosis experiment 1. Objective of the experiment: The antibody endocytosis activity of the HU6DL mutant was evaluated by flow cytometry against tumor cell lines expressing the TROP-2 antigen.

[0153] 2. Experimental procedure: Proliferating gastric cancer cells NCI-N87 (purchased from the Chinese Academy of Sciences Cell Bank, TCHu130) were treated with Accutase (Sigma, cat:A6964) digestion solution. Single-cell suspensions were prepared in 2% FBS (diluted in DBPS, pH 7.4) solution, and the cell concentration was increased to 1 × 10⁻⁶. 7 The suspension was adjusted to the required cell / mL concentration. The suspension was aliquoted at 100 μL / well into a 96-well V-bottom plate. Candidate antibody solution was added at a final concentration of 20 μg / mL, thoroughly mixed, and incubated at 4°C for 1 hour.

[0154] The plate was centrifuged at 4°C at 300g for 5 minutes. 5μL / 10 6 A mouse anti-human IgG Fc, PE-labeled secondary antibody (Biolegend, cat:409304), diluted to the cell ratio, was added at a rate of 100 μL / well and incubated at 4°C for 1 hour.

[0155] The plates were centrifuged at 4°C at 300g for 5 minutes and washed twice. The cell pellet was resuspended in 1 mL of preheated complete medium and aliquoted into four groups: a 0-minute group, a blank group, a 30-minute group, and a 120-minute group. The 0-minute group and the blank control were placed on ice. The remainder were placed in a 37°C incubator for 30 minutes and 120 minutes, respectively, for endocytosis. The groups were removed at the corresponding time points and pre-cooled on ice for 5 minutes. All treatment groups were centrifuged and the supernatant was discarded (4°C, 1500 rpm for 5 minutes). The cells were washed once with FACS buffer and the supernatant was discarded. Except for the 0-minute group, 250 μL of strip buffer was added to all treatment groups, incubated at room temperature for 8 minutes, centrifuged (4°C, 1500 rpm for 5 minutes), and the supernatant was discarded. The cells were washed twice with FACS buffer and the supernatant was discarded. 80 μL of 2% FBS was added to each sample group to resuspend the cells. The fluorescence signal of the sample under test was detected by ZE5 flow cytometry (Bio-Rad, ZE5).

[0156] 3. Data processing: The endocytosis rate for each candidate antibody was calculated according to the following equation: Antibody endocytosis rate (%) = (MFI of the treatment group - MFI of the blank control group) / (MFI of the 0 min group - MFI of the blank control group) × 100%. The endocytosis rate of the HU6DL variant was detected by the above method and is shown in Table 9.

[0157] [Table 9]

[0158] 4. Conclusion of the experiment: The data above demonstrates that the HU6DL variant of this disclosure is fully endocytosizable in NCI-N87 gastric cancer cells mediated by the TROP-2 protein. [Examples]

[0159] Detection of impurity components in the HU6DL mutant 1. Experimental Objective: We detected and compared changes in the content and level of impurity peaks of mutant antibodies using capillary electrophoresis based on the Maurice-nrCE-SDS method.

[0160] 2. Experimental Procedure 2.1. Sample preparation: (1) Buffer exchange and concentration of the sample: When the protein concentration of the sample was less than 5 mg / ml, or when the salt concentration in the sample buffer was high, it was necessary to exchange the buffer and concentrate the sample to ensure a protein concentration of approximately 5 mg / ml and a salt concentration in the sample of less than 50 mM.

[0161] (2) Non-reducing treatment of CE samples: Samples were added to EP tubes in amounts of 50 μg of protein taken from each sample. 1 μl of 10 kD internal standard (Protein Simple, 046-144) was added, followed by 2.5 μl of 250 mM IAM (Sigma, I1149-5G), and finally 50 μl of 1× sample buffer (Protein Simple, 046-567) was added.

[0162] (3) The sample was thoroughly mixed by shaking, heated, incubated at 70°C for 10 minutes, and then removed. The sample was incubated on ice for 5 minutes until cooled, and then centrifuged at 12,000 rpm for 5 minutes. After centrifugation, 35 μl of supernatant was transferred to a 96-well sample plate suitable for the instrument, and then centrifuged at 1,000 rpm for 5 minutes. The 96-well sample plate was placed in a Maurice (Protein Simple) for sample loading and analysis.

[0163] 2.2. Detection by equipment I turned on the instrument and opened the software. I performed the instrument's self-check according to the instrument's operating procedure. I installed the capillary cartridge and placed the prepared corresponding reagents in their corresponding positions within the instrument. I set the corresponding parameters and performed the non-reducing CE analysis according to the instrument's operating procedure. I set the sample sequence by editing the corresponding sequence according to the sample name, ensuring that the number of samples for each sequence did not exceed 48. After completing the sequence editing, I started the sequence detection by clicking Start.

[0164] The main peak and impurity peak content of the sample were calculated using the following formula.

[0165]

number

[0166] Note: In this formula, the non-reducing purity is the percentage of the corrected main peak area. CA main peak This represents the corrected main peak area. CA total This represents the sum of the corrected main peak and impurity peak areas.

[0167] 3. Experimental results:

[0168] [Table 10]

[0169] The above data shows that the main peak content of sample HU6DL.T54 reached 93.62%, and no impurities were detected. The experimental results indicate that N in HCDR2 of HU6DL 54 D 55 S 56 This study demonstrates that the motif is susceptible to glycosylation, and that site-directed mutation at N54 effectively reduces glycosylated impurities, leading to higher purity. [Examples]

[0170] Competitive binding of humanized antibodies to antigens 1. Experimental Objective: To study the binding modes and sites of different antibodies to an antigen through competitive binding experiments.

[0171] 2. Experimental Procedure Coated antibodies hRS7 and HU6DL variants were diluted to a concentration of 1 μg / ml in pH 7.4 PBS and added to 96-well high-affinity ELISA plates at a rate of 100 μl / well. The plates were incubated overnight (16-20 hours) at 4°C in a refrigerator. After washing the plates four times with PBST (pH 7.4 PBS containing 0.05% Tween-20), 200 μl / well of 2% bovine serum albumin (BSA) blockade solution diluted with PBST was added, and the plates were incubated at room temperature for 1 hour for blockade. After blockade was complete, the blockade solution was discarded, and the plates were washed four times with PBST buffer.

[0172] The competitive antibodies hRS7 and HU6DL were diluted to 100 μg / ml in PBST containing 0.5% BSA and added to ELISA plates at a rate of 50 μg / well. The affinity of TROP-2His protein (Acro Biosystems, cat# TR2-H5223) to the coated antibody was measured at 2×EC. 80 or 2 × EC 90Each sample was diluted to the corresponding concentration in PBST containing 0.5% BSA and added to an ELISA plate at 50 μl / well. The ELISA plate was incubated at room temperature for 1 hour. After incubation was complete, the plate was washed four times with PBST. 100 μl / well of anti-His HRP-labeled secondary antibody (Sino Biological, cat# 105327-MM02T-H), diluted 1:5000 in PBST containing 0.5% BSA, was added and incubated at room temperature for 1 hour. The plate was washed four times with PBST, then 100 μl / well of TMB chromogenic substrate (Cell Signaling Technology, cat# 7004S) was added and incubated in the dark at room temperature for 1 minute. The reaction was stopped by adding 50 μl / well of stop solution (Cell Signaling Technology, cat# 7002S).

[0173] 3. Data Processing The absorbance at 450 nm was read using a microplate reader (Thermo, model Varioskan LUX), and the data was analyzed as shown in Table 11 below.

[0174] [Table 11]

[0175] 4. Experimental Results The humanized antibody modified by the mutation of the present invention exhibits a very low inhibition rate against the binding of the hRS7 antibody to the TROP2 protein, suggesting that the humanized antibody modified by the mutation of the present invention and the hRS7 antibody do not competitively bind to the same epitope.

Claims

1. An anti-TROP-2 antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, 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. Anti-TROP-2 antibody or its antigen-binding fragment.

2. 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 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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. The anti-TROP-2 antibody or its antigen-binding fragment according to claim 1.

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

4. The anti-TROP-2 antibody or antigen-binding fragment according to any one of claims 1 to 3, further comprising a constant region derived from human IgG1, IgG2, IgG3, or IgG4.

5. The anti-TROP-2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises an IgG1 heavy chain constant region into which 239D and 241L mutations have been introduced, and the sites of the mutations are determined by SEQ ID NO:

12.

6. The anti-TROP-2 antibody or antigen-binding fragment according to any one of claims 1 to 5, wherein the anti-TROP-2 antibody or antigen-binding fragment comprises the heavy chain constant region of SEQ ID NO:

12.

7. The anti-TROP-2 antibody or antigen-binding fragment according to any one of claims 4 to 6, wherein the anti-TROP-2 antibody or antigen-binding fragment further comprises a light chain constant region derived from a human κ chain or λ chain.

8. The anti-TROP-2 antibody or antigen-binding fragment according to any one of claims 4 to 7, wherein the anti-TROP-2 antibody or antigen-binding fragment comprises the light chain constant region of SEQ ID NO:

13.

9. The anti-TROP-2 antibody or antigen-binding fragment according to any one of claims 1 to 8, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof includes a heavy chain variable region selected from the group consisting of SEQ ID NOs. 20, SEQ ID NOs. 16, SEQ ID NOs. 17, SEQ ID NOs. 18, SEQ ID NOs. 19, SEQ ID NOs. 31, SEQ ID NOs. 32, SEQ ID NOs. 33, and SEQ ID NOs. 34, or a heavy chain variable region having at least 90%, 95%, or 99% identity thereto.

10. The anti-TROP-2 antibody or antigen-binding fragment according to any one of claims 1 to 3 and 9, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof includes the light chain variable region of SEQ ID NO: 11, or a light chain variable region having at least 90%, 95%, or 99% identity therewith.

11. The anti-TROP-2 antibody or antigen-binding fragment thereof according to claim 9 or 10, wherein the anti-TROP-2 antibody comprises a heavy chain selected from the group consisting of SEQ ID NOs. 25, SEQ ID NOs. 21, SEQ ID NOs. 22, SEQ ID NOs. 23, SEQ ID NOs. 24, SEQ ID NOs. 35, SEQ ID NOs. 36, SEQ ID NOs. 37, and SEQ ID NOs. 38, or a heavy chain having at least 90%, 95%, or 99% identity thereto.

12. The anti-TROP-2 antibody or antigen-binding fragment thereof according to claim 9 or 10, wherein the anti-TROP-2 antibody comprises the light chain of SEQ ID NO: 15, or a light chain having at least 90%, 95%, or 99% identity therewith.

13. The anti-TROP-2 antibody or its antigen-binding fragment includes the heavy chain variable region shown in SEQ ID NO: 20, or a heavy chain variable region having at least 90%, 95%, or 99% identity thereto, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody or its antigen-binding fragment includes the heavy chain variable region shown in SEQ ID NO: 16, or a heavy chain variable region having at least 90%, 95%, or 99% identity thereto, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody or its antigen-binding fragment includes the heavy chain variable region shown in SEQ ID NO: 17, or a heavy chain variable region having at least 90%, 95%, or 99% identity thereto, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody or its antigen-binding fragment includes the heavy chain variable region shown in SEQ ID NO: 18, or a heavy chain variable region having at least 90%, 95%, or 99% identity thereto, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody or its antigen-binding fragment includes the heavy chain variable region shown in SEQ ID NO: 19, or a heavy chain variable region having at least 90%, 95%, or 99% identity thereto, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody or its antigen-binding fragment includes the heavy chain variable region shown in SEQ ID NO: 31, or a heavy chain variable region having at least 90%, 95%, or 99% identity thereto, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody or its antigen-binding fragment includes the heavy chain variable region shown in SEQ ID NO: 32, or a heavy chain variable region having at least 90%, 95%, or 99% identity thereto, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody or its antigen-binding fragment includes the heavy chain variable region shown in SEQ ID NO: 33, or a heavy chain variable region having at least 90%, 95%, or 99% identity thereto, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody or its antigen-binding fragment includes the heavy chain variable region shown in SEQ ID NO: 34, or a heavy chain variable region having at least 90%, 95%, or 99% identity thereto, and the light chain variable region shown in SEQ ID NO: 11, or a light chain variable region having at least 90%, 95%, or 99% identity thereto. An anti-TROP-2 antibody or its antigen-binding fragment according to any one of claims 1 to 12.

14. The anti-TROP-2 antibody contains the heavy chain shown in SEQ ID NO: 25, or a heavy chain having at least 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody contains the heavy chain shown in SEQ ID NO: 21, or a heavy chain having at least 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody contains the heavy chain shown in SEQ ID NO: 22, or a heavy chain having at least 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody contains the heavy chain shown in SEQ ID NO: 23, or a heavy chain having at least 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody contains the heavy chain shown in SEQ ID NO: 24, or a heavy chain having at least 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody contains the heavy chain shown in SEQ ID NO: 35, or a heavy chain having at least 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody contains the heavy chain shown in SEQ ID NO: 36, or a heavy chain having at least 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody contains the heavy chain shown in SEQ ID NO: 37, or a heavy chain having at least 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 90%, 95%, or 99% identity thereto, or The anti-TROP-2 antibody comprises the heavy chain shown in SEQ ID NO: 38, or a heavy chain having at least 90%, 95%, or 99% identity thereto, and the light chain shown in SEQ ID NO: 15, or a light chain having at least 90%, 95%, or 99% identity thereto. An anti-TROP-2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.

15. A polynucleotide encoding an anti-TROP-2 antibody or an antigen-binding fragment thereof, as described in any one of claims 1 to 14.

16. An expression vector comprising the polynucleotide described in claim 15.

17. A host cell transformed with the expression vector described in claim 16, the host cell being selected from the group consisting of bacteria, yeast, and mammalian cells.

18. The host cell according to claim 17, wherein the host cell is selected from the group consisting of Escherichia coli, Pichia pastrius, CHO cells, or HEK293 cells.

19. A method for producing an anti-TROP-2 antibody or its antigen-binding fragment according to any one of claims 1 to 14, comprising the following steps: A step of culturing host cells according to claim 17 or 18, The process of isolating antibodies from the culture, The process of purifying the aforementioned antibody and Methods that include...

20. The method comprises the following steps: The process of culturing HEK293 cells, The process of isolating antibodies from cell culture medium, A step of purifying the antibody by chromatography and The method according to claim 19, including the method described in claim 19.

21. A pharmaceutical composition comprising an anti-TROP-2 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 14, and a pharmaceutically acceptable excipient, diluent, or carrier.

22. A detection or diagnostic reagent comprising an anti-TROP-2 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 14.

23. Use in the preparation of a pharmaceutical product, wherein the use of an anti-TROP-2 antibody or its antigen-binding fragment according to any one of claims 1 to 14, or the pharmaceutical composition according to claim 21, wherein the pharmaceutical product is used to treat or prevent a TROP-2-mediated disease or condition.

24. Use in the preparation of a kit, wherein the kit is for the detection, diagnosis, or prognosis of a TROP-2 mediated disease or condition, using an anti-TROP-2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, or a detection or diagnostic reagent according to claim 22.

25. The use according to claim 23 or 24, wherein the TROP-2-mediated disease or condition is cancer.

26. The TROP-2-mediated disease or condition is a cancer that expresses TROP-2, The use according to claim 25, wherein the cancer is selected from the group consisting of 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.

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