Anti human trop-2 antibody and use for the same

Abstract

To provide a method for preparing an antibody that binds to a human tumor-associated calcium signal sensor 2 (Trop-2) protein, the antibody, and a use thereof.SOLUTION: A method for preparing an anti Trop-2 monoclonal antibody includes: (1) a step of preparing a hybridoma cell by immunizing an animal by using a recombinant Trop-2 protein as an immunogen; (2) a step of screening a positive hybridoma cell which secretes an anti Trop-2 monoclonal antibody by using a recombinant Trop-2 protein as a coating antigen; and (3) a step of re-screening the positive hybridoma cell obtained in the step (2) by using a positive cell regarding Trop-2 on a cell membrane surface, wherein the anti Trop-2 monoclonal antibody specifically recognizes a natural epitope of a Trop-2 extracellular domain and binds to the epitope.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims the benefit of priority from Chinese Patent Application No. 201910962965.1, filed on October 11, 2019, the entire contents of which are incorporated herein by reference.

[0002] Technical Field The present invention relates to a novel anti-human Trop-2 antibody or a functional fragment thereof, which is in the field of biomedicine. The present invention also relates to uses of the antibody or functional fragment thereof. [Background technology]

[0003] Background of the Invention The human trophoblast cell surface antigen Trop-2, also known as tumor-associated calcium signaling complex (TACSTD2), epithelial glycoprotein-1 antigen (EGP-1), and gastrointestinal tumor-associated antigen 1 (GA733-1), is a cell surface glycoprotein encoded by the TACSTD2 gene. Trop-2 has a total length of 323 amino acids and consists of three extracellular domains, and has been confirmed to exist as a dimer.

[0004] Trop-2 is a transmembrane glycoprotein. Unlike other proto-oncogenes, Trop-2 is mutation-free, meaning there are no genetic changes that lead to its overexpression. Trop-2 stimulates cell proliferation via the ERK / MAPK and cyclin D1 pathways, thereby promoting tumor invasion, angiogenesis, tumor progression, drug resistance, and other mechanisms. Trop-2 expression has been found to be highly elevated in various tumors, particularly triple-negative breast cancer and non-small cell lung cancer, and has been associated with poor prognosis. In contrast, Trop-2 expression is extremely low in normal tissues, making it an ideal target for ADC drugs.

[0005] Antibody drugs currently under development targeting Trop-2 are primarily antibody-drug conjugates (ADCs). According to incomplete statistics, more than three are currently under clinical investigation. Small molecule drugs include irinotecan derivatives and tubulin inhibitors. Currently, the use of SN-38, a novel and less toxic metabolite of a topoisomerase inhibitor, is considered desirable. SN-38 has a different tumor inhibition mechanism from existing microtubule inhibitors and DNA alkylating agents, making it particularly suitable for tumors with high tumor heterogeneity and multidrug resistance, such as triple-negative breast cancer, pancreatic cancer, and gastric cancer. The ADC making the most rapid progress in clinical trials is IMMU-132, developed by Immunomedics, which is participating in a Phase III clinical trial (ASCENT-Study) for the treatment of recurrent and metastatic triple-negative breast cancer, a Phase II clinical trial for the treatment of triple-negative breast cancer alone or in combination with carboplatin (NCT02161679), a Phase II clinical trial for the treatment of urothelial carcinoma (NCT03547973), and a Phase I / II clinical trial for the treatment of solid tumors, including gastric cancer, cervical cancer, and small cell lung cancer (NCT01631552). Other antibody-drug conjugates using similar technology have been developed by Daiichi Sankyo Company Limited, Pfizer Inc., and other pharmaceutical companies with foundations in ADC development.

[0006] Currently, few anti-Trop-2 antibodies are being clinically investigated. Therefore, there is still a need in the art to discover new anti-Trop-2 antibodies that are particularly suitable for the development of ADCs. Initial studies of Trop-2 have shown promising potential for use as a tumor therapeutic target. However, because the structure and biological function of the epitope sequence of Trop-2 have not been elucidated, high-affinity monoclonal antibodies obtained in early in vitro screening often exhibit low tumor targeting activity and poor cytotoxin delivery capability in in vivo testing. This has directly led to the situation where anti-Trop-2 monoclonal antibody drugs have not yet completed clinical efficacy testing. Summary of the Invention

[0007] Based on the mechanisms of using Trop-2 as a target for tumor therapy in the prior art and the progress of clinical trials of anti-Trop-2 antibody drugs, the present inventors have found through in-depth research and analysis that the reason why anti-Trop-2 antibodies do not achieve the theoretically predicted effects in preclinical and clinical trials is not simply due to insufficient affinity for Trop-2, but also due to the fact that the best epitopes bound by antibodies are typically located on recombinant Trop-2 protein or fragments thereof, rather than on Trop-2 in its native conformation. Therefore, although high-affinity antibodies against Trop-2 can be screened and obtained in vitro, their effective affinity for targeting the native conformation of the Trop-2 extracellular domain in vivo remains insufficient.

[0008] On the other hand, using recombinant proteins as immunogens and coating antigens offers many advantages, including easy purification of the immunogen, a uniform structure of the exogenous antigen, and easy screening of monoclonal antibodies directed against the recombinant protein. However, the modifications (e.g., glycosylation), folding pattern, etc. of recombinantly expressed proteins are often affected by the recombinant expression system. Even when a mammalian expression system is employed, purified free recombinant Trop-2 or its extracellular domain differs from the Trop-2 extracellular domain bound to the cell membrane surface in its native conformation. This difference in the conformation or spatial structure of the protein results in differences in the specificity of antibodies generated by immunization and in the effective affinity of the antibodies for the native conformation of the Trop-2 extracellular domain in vivo.

[0009] On the other hand, using cells positive for Trop-2 on the cell membrane surface as the immunogen and screening antigen has the advantage that the Trop-2 extracellular domain is in its native conformation, and a monoclonal antibody with high affinity for the Trop-2 extracellular domain in vitro is predicted to have high affinity for the Trop-2 extracellular domain in its native conformation in vivo. However, cells positive for Trop-2 on the cell membrane surface are too complex, and using such cells as an immunogen can easily mask the immunogenicity of the Trop-2 extracellular domain. Furthermore, performing flow cytometry-based screening using cells positive for Trop-2 on the cell membrane surface as a screening antigen is costly and has low screening efficiency.

[0010] Based on previous research reports and considering the advantages and disadvantages of various technical routes for preparing monoclonal antibodies using recombinant Trop-2 protein and Trop-2-positive cells, the present invention proposes a new technical route: using recombinant Trop-2 protein as an immunogen to immunize animals and prepare hybridoma cells, thereby circumventing the shielding effect of Trop-2 when complex antigens are used as immunogens. Furthermore, using recombinant Trop-2 protein as a coating antigen and performing primary screening through ELISA screening, the screening is high-throughput. Cells positive for Trop-2 on the membrane surface are then used to rescreen hybridomas that test positive in the primary screening, ensuring that the resulting positive antibodies can bind to the native conformation of the Trop-2 extracellular domain.

[0011] The present invention also aims to solve the technical problem of how to improve the performance of anti-Trop-2 monoclonal antibodies in preclinical animal studies to match the actual in vivo test results in humans. To this end, the present disclosure proposes a technical route to obtain monoclonal antibodies that can specifically bind to the common structure between the human Trop-2 extracellular domain and the cynomolgus monkey Trop-2 extracellular domain through species cross-reactivity screening based on the common structure between the human Trop-2 extracellular domain and the cynomolgus monkey Trop-2 extracellular domain. To ensure high specificity of the anti-Trop-2 MAB, antibodies that specifically bind to the mouse Trop-2 extracellular domain are excluded.

[0012] In response to the above technical challenges, the present disclosure provides, through hybridoma screening and humanization techniques, an anti-Trop-2 antibody that has high affinity for human Trop-2, specific killing effect on cancer cells, and high internalization ability, making it particularly suitable for the development of ADC drugs.

[0013] Specifically, the present disclosure provides the following technical solutions:

[0014] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (1) preparing hybridoma cells by immunizing an animal with a recombinant Trop-2 protein as an immunogen; (2) screening for positive hybridoma cells secreting anti-Trop-2 monoclonal antibodies using recombinant Trop-2 protein as a coating antigen; (3) rescreening the positive hybridoma cells obtained in step (2) using cells that are positive for Trop-2 on the cell membrane surface. 1. A method for preparing an anti-Trop-2 monoclonal antibody, comprising: A method for anti-Trop-2 monoclonal antibodies to specifically recognize and bind to a native epitope in the Trop-2 extracellular domain to provide.

[0015] Preferably, in the method for preparing an anti-Trop-2 monoclonal antibody according to the present disclosure, the cells positive for Trop-2 on their cell membrane surface in step (3) are recombinant animal cells derived from the same species as the animal immunized in preparing the hybridoma cells in step (1).

[0016] More preferably, in the method for preparing an anti-Trop-2 monoclonal antibody according to the present disclosure, in step (1), hybridoma cells are prepared by immunizing a mouse, and in step (3), the cells positive for Trop-2 on the cell membrane surface are recombinant mouse cells expressing exogenous Trop-2 protein.

[0017] Preferably, in the method for preparing anti-Trop-2 monoclonal antibodies according to the present disclosure, in step (2), positive hybridoma cells secreting anti-Trop-2 monoclonal antibodies are screened using enzyme-linked immunosorbent assay (ELISA), and in step (3), rescreening is performed by flow cytometry (FACS) analysis to obtain hybridomas secreting antibodies that specifically recognize and bind to native epitopes of the Trop-2 extracellular domain.

[0018] Preferably, the method for preparing an anti-Trop-2 monoclonal antibody according to the present disclosure further comprises step (4): identifying an antibody that specifically recognizes and binds to a native epitope of the Trop-2 extracellular domain, wherein the identifying step includes affinity identification and specificity identification.

[0019] Preferably, in the method for preparing an anti-Trop-2 monoclonal antibody according to the present disclosure, in step (4), a monoclonal antibody is selected that has specific binding ability to human Trop-2 and cynomolgus monkey Trop-2, but not to mouse Trop-2.

[0020] In another aspect, the present disclosure provides an antibody or fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) comprise a combination of CDRs (HCDR1, HCDR2, HCDR3; and LCDR1, LCDR2, LCDR3) selected from the group consisting of: TIFF2025163135000001.tif247161

[0021] The antibody or fragment thereof binds to human Trop-2.

[0022] Preferably, the heavy chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 17, or an amino acid sequence having at least 75% identity to said amino acid sequence, and / or the light chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 18 to 36, or an amino acid sequence having at least 75% identity to said amino acid sequence.

[0023] According to certain embodiments of the present disclosure, the heavy chain variable region and the light chain variable region comprised by an antibody or fragment thereof of the present disclosure comprise one of the following:

[0024] (1) an amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 1; and an amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 18; (2) an amino acid sequence set forth in SEQ ID NO:2, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:2; and an amino acid sequence set forth in SEQ ID NO:19, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:19; (3) an amino acid sequence set forth in SEQ ID NO:3, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:3; and an amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:20; (4) an amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:4; and an amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:20; (5) an amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 3; and an amino acid sequence set forth in SEQ ID NO: 21, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 21; (6) an amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 4; and an amino acid sequence set forth in SEQ ID NO: 21, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 21; (7) the amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 3; and the amino acid sequence set forth in SEQ ID NO: 22, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 22; (8) an amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 3; and an amino acid sequence set forth in SEQ ID NO: 23, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 23; (9) the amino acid sequence set forth in SEQ ID NO: 5, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 5; and the amino acid sequence set forth in SEQ ID NO: 24, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 24; (10) The amino acid sequence set forth in SEQ ID NO: 6, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 6; and the amino acid sequence set forth in SEQ ID NO: 25, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 25; (11) The amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 7; and the amino acid sequence set forth in SEQ ID NO: 26, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 26; (12) The amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 7; and the amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 27; (13) The amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 8; and the amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 27; (14) The amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 9; and the amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 27; (15) The amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 10; and the amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 27; (16) The amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 7; and the amino acid sequence set forth in SEQ ID NO: 28, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 28; (17) The amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 8; and the amino acid sequence set forth in SEQ ID NO: 28, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 28; (18) The amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 9; and the amino acid sequence set forth in SEQ ID NO: 28, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 28; (19) The amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 10; and the amino acid sequence set forth in SEQ ID NO: 28, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 28; (20) The amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 7; and the amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 29; (21) The amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 8; and the amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 29; (22) The amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 9; and the amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 29; (23) The amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 10; and the amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 29; (24) The amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 11; and the amino acid sequence set forth in SEQ ID NO: 30, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 30; (25) The amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 12; and the amino acid sequence set forth in SEQ ID NO: 31, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 31; (26) The amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 13; and the amino acid sequence set forth in SEQ ID NO: 32, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 32; (27) The amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 16; and the amino acid sequence set forth in SEQ ID NO: 32, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 32; (28) The amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 14; and the amino acid sequence set forth in SEQ ID NO: 33, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 33; (29) The amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 16; and the amino acid sequence set forth in SEQ ID NO: 33, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 33; (30) The amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 14; and the amino acid sequence set forth in SEQ ID NO: 34, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 34; (31) The amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 14; and the amino acid sequence set forth in SEQ ID NO: 35, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 35; (32) The amino acid sequence set forth in SEQ ID NO: 15, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 15; and the amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 36; (33) The amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 14; and the amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 36; and (34) An amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 16; and an amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 36.

[0025] Typically, the antibody or fragment thereof is in any form, such as a monoclonal antibody, a single chain antibody, a diabody, a single domain antibody, a nanobody, a fully or partially humanized antibody, or a chimeric antibody, or the antibody or fragment thereof is a half-antibody, or an antigen-binding fragment of a half antibody, such as a single-chain variable fragment (scFv), a bivalent single-chain variable fragment (BsFv), a disulfide-stabilized Fv fragment (dsFv), a (disulfide-stabilized Fv fragment)2 (dsFv)2, an antigen-binding fragment (Fab), a Fab' fragment, a F(ab')2 fragment, or a variable fragment (Fv), and the antibody or fragment thereof may be of murine, rat, human, or any other origin.

[0026] Preferably, the antibody or fragment thereof further comprises a human or mouse constant region, preferably a human or mouse light chain constant region (CL) and / or heavy chain constant region (CH), and more preferably the antibody or fragment thereof comprises a heavy chain constant region selected from the group consisting of IgG, IgA, IgM, IgD and IgE, and / or a kappa-type or lambda-type light chain constant region.

[0027] According to certain aspects of the present disclosure, the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody, and more preferably, the heavy chain constant region of the monoclonal antibody is of the IgG1 or IgG4 subtype and the light chain constant region of the monoclonal antibody is of the κ type.

[0028] According to certain embodiments of the present disclosure, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO:37, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth, and preferably, the light chain constant region of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO:38, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth.

[0029] In the present disclosure, at least 75% identity as defined above refers to any percent identity of 75% or greater, such as at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or even 99% identity.

[0030] Based on the antibodies or fragments thereof of the present disclosure, in a further aspect, the present disclosure provides nucleic acid molecules comprising nucleotide sequences encoding the heavy chain CDRs, light chain CDRs, heavy chain variable regions, light chain variable regions, heavy chains, or light chains contained in the antibodies or fragments thereof according to the present disclosure.

[0031] In yet another aspect, the present disclosure provides a vector comprising a nucleic acid molecule of the present disclosure. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, etc.

[0032] The vectors or nucleic acid molecules of the present disclosure may be used to transform or transfect host cells, or may be introduced into host cells by any method, such as for storage or expression of antibodies.

[0033] Thus, in a further aspect, the present disclosure provides a host cell comprising, or transformed or transfected with, a nucleic acid molecule and / or vector according to the present disclosure. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial cell or an insect cell, a fungal cell, a plant cell, or an animal cell.

[0034] According to the disclosure of this aspect, the antibodies or fragments thereof, nucleic acid molecules, vectors, and / or host cells provided by the present disclosure can be obtained using any conventional techniques known in the art. The antibodies or fragments thereof, nucleic acid molecules, vectors, and / or host cells provided by the present disclosure can be contained in pharmaceutical compositions, more specifically pharmaceutical preparations, and used for various purposes as needed.

[0035] Thus, in yet a further aspect, the present disclosure also provides a pharmaceutical composition comprising an antibody or fragment thereof according to the present disclosure, a nucleic acid molecule, a vector and / or a host cell, and optionally a pharmaceutically acceptable excipient.

[0036] The antibody or fragment thereof of the present disclosure can be used in combination with other antibody-based drugs that can induce phagocytosis by macrophages. Preferably, the antibody-based drug promotes phagocytosis of cells by macrophages through binding to proteins expressed on the surface of cells. Therefore, the pharmaceutical composition provided by the present disclosure can include an additional antibody-based drug, preferably an antibody against a macrophage-associated immune checkpoint. According to a specific embodiment of the present disclosure, the antibody is an anti-CD47 antibody.

[0037] The present disclosure also provides related uses of the above subject matter with antibodies that bind to human Trop-2 or any portion thereof.

[0038] In particular, in a further aspect, the present disclosure provides use of the antibody or fragment thereof, nucleic acid molecule, vector, host cell and / or pharmaceutical composition in the manufacture of a medicament for treating a cancer that overexpresses Trop-2, preferably gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous cell lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial cancer, triple-negative breast cancer or cervical cancer.

[0039] In this regard, use encompasses the use of an antibody of the present disclosure or a fragment thereof in combination with other antibody-based drugs as described above in the manufacture of a medicament.

[0040] The antibodies or fragments thereof provided by the present disclosure may also be fused or conjugated to other moieties, for example, the present disclosure provides fusion proteins or conjugates comprising an antibody or fragment thereof according to the present disclosure.

[0041] With respect to fusion proteins, the fusion protein may include any other moiety, such as an amino acid, polypeptide, or protein, that modifies an antibody of the present disclosure or a fragment thereof.

[0042] With respect to conjugates, the conjugate may comprise an antibody of the present disclosure or a fragment thereof and a drug conjugated thereto, where the drug is, for example, a cytotoxic agent.

[0043] Preferably, the conjugate is an antibody drug conjugate (ADC) represented by the formula: (antibody or fragment thereof according to the present disclosure)-(linker)-(cytotoxic agent), and preferably, the cytotoxic agent is a tubulin inhibitor (e.g., paclitaxel, docetaxel, etc.) or a DNA replication inhibitor (e.g., irinotecan or its active metabolite SN-38, etc.).

[0044] According to certain embodiments of the present disclosure, the conjugate is an "anti-TROP-2 antibody-linker-SN-38 antibody drug conjugate."

[0045] The present disclosure also provides use of the antibody or fragment thereof, nucleic acid molecule, vector, host cell and / or pharmaceutical composition in the manufacture of an antibody-drug conjugate (ADC) for treating a cancer that overexpresses Trop-2, preferably gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous cell lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial cancer, triple-negative breast cancer or cervical cancer.

[0046] Furthermore, the present disclosure provides a method for preventing and / or treating a disease, comprising administering to a subject in need thereof an antibody or fragment thereof, nucleic acid molecule, vector, host cell, pharmaceutical composition, fusion protein, or conjugate according to the present disclosure, and optionally, other drugs or means. The term "any other drugs or means" refers to other drugs or means that may be administered in combination with the antibody or fragment thereof, nucleic acid molecule, vector, host cell, pharmaceutical composition, fusion protein, or conjugate of the present disclosure, such as small molecule drugs, targeted drugs, recombinant protein drugs, e.g., antibodies, vaccines, ADCs, oncolytic viruses, gene therapy drugs, or nucleic acid therapy drugs, and radiation therapy. The co-administration of the two may be in any manner, including simultaneously, sequentially, or at intervals.

[0047] Preferably, the disease is a cancer with high Trop-2 expression, and more preferably, the cancer with high Trop-2 expression is gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous cell lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial cancer, triple-negative breast cancer, or cervical cancer. The subject is a mammal, and preferably, the subject is human.

[0048] Unless otherwise indicated, the term "immunoglobulin sequence" is used as a general term to encompass a full-size antibody, its individual chains, and any portion thereof, domain or fragment thereof (including, but not limited to, an antigen-binding domain or fragment, e.g., a VHH domain or a VH / VL domain, respectively).

[0049] The term "antibody" is understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., "intact antibody molecules") as well as antigen-binding fragments thereof. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like encompass any naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" of an antibody or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to Trop-2. Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing CDRs, or isolated CDRs. Antigen-binding fragments of antibodies can be derived from intact antibody molecules using, for example, any suitable standard techniques, such as proteolytic or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding the variable and (optionally) constant domains of the antibody. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains in a suitable configuration, or to introduce codons, to generate cysteine ​​residues, to modify, add, or delete amino acids.

[0050] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed by the term "antigen-binding fragment" as used herein.

[0051] Antigen-binding fragments of antibodies typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in-frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be positioned relative to each other in any suitable configuration. For example, the variable region may be dimeric, including a VH-VH dimer, a VH-VL dimer, or a VL-VL dimer. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0052] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody according to the present disclosure include: (i) VH-CH1, (ii) VH-CH2, (iii) VH-CH3, (iv) VH-CH1-CH2, (v) VH-CH1-CH2-CH3, (vi) VH-CH2-CH3, (vii) VH-CL, (viii) VL-CH1, (ix) VL-CH2, (x) VL-CH3, (xi) VL-CH1-CH2, (xii) VL-CH1-CH2-CH3, (xiii) VL-CH2-CH3, and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations described above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, providing a flexible or semi-flexible linkage between adjacent variable and / or constant domains within a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present disclosure may comprise homodimers or heterodimers (or other multimers) of any of the above variable and constant domain configurations non-covalently associated (e.g., via disulfide bonds) with each other and / or with one or more monomeric VH or VL domains.

[0053] As with intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, each capable of specifically binding to a separate antigen or to different epitopes on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of antigen-binding fragments of antibodies according to the present disclosure using routine techniques available in the art.

[0054] The term "chimeric antibody" refers to an antibody in which (a) the constant region or a portion thereof has been altered, substituted, or exchanged so that the antigen-binding site (variable region, CDR, or a portion thereof) is linked to a constant region of a different or altered class, effector function, and / or species, or (b) the variable region or a portion thereof has been altered, substituted, or exchanged with a variable region having a different or altered antigen specificity (e.g., CDR and framework regions from a different species). Chimeric antibodies can include recombinant antibodies containing variable region fragments, such as two Fab or Fv regions or scFv. As noted above, chimeric antibodies can also contain an Fc region derived from a source different from the combined Fv region. In some cases, chimeric antibodies contain a chimeric region within the Fv region. An example of such a chimeric antibody is a humanized antibody in which the Fv and CDRs are derived from different sources.

[0055] The term "humanized antibody" refers to an antibody in which the antigen binding loops, i.e., CDRs, obtained from the VH and VL regions of a non-human antibody are grafted onto a human framework sequence.Humanization, i.e., the replacement of non-human CDR sequences with the corresponding sequences of a human antibody, can be carried out according to the methods described in, for example, U.S. Patent No. 5,545,806, U.S. Patent No. 5,569,825, U.S. Patent No. 5,633,425, U.S. Patent No. 5,661,016, Riechmann et al., Nature 332:323-327 (1988); Marks et al., Bio / Technology 10:779-783 (1992); Morrison, Nature 368:812-13 (1994); and Fishwild et al., Nature Biotechnology 14:845-51 (1996). As disclosed in US Pat. No. 6,673,986, transgenic mice, or other organisms, such as other mammals, may be used to express humanized or human antibodies.

[0056] As used herein, the term "percent (%) identity" refers to the percentage of amino acid residues (or nucleic acid bases) of a candidate sequence, e.g., an isolated anti-Trop-2 antibody of the present disclosure, that are identical to the amino acid residues (or nucleic acid bases) of a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Alignment to determine percent identity can be achieved using BLAST 2.0 software with standard settings. Alignment can be performed to achieve maximum alignment over the entire length of the sequences being compared. In some embodiments, the percent amino acid (or nucleic acid) sequence identity of a given candidate sequence to, with, or relative to a given reference sequence (which may alternatively be expressed as a given candidate sequence having or containing a particular percent amino acid (or nucleic acid) sequence identity to, with, or relative to a given reference sequence) is calculated as follows: 100 x (A / B ratio) where A is the number of amino acid residues (or nucleobases) scored as identical in the alignment of the candidate sequence with the reference sequence, and B is the total number of amino acid residues (or nucleobases) in the reference sequence. In some embodiments where the length of the candidate sequence is not equal to the length of the reference sequence, the percent amino acid (or nucleic acid) sequence identity of the candidate sequence to the reference sequence is not equal to the percent amino acid (or nucleic acid) sequence identity of the reference sequence to the candidate sequence.

[0057] In certain embodiments, a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits 50% to 100% identity over the entire length of the candidate sequence or over a selected portion of consecutive amino acid residues (or nucleic acid bases) of the candidate sequence. The length of the candidate sequence aligned for comparison is at least 30%, e.g., at least 40%, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the length of the reference sequence. If a position in the candidate sequence is occupied by the same amino acid residue (or nucleic acid base) as the corresponding position in the reference sequence, the molecules are identical at that position.

[0058] As used herein, the terms "antigen," "immunogen," "antibody target," "target analyte," etc. refer to molecules, compounds, or complexes that can be recognized by antibodies, i.e., that can be specifically bound by antibodies. These terms can refer to any molecule that can be specifically recognized by antibodies, such as polypeptides, polynucleotides, carbohydrates, lipids, chemical moieties, or combinations thereof (e.g., phosphorylated polypeptides or glycosylated polypeptides, etc.). Those skilled in the art will understand that these terms do not indicate that a molecule is immunogenic in all circumstances, but simply indicate that it can be targeted by antibodies.

[0059] As used herein, the term "isolated" refers to a state obtained from a natural state by artificial means.If a certain "isolated" substance or component exists in nature, it is possible because its natural environment is changed, or the substance is isolated from the natural environment, or both.For example, a certain non-isolated polynucleotide or non-isolated polypeptide naturally exists in the body of a certain living animal, and the same highly purified polynucleotide or polypeptide isolated from such a natural state is called an isolated polynucleotide or polypeptide.The term "isolated" does not exclude artificial or synthetic substances mixed therewith, or other non-pure substances that do not affect the activity of the isolated substance.

[0060] The term "host cell" refers to a cell into which a vector can be introduced, including, but not limited to, prokaryotic cells such as Escherichia coli cells, fungal cells such as yeast cells, insect cells such as Drosophila melanogaster (S2) cells or Spodoptera frugiperda (Sf9) cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

[0061] The term "KD" refers to the equilibrium dissociation constant (KD) of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the closer the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Generally, antibodies have a dissociation constant of about 10, as determined, for example, by surface plasmon resonance (SPR) in a BIACORE instrument. -5 Less than m, e.g., about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M or about 10 -10 It binds to the antigen with an equilibrium dissociation constant of less than or equal to M. For example, the affinity of an antibody to a cell is detected using the KINEXA method with a KINEXA 400 instrument.

[0062] The term "specific binding" refers to the ability of an antibody to react with one or more antigenic determinants of an antigen but not with other polypeptides, or with very low affinity (Kd>10 -6) to bind to other polypeptides. Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibodies), single chain, Fab, Fab' and F(ab')2 fragments, Fv, scFv and Fab expression libraries. A monoclonal antibody (mAb) is an antibody obtained from a monoclonal cell line, which may be, but is not limited to, a eukaryotic cell line, a prokaryotic cell line, or a phage clone cell line. Monoclonal antibodies or antigen-binding fragments thereof may be obtained, for example, by hybridoma technology, recombinant technology, phage display technology, and synthetic techniques such as CDR grafting, or recombinantly using other known techniques.

[0063] The term "Trop-2" refers to TROP2, a cell surface glycoprotein that belongs to the TACSTD family and is encoded and expressed by the TACSTD2 gene. It is also known as tumor-associated calcium signaling substrate 2 (TACSTD2), epidermal glycoprotein 1 (EGP-1), gastrointestinal tumor-associated antigen (GA733-1), and surface marker 1 (M1S1). Trop-2 is an oncogene that is overexpressed in various malignant tumors and is associated with the development, invasion, and metastasis of malignant tumors.

[0064] The Trop-2 gene is located on the short arm of chromosome 1, specifically at 1p32.1. The gene is 9072 bp long, contains no introns, and has only one exon. The similarity of mouse Trop-2 to the human homologous gene sequence was 87.4%. The primary structure of Trop-2 protein is a 36-kD polypeptide consisting of 323 amino acids, a single transmembrane surface glycoprotein. Trop-2 consists of a hydrophobic precursor peptide (AA1-26), an extracellular domain (AA27-274), one transmembrane domain (AA275-297), and one cytoplasmic tail (AA298-323). The N-terminus of Trop-2 protein is the extracellular domain (ECD), which is connected to a short intracellular tail (IC) by a unidirectional transmembrane helix (TM), thereby anchoring it to the membrane. The cytoplasmic tail of Trop-22 contains a highly conserved phosphatidylinositol 4,5-bisphosphate (PIP2)-binding sequence, suggesting that PIP2 plays an important role in Trop-2 signaling. In addition to the PIP2-binding motif, the cytoplasmic tail of Trop-22 also contains conserved tyrosine and serine phosphorylation sites. Mutation of the serine residue at position 303 abolished the ability of Trop-2 to stimulate tumor growth. Phosphorylation of this residue is the role of protein kinase C (PKC).

[0065] The term "antibody-drug conjugate (ADC)" refers to a conjugate in which a biologically active small molecule drug is linked via a chemical linker to a monoclonal antibody, which acts as a carrier for targeted transport of the small molecule drug to target cells.

[0066] The antibody molecules in ADC drugs are usually humanized monoclonal antibodies, and their fragment crystallizable (Fc) regions are modified to reduce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). First, because antibody molecules are biological macromolecules, they generally suffer from the toxicity risks associated with biological macromolecules, such as immunogenicity and immunotoxicity, and in the case of monoclonal antibodies, ADCC, CDC, and renal basement membrane immune complex deposition. Second, the most important role of antibody molecules in ADC drugs is targeting, i.e., targeted delivery of small molecule compounds to the antigen-antibody binding site. If the antibody has low selectivity or if the antigen is present in normal tissues, the cytotoxic drug will be delivered to normal cells, resulting in targeted toxicity. Third, in addition to targeted toxicity, the excretion of small molecules in the circulation can result in some degree of off-target toxicity. If the Fc in an antibody molecule has the ability to bind to Fc receptors on immune cells, such as FcγR / FcRN, it can readily bind to immune cells, thereby causing their death. Finally, ADC drugs as exogenous biopolymers can also be taken up into the circulation and enter cells via pinocytosis, resulting in cell death.

[0067] Linkers commonly used in ADC drugs mainly include hydrazone bonds, disulfide bonds, and peptide bonds. Hydrazone bonds are relatively unstable bonds that undergo hydrolysis under acidic conditions. Hydrazone linkers are used in Mylotarg®, and researchers believe that the hydrazone linker is a significant cause of the failure of Mylotarg®. Disulfide bonds are hydrolyzed by high concentrations of glutathione within cells and are not easily degraded outside the cell. Peptide bonds are the tightest bond and are only cleaved by lysosomal proteases. Linker stability directly affects the unintended dissociation of cytotoxic drugs, and cleavage leads to in vivo exposure of small molecule cytotoxic drugs, i.e., off-target toxicity.

[0068] The cytotoxic drugs commonly used in ADC drugs are conventionally used chemotherapeutic agents, which determine the primary toxic profile of ADC drugs. Because of their widespread clinical use, these drugs generally have distinct toxicity profiles. The risk of toxicity can be significantly determined depending on the type of drug, such as tubulin polymerization inhibitors or DNA damaging agents / DNA replication inhibitors. Among these drugs, tubulin inhibitors include their dolastatin and auristatin derivatives (MMAE, MMAF, and MMAD), maytansine and maytansinoid derivatives (DM1, DM2, DM3, and DM4), paclitaxel and paclitaxel derivatives (docetaxel), docetaxel, vincristine, etc., while DNA damaging agents / DNA replication inhibitors include irinotecan or its active metabolite SN-38, etc.

[0069] "Optional," "optionally," "any," or "any one" means that the following event or circumstance may, but does not necessarily, occur, and the description includes cases where the event or circumstance occurs or does not occur. For example, "optionally comprising one antibody heavy chain variable region" means that an antibody heavy chain variable region having a particular sequence may, but does not necessarily, be present.

[0070] The term "pharmaceutical composition" refers to a mixture containing one or more compounds of the present disclosure, or physiologically / pharmaceutically acceptable salts or prodrugs thereof, together with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. Pharmaceutical compositions are used to facilitate administration to an organism and promote absorption of the active ingredient to exert its biological activity. Therapeutic compositions should generally be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high antibody concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antibody or antibody portion) in the required amount in an appropriate solvent containing one or a combination of the above ingredients, followed by filtration for sterilization, if necessary.

[0071] The methods, compositions, and combination therapies of the present disclosure can be combined with other active agents or therapeutic methods. The methods include administering to a subject an amount of an anti-Trop-2 antibody molecule of the present disclosure effective for treating or preventing a disease (e.g., cancer), optionally in combination with one or more inhibitors selected from PD-1, PD-L1, PD-L2, LAG-3, CTLA-4, or Tim-3 antibodies (immunotherapy) or other tumor-treating antibodies, such as Her-2, EGFR, VEGF, or VEGFR antibodies, as well as ADCs (antibody-drug conjugates such as T-DM1), bispecific antibodies, chemotherapeutic agents, and the like. The methods may also include the administration of additional active agents, or may be administered in amounts or doses that are higher, lower, or equal to the amounts or doses of each active agent used alone (e.g., as monotherapy). The dosage or dose of the or all of the additional active agents is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dose of each active agent used alone (e.g., as monotherapy).

[0072] The present disclosure provides novel antibodies against human Trop-2 with good biological activity: the antibodies provided by the present disclosure (including chimeric and humanized antibodies) can effectively bind to both recombinant Trop-2 protein or Trop-2 antigen expressed on the surface of cells, similar to the control antibody sacituzumab. Meanwhile, the antibodies provided by the present disclosure have high affinity for human Trop-2: the humanized antibodies of the present disclosure exhibited even higher specific binding ability to human Trop-2 protein compared to the control antibody sacituzumab, and higher affinity than sacituzumab. Therefore, the antibodies of the present disclosure have good therapeutic efficacy.

[0073] Experiments have demonstrated that the antibodies of the present disclosure have good internalization capabilities: the humanized antibodies have similar internalization rates to the control antibody sacituzumab, and when formulated into ADCs, their internalization capabilities are significantly enhanced. Therefore, the antibodies of the present disclosure have potential for developing ADC drugs. The anti-Trop-2 antibodies of the present disclosure may also have synergistic effects with other antibodies. For example, the antibodies of the present disclosure can be used in combination with anti-CD47 antibodies to further promote phagocytosis of tumor cells by macrophages.

[0074] Furthermore, the antibodies of the present disclosure have been demonstrated to have good in vivo therapeutic effects. When ADCs were prepared using the antibodies of the present disclosure, the anti-Trop2 ADCs exhibited a dose-dependent inhibitory effect on tumor growth, and at high doses (10 mg / kg), the therapeutic effect of each ADC was comparable to that of the control antibody sacituzumab. No obvious toxic effects of the small molecule SN38 in the ADC were observed, and the body weight of animals in each experimental group steadily increased without any obvious difference from the control. [The present invention 1001] (1) preparing hybridoma cells by immunizing an animal with a recombinant Trop-2 protein as an immunogen; (2) screening for positive hybridoma cells secreting anti-Trop-2 monoclonal antibodies using recombinant Trop-2 protein as a coating antigen; (3) rescreening the positive hybridoma cells obtained in step (2) using cells that are positive for Trop-2 on the cell membrane surface. 1. A method for preparing an anti-Trop-2 monoclonal antibody, comprising: A method in which an anti-Trop-2 monoclonal antibody specifically recognizes and binds to a native epitope in the Trop-2 extracellular domain. [The present invention 1002] A method for preparing an anti-Trop-2 monoclonal antibody of the present invention, wherein the cells positive for Trop-2 on the cell membrane surface in step (3) are recombinant animal cells derived from the same species as the animal immunized in preparing the hybridoma cells in step (1). [The present invention 1003] A method for preparing an anti-Trop-2 monoclonal antibody of the present invention, wherein in step (1), hybridoma cells are prepared by immunizing a mouse, and in step (3), the cells positive for Trop-2 on the cell membrane surface are recombinant mouse cells expressing exogenous Trop-2 protein. [The present invention 1004] The method for preparing an anti-Trop-2 monoclonal antibody of the present invention 1001, wherein in step (2), positive hybridoma cells secreting anti-Trop-2 monoclonal antibodies are screened using enzyme-linked immunosorbent assay (ELISA); and in step (3), rescreening is performed by flow cytometry (FACS) analysis to obtain hybridomas secreting antibodies that specifically recognize and bind to native epitopes of the Trop-2 extracellular domain. [The present invention 1005] Process (4): Identifying antibodies that specifically recognize and bind to native epitopes of the Trop-2 extracellular domain. The method for preparing an anti-Trop-2 monoclonal antibody of the present invention further comprises: [The present invention 1006] A method for preparing an anti-Trop-2 monoclonal antibody of the present invention, wherein in step (4), a monoclonal antibody having specific binding ability to human Trop-2 and cynomolgus monkey Trop-2 but not to mouse Trop-2 is selected. [The present invention 1007] An anti-Trop-2 monoclonal antibody or a fragment thereof obtained by the method for preparing an anti-Trop-2 monoclonal antibody of any one of claims 1001 to 1006 of the present invention. [The present invention 1008] The monoclonal antibody is 1×10 -8 Less than M, 5 x 10 -9 Less than M, 1 x 10 -10 Less than M or 5 x 10 -10 1007. The anti-Trop-2 monoclonal antibody or fragment thereof of the present invention, which binds to the recombinant human Trop-2 extracellular domain with an affinity measured by a KD value of less than M. [The present invention 1009] An antibody or fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL) comprising a combination of CDRs (HCDR1, HCDR2, HCDR3; and LCDR1, LCDR2, LCDR3) selected from the group consisting of: TIFF2025163135000002.tif247161. [The present invention 1010] The antibody or fragment thereof of the present invention, wherein the heavy chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 17, or an amino acid sequence having at least 75% identity to said amino acid sequence set forth, and / or the light chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 18 to 36, or an amino acid sequence having at least 75% identity to said amino acid sequence set forth. [The present invention 1011] The antibody or fragment thereof of the present invention 1009 or 1010, wherein the heavy chain variable region and the light chain variable region comprised by said antibody or fragment thereof comprise one of the following: (1) an amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 1; and an amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 18; (2) an amino acid sequence set forth in SEQ ID NO:2, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:2; and an amino acid sequence set forth in SEQ ID NO:19, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:19; (3) an amino acid sequence set forth in SEQ ID NO:3, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:3; and an amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:20; (4) an amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:4; and an amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:20; (5) an amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 3; and an amino acid sequence set forth in SEQ ID NO: 21, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 21; (6) an amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 4; and an amino acid sequence set forth in SEQ ID NO: 21, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 21; (7) the amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 3; and the amino acid sequence set forth in SEQ ID NO: 22, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 22; (8) an amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 3; and an amino acid sequence set forth in SEQ ID NO: 23, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 23; (9) the amino acid sequence set forth in SEQ ID NO: 5, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 5; and the amino acid sequence set forth in SEQ ID NO: 24, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 24; (10) The amino acid sequence set forth in SEQ ID NO: 6, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 6; and the amino acid sequence set forth in SEQ ID NO: 25, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 25; (11) The amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 7; and the amino acid sequence set forth in SEQ ID NO: 26, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 26; (12) The amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 7; and the amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 27; (13) The amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 8; and the amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 27; (14) The amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 9; and the amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 27; (15) The amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 10; and the amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 27; (16) The amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 7; and the amino acid sequence set forth in SEQ ID NO: 28, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 28; (17) The amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 8; and the amino acid sequence set forth in SEQ ID NO: 28, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 28; (18) The amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 9; and the amino acid sequence set forth in SEQ ID NO: 28, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 28; (19) The amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 10; and the amino acid sequence set forth in SEQ ID NO: 28, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 28; (20) The amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 7; and the amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 29; (21) The amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 8; and the amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 29; (22) The amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 9; and the amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 29; (23) The amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 10; and the amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 29; (24) The amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 11; and the amino acid sequence set forth in SEQ ID NO: 30, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 30; (25) The amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 12; and the amino acid sequence set forth in SEQ ID NO: 31, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 31; (26) The amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 13; and the amino acid sequence set forth in SEQ ID NO: 32, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 32; (27) The amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 16; and the amino acid sequence set forth in SEQ ID NO: 32, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 32; (28) The amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 14; and the amino acid sequence set forth in SEQ ID NO: 33, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 33; (29) The amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 16; and the amino acid sequence set forth in SEQ ID NO: 33, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 33; (30) The amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 14; and the amino acid sequence set forth in SEQ ID NO: 34, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 34; (31) The amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 14; and the amino acid sequence set forth in SEQ ID NO: 35, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 35; (32) The amino acid sequence set forth in SEQ ID NO: 15, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 15; and the amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 36; (33) The amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 14; and the amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 36; and (34) An amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 16; and an amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 36. [The present invention 1012] any form, such as a monoclonal antibody, a single chain antibody, a diabody, a single domain antibody, a nanobody, a fully or partially humanized antibody, or a chimeric antibody, or a half antibody or an antigen-binding fragment of a half antibody, such as an scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv; Preferably, it further comprises a human or mouse constant region, preferably a human or mouse light chain constant region (CL) and / or a heavy chain constant region (CH), More preferably, the antibody comprises a heavy chain constant region selected from the group consisting of IgG, IgA, IgM, IgD and IgE, and / or a kappa or lambda light chain constant region. Any one of the antibodies or fragments thereof according to the present invention 1001 to 1011. [The present invention 1013] the antibody is a monoclonal antibody, preferably a mouse monoclonal antibody, a chimeric monoclonal antibody or a humanized monoclonal antibody, and preferably the heavy chain constant region of the monoclonal antibody is of the IgG1 or IgG4 subtype, and the light chain constant region of the monoclonal antibody is of the κ type; Preferably, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 37, or an amino acid sequence having at least 75% identity to the amino acid sequence shown, and preferably, the light chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 38, or an amino acid sequence having at least 75% identity to the amino acid sequence shown. Any one of the antibodies 1001 to 1012 of the present invention or a fragment thereof. [The present invention 1014] A nucleic acid molecule comprising a nucleotide sequence encoding any one of the antibodies 1001 to 1013 of the present invention or a fragment thereof, or encoding the heavy chain CDR, light chain CDR, heavy chain variable region, light chain variable region, heavy chain or light chain contained in said antibody or fragment thereof. [The present invention 1015] A vector comprising a nucleic acid molecule of the present invention. [The present invention 1016] A host cell comprising a nucleic acid molecule of the invention 1014 and / or a vector of the invention 1015, or transformed or transfected with a nucleic acid molecule of the invention 1014 and / or a vector of the invention 1015. [The present invention 1017] A pharmaceutical composition comprising any one of the antibodies or fragments thereof of the present invention 1001 to 1013, the nucleic acid molecule of the present invention 1014, the vector of the present invention 1015, or the host cell of the present invention 1016, and optionally a pharmaceutically acceptable excipient. [The present invention 1018] the pharmaceutical composition comprises an additional antibody-based drug; Preferably, the antibody-based further drug is an antibody against a macrophage-associated immune checkpoint, more preferably an anti-CD47 antibody. The pharmaceutical composition of the present invention 1017. [The present invention 1019] Use of an antibody or fragment thereof according to any one of claims 1001 to 1013, a nucleic acid molecule according to claim 1014, a vector according to claim 1015, a host cell according to claim 1016, and / or a pharmaceutical composition according to claim 1017 or 1018 in the manufacture of a pharmaceutical, Preferably, the medicament is for treating a cancer with high Trop-2 expression, Preferably, the Trop-2 highly expressing cancer is gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous cell lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial cancer, triple-negative breast cancer or cervical cancer. use. [The present invention 1020] A kit comprising an antibody or fragment thereof of any of the present inventions 1001 to 1013, a nucleic acid molecule of the present invention 1014, a vector of the present invention 1015, a host cell of the present invention 1016, or a pharmaceutical composition of the present invention 1017 or 1018. [The present invention 1021] A fusion protein comprising any one of the antibodies of the present invention 1001 to 1013 or a fragment thereof. [The present invention 1022] A conjugate comprising any one of the antibodies or fragments thereof of the present inventions 1001 to 1013 and a drug conjugated thereto, wherein the drug is a cytotoxic agent. [The present invention 1023] The conjugate has the formula: (Antibody or fragment thereof of any of the present inventions 1001 to 1013)-(linker)-(cytotoxic agent) is an antibody drug conjugate (ADC) represented by Preferably, the cytotoxic agent is a tubulin inhibitor (e.g., paclitaxel, docetaxel, etc.) or a DNA replication inhibitor (e.g., irinotecan or its active metabolite SN-38, etc.). A conjugate of the present invention 1022. [The present invention 1024] Use of any one of the antibodies or fragments thereof according to 1001 to 1013 of the present invention, the nucleic acid molecule according to 1014 of the present invention, the vector according to 1015 of the present invention, or the host cell according to 1016 of the present invention in the manufacture of an antibody-drug conjugate (ADC), Preferably, the ADC is for treating a cancer with high Trop-2 expression, Preferably, the Trop-2 highly expressing cancer is gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous cell lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial cancer, triple-negative breast cancer or cervical cancer. use. [The present invention 1025] A method for preventing and / or treating a disease, comprising the step of administering to a subject in need thereof an antibody or fragment thereof of any of the present inventions 1001 to 1013, a nucleic acid molecule of the present invention 1014, a vector of the present invention 1015, a host cell of the present invention 1016, a pharmaceutical composition of the present invention 1017 or 1018, a fusion protein of the present invention 1021, or a conjugate of the present invention 1022 or 1023, and optionally other drugs or means, Preferably, the disease is a cancer with high Trop-2 expression, Preferably, the Trop-2 highly expressing cancer is gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous cell lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial cancer, triple-negative breast cancer or cervical cancer. The method. [Brief explanation of the drawings]

[0075] Aspects of the present invention are described in detail below with reference to the accompanying drawings.

[0076] [Figure 1] Figure 1 shows the results of screening for binding of positive hybridoma supernatants to Trop-2 on the surface of CHO cells. [Figure 2] Figure 1 shows the results of screening for binding of positive hybridoma supernatants to Trop-2 on the surface of CHO cells. [Figure 3] Figure 1 shows the results of ELISA detection of cross-reactivity of positive hybridoma supernatants with recombinant Trop-2 proteins of different species. [Figure 4A] ELISA results showing the binding activity of anti-human Trop-2 chimeric antibodies to recombinant Trop-2 protein: Panel 4A: ch3-11; Panel 4B: ch4-3; Panel 4C: ch23-12; Panel 4D: ch11-4; Panel 4E: ch17-1. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E] See legend to Figure 4A. [Figure 5A] Figure 5 shows the results of FACS detection of the binding activity of anti-human Trop-2 chimeric antibodies to recombinant Trop-2 protein on the cell surface: Panel 5A: ch3-11; Panel 5B: ch23-12; Panel 5C: ch11-4; Panel 5D: ch4-3; Panel 5E: ch17-1. [Figure 5B] See the legend to Figure 5A. [Figure 5C] See the legend to Figure 5A. [Figure 5D] See the legend to Figure 5A. [Figure 5E] See the legend to Figure 5A. [Figure 6A] ELISA detection of species-specific binding of anti-human Trop-2 antibodies to Trop-2: Panel 6A: h23-12; Panel 6B: h4-3; Panel 6C: sacituzumab. [Figure 6B]See the legend to Figure 6A. [Figure 6C] See the legend to Figure 6A. [Figure 7A] Figure 7 shows the results of an analysis of the affinity of anti-human Trop-2 antibodies to the recombinant human Trop-2 extracellular domain: Panel 7A: sacituzumab; Panel 7B: h23-12; Panel 7C: h4-3. [Figure 7B] See the legend to Figure 7A. [Figure 7C] See the legend to Figure 7A. [Figure 8] 1 shows the observed internalization of anti-Trop-2 humanized antibodies after binding to Trop-2 on the surface of N87 cells. [Figure 9] Figure 9 shows the drug concentration versus time curves after a single dose of anti-Trop-2 humanized antibody in nude mice (detected by Trop-2). Panel 9A: h23-12; Panel 9B: h4-3. [Figure 10] 1 shows the inhibition rate of anti-Trop-2 ADC on cell proliferation. [Figure 11A] 1 shows the curves of body weight change in tumor-bearing BALB / c nu mice in the N87 gastric cancer mouse model: Panel 11A: after administration of ch4-3-SN38; Panel 11B: after administration of h23-12-SN38; Panel 11C: after administration of an isotype control antibody; Panel 11D: after administration of high doses of ch4-3-SN38 and h23-12-SN38. [Figure 11B] See the legend to Figure 11A. [Figure 11C] See the legend to Figure 11A. [Figure 11D] See the legend to Figure 11A. [Figure 12A] Curves showing tumor volume changes in tumor-bearing BALB / c nu mice in the N87 gastric cancer mouse model: Panel 12A: after administration of ch4-3-SN38; Panel 12B: after administration of h23-12-SN38; Panel 12C: after administration of an isotype control antibody; Panel 12D: after administration of high doses of ch4-3-SN38 and h23-12-SN38. [Figure 12B]See the legend to Figure 12A. [Figure 12C] See the legend to Figure 12A. [Figure 12D] See the legend to Figure 12A. [Figure 13] 1 shows the curve of changes in mouse body weight in a SKOV3 subcutaneous xenograft mouse model following simultaneous administration of anti-Trop-2 antibody and anti-CD47 antibody. [Figure 14] 1 shows the curve of changes in tumor volume in mice in a SKOV3 subcutaneous xenograft mouse model following simultaneous administration of anti-Trop-2 antibody and anti-CD47 antibody. [Figure 15] 1 shows the curves of tumor volume changes in tumor-bearing BALB / c nu mice in a subcutaneous xenograft mouse model of NCI-N87 gastric cancer. [Figure 16] 1 shows the curves of body weight changes in tumor-bearing BALB / c nu mice in a subcutaneous xenograft mouse model of NCI-N87 gastric cancer. DETAILED DESCRIPTION OF THE INVENTION

[0077] Detailed Description of the Preferred Embodiments The present invention will now be described with reference to specific examples, which will be understood by those skilled in the art as being merely illustrative of the present invention and in no way limiting the scope of the present invention.

[0078] All experimental procedures in the following examples are conventional unless otherwise specified. All materials and reagents used in the following examples are commercially available unless otherwise specified. [Example]

[0079] Example 1 Preparation of hybridoma cells secreting anti-human Trop-2 antibodies Immunization: Balb / c mice were immunized with recombinant human Trop-2 protein (accession number: NP_002344.2, AA 1-274), and serum titers were detected via ELISA in 96-well ELISA plates coated with recombinant human Trop-2-his protein (accession number: NP_002344.2, AA 1-274). Mice with serum titers that met the requirements for cell fusion were used for the next step, cell fusion.

[0080] Cell fusion and hybridoma preparation: Mice with the required titer were selected and subjected to rush immunization. Three days later, spleens were aseptically harvested from the mice. B lymphocyte suspensions were prepared and mixed with SP2 / 0 myeloma cells at a 4:1 ratio, and the cells were fused in the presence of PEG. The fused cells were resuspended in HAT medium, seeded into 96-well cell culture plates, and cultured in an incubator at 37°C with 5% CO2.

[0081] Example 2 Screening of positive hybridoma cell lines secreting anti-human Trop-2 antibodies 1. Screening for positive hybridoma binding Ten to 14 days after fusion, ELISA plates were coated with recombinant human Trop-2-his protein (accession number: NP_002344.2, AA 1-274) (20 ng / ml) overnight at 4°C. After washing three times with PBS, the plates were blocked with 4% nonfat dry milk in PBS for 1 hour at room temperature. The plates were then washed three times with PBS, and culture supernatants of hybridoma clones were added to the plates and incubated for 1 hour at room temperature. The following controls were set up: (1) positive control (PC): serum from immunized mice (diluted 1:1000 in PBS); (2) negative control (NC): fusion wells without cell growth. The plates were washed three times with PBST (0.05% Tween-PBS) and twice with PBS. Then, HRP-goat anti-mouse IgG (Fcγ) was added to the plates and incubated for 0.5 hours at 37°C. The plate was then washed three times with PBST (0.05% Tween 20-PBS), and TMB substrate was added. The color was developed in the dark for 15-30 minutes. ELISA stop solution was then added to stop the reaction. The A450 values ​​were read using a microplate reader.

[0082] Clones were ranked from highest to lowest readings, and the top 95 clones with high readings were selected for double-checking by ELISA. A pool of 25 positive antibody-secreting cells was subcloned by limiting dilution. Ten days after plating, culture supernatants of monoclonal cells were selected for subsequent screening of positive clones by ELISA, as described above. The clones were re-ordered from highest to lowest read value, and the top 21 clones with high read values, namely m1-1, m3-11, m4-3, m5-5, m6-6, m7-13, m11-4, m12-2, m12-4, m13-2, m14-2, m15-3, m16-7, m17-1, m18-4, m19-5, m20-4, m21-1, m22-1, m23-12, and m24-3, were selected for the next step of screening for binding by FACS.

[0083] 2. Screening for binding of positive hybridomas to Trop-2 on the surface of CHO cells The Trop-2 gene reading frame was cloned by PCR from a vector containing Trop-2 cDNA (catalog number HG10428-M, Beijing Yiqiao Shenzhou Science and Technology Co., Ltd.), and then cloned into a stable expression vector containing the glutamine synthetase (GS) gene by enzymatic digestion. Suspension-cultured CHO-K1 cells were electrotransfected (Nucleofector IIb, Lonza), transferred to CD CHO AGT™ medium (catalog number 12490-025, Gibco) containing 50 μM MSX (catalog number M5379, Sigma), and seeded into 96-well cell culture plates. After incubation at 37°C and 5% CO for 2-3 weeks, 22 wells containing cells were obtained through pre-screening by MSX pressure screening, and the cells were expanded in a 24-well cell culture plate. Finally, clone No. 1-T-21 (CHO / Trop-2 cells) was selected through flow cytometry (FACS) analysis. A scale-up culture of the clone was performed, and the cells were cryopreserved and used for FACS detection.

[0084] Based on the ELISA results, the supernatants of the 21 selected hybridoma clones were diluted 100-fold and incubated with a suspension of engineered CHO cells (CHO / Trop-2 cells) at 37°C for 30 minutes. The following controls were set up: (1) positive control (PC): sacituzumab, a version containing a mouse IgG constant region, 1 μg / ml; (2) negative control (NC): an unrelated mouse antibody, 1 μg / ml. The cells were washed three times with PBS, and a 1:200 dilution of goat anti-mouse IgG-FITC (catalog number F9006, Sigma) was added to the cells and incubated for 30 minutes. The cells were then washed three times with PBS, and the mean fluorescence intensity (MFI) of the cells was measured using a flow cytometer (model number B49007AD, SNAW31211, Beckman Coulter) to confirm whether the antibodies secreted by each hybridoma could bind to Trop-2 on the surface of CHO cells. The results are shown in Figure 1.

[0085] Based on Figure 1 and cell conditions, clones m1-1, m3-11, m4-3, m6-6, m7-13, m11-4, m12-2, m12-4, m13-2, m14-2, m16-7, m17-1, m19-5, m21-1, and m23-12 were selected, and their hybridoma supernatants were purified through a ProA affinity chromatography column. The binding of the purified mouse antibodies was confirmed again. The antibodies were diluted to 13 nM and 0.66 nM, respectively, and then incubated with a suspension of CHO cells recombinantly expressing human Trop-2 (CHO / Trop-2 cells) at 37°C for 30 minutes. The following controls were set up: (1) positive control (PC): sacituzumab, a version containing a mouse IgG constant region, 1 μg / ml; (2) negative control (NC): an unrelated mouse antibody, 1 μg / ml. The cells were washed three times with PBS, and a 1:200 dilution of goat anti-mouse IgG-FITC (catalog: F9006, Sigma) was added to the cells and then incubated for 30 minutes. The cells were then washed three times with PBS, and the mean fluorescence intensity (MFI) of the cells was measured using a flow cytometer (model number B49007AD, SNAW31211, BECKMAN COULTER) to confirm whether the antibodies secreted by each hybridoma could bind to Trop-2 on the surface of CHO cells. As shown in Figure 2, all of the antibodies derived from the supernatants of the 15 clones bound well to Trop-2 on the surface of CHO cells.

[0086] Clones m3-11, m4-3, m11-4, m17-1 and m23-12 were selected as candidate clones for further screening.

[0087] 3. Screening of positive hybridoma clones for cross-reactivity by ELISA Plates were coated overnight at 4°C with recombinant human Trop-2-his protein (accession number: NP_002344.2, AA 1-274), recombinant cynomolgus monkey Trop-2-his protein (accession number: UniProtKB-A0A2K5UE71, AA 1-272), and recombinant mouse Trop-2-his protein (catalog number: 50922-M08H, Beijing Yiqiao Shenzhou Science and Technology Co., Ltd.) at coating concentrations of 0.2 and 1 μg / ml, respectively. After washing three times with PBS, the plates were blocked with 5% BSA PBS at 37°C for 60 minutes and then washed three times with PBST. Fifteen purified mouse antibodies were diluted to 1 μg / ml in PBS. The following controls were set up: (1) positive control (PC): sacituzumab (WHO Drug Information (Vol. 31, No. 1, 2017), SEQ ID NO: 39 and SEQ ID NO: 40), a version with a mouse IgG constant region, 1 μg / ml; (2) negative control (NC): an antibody derived from an unrelated hybridoma, 1 μg / ml; (3) blank control: PBS. After incubation at 37°C for 60 minutes, the plate was washed four times with PBST. A 1:5000 dilution of HRP-goat anti-mouse IgG (Fcγ) (catalog: 115-035-071; Jackson ImmunoResearch) was added to the plate and incubated at 37°C for 30 minutes. The plate was then washed four times with PBST, and TMB substrate was added and developed at 37°C for 10 minutes. The reaction was then stopped by adding 2 M HCl. The absorbance was measured at 450 nm and 630 nm (as reference wavelengths), and the A450-630 nm values ​​for each well in the plate were recorded. Except for antibodies derived from clones m12-4, m17-1, m19-5, and m21-1, which showed cross-reactivity with mouse Trop-2, the other antibodies did not. All hybridoma antibodies were able to specifically bind to recombinant human Trop-2 and recombinant cynomolgus monkey Trop-2 (Figure 3).

[0088] Example 3 Sequencing of mouse anti-human Trop-2 antibodies After the growth culture of the anti-human Trop-2 antibody-secreting hybridomas m3-11, m4-3, m11-4, m17-1, and m23-12, the antibody subtypes were detected using the Mouse Monoclonal Antibody IgG Subclass Test Card (Catalog: A12403, VicNovo) and the Mouse Monoclonal Antibody Light / Heavy Chain Test Card (Catalog: A12401, VicNovo) according to the reagent protocol. The subtypes were identified as follows: the heavy chain of the antibody was IgG1 and the light chain was κ. The results provided the basis for gene cloning of the antibodies derived from m3-11, m4-3, m11-4, m17-1, and m23-12.

[0089] Total RNA was extracted from hybridoma cells m3-11, m4-3, m11-4, m17-1, and m23-12 using TRIzol (catalog number 15596026, Invitrogen) according to the manufacturer's instructions. Total RNA from hybridoma cells was reverse transcribed into cDNA using M-MuLV reverse transcriptase (catalog number M0253S, NEB). Degenerate primers (see Zhiwei DONG and Yan Wang, Antibody Engineering (2nd Edition), Peking University Medical Press, 2001, pages 313-314) and the Phusion kit (catalog number E0553L, NEB) were used to amplify the light chain variable region (IgVL) and heavy chain variable region (V) of the antibody. HThe sequence of the mAb was amplified. The PCR product was purified using a gel extraction kit (catalog: AP-GX-250, Axygen). The purified PCR product was ligated into a T vector according to the instructions of a T vector cloning kit (catalog: ZC205, Beijing Zoman Biotechnology Co., Ltd.). The resulting vector was then transformed into competent E. coli cells and amplified. The plasmid was then extracted for DNA sequencing to obtain the variable region sequences of the monoclonal antibody.

[0090] Sequencing results showed the following: The nucleotide sequence (DNA) of the heavy chain variable region of the mouse antibody derived from clone m3-11 is shown as SEQ ID NO: 41, and the amino acid sequence of the heavy chain variable region of the mouse antibody derived from clone m3-11 deduced from the nucleotide sequence is shown as SEQ ID NO: 1. The nucleotide sequence (DNA) of the light chain variable region of the mouse antibody derived from clone m3-11 is shown as SEQ ID NO: 42, and the amino acid sequence of the light chain variable region of the mouse antibody derived from clone m3-11 deduced from the nucleotide sequence is shown as SEQ ID NO: 18. TIFF2025163135000003.tif59162

[0091] The nucleotide sequence (DNA) of the heavy chain variable region of the mouse antibody derived from clone m4-3 is shown as SEQ ID NO: 43, and the amino acid sequence of the heavy chain variable region of the mouse antibody derived from clone m4-3 deduced from the nucleotide sequence is shown as SEQ ID NO: 2. The nucleotide sequence (DNA) of the light chain variable region of the mouse antibody derived from clone m4-3 is shown as SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the mouse antibody derived from clone m4-3 deduced from the nucleotide sequence is shown as SEQ ID NO: 19. TIFF2025163135000004.tif58162

[0092] The nucleotide sequence (DNA) of the heavy chain variable region of the mouse antibody derived from clone m11-4 is shown as SEQ ID NO: 47, and the amino acid sequence of the heavy chain variable region of the mouse antibody derived from clone m11-4 deduced from the nucleotide sequence is shown as SEQ ID NO: 5. The nucleotide sequence (DNA) of the light chain variable region of the mouse antibody derived from clone m11-4 is shown as SEQ ID NO: 48, and the amino acid sequence of the light chain variable region of the mouse antibody derived from clone m11-4 deduced from the nucleotide sequence is shown as SEQ ID NO: 24. TIFF2025163135000005.tif58162

[0093] The nucleotide sequence (DNA) of the heavy chain variable region of the mouse antibody derived from clone m17-1 is shown as SEQ ID NO: 51, and the amino acid sequence of the heavy chain variable region of the mouse antibody derived from clone m17-1 deduced from the nucleotide sequence is shown as SEQ ID NO: 11. The nucleotide sequence (DNA) of the light chain variable region of the mouse antibody derived from clone m17-1 is shown as SEQ ID NO: 52, and the amino acid sequence of the light chain variable region of the mouse antibody derived from clone m17-1 deduced from the nucleotide sequence is shown as SEQ ID NO: 30. TIFF2025163135000006.tif59161

[0094] The nucleotide sequence (DNA) of the heavy chain variable region of the mouse antibody derived from clone m23-12 is shown as SEQ ID NO: 53, and the amino acid sequence of the heavy chain variable region of the mouse antibody derived from clone m23-12 deduced from the nucleotide sequence is shown as SEQ ID NO: 12. The nucleotide sequence (DNA) of the light chain variable region of the mouse antibody derived from clone m23-12 is shown as SEQ ID NO: 54, and the amino acid sequence of the light chain variable region of the mouse antibody derived from clone m23-12 deduced from the nucleotide sequence is shown as SEQ ID NO: 31. TIFF2025163135000007.tif58162

[0095] Example 4 Preparation of anti-human Trop-2 chimeric antibody and control antibody The entire light and heavy chain sequences of the control antibody (sacituzumab) were synthesized and cloned into eukaryotic transient expression vectors to obtain expression plasmids expressing the light and heavy chains of the control antibody. The expression plasmids were transformed into E. coli cells for amplification, and multiple plasmids containing the light and heavy chains of the control antibody were obtained by plasmid recovery. For recombinant expression, the plasmids containing the light and heavy chains of the control antibody were transfected into HEK293 cells using 293fectin (Cat. No. 12347019, Gibco) transfection reagent according to the manufacturer's instructions. Five to six days after cell transfection, the culture supernatant was collected and purified through a ProA affinity chromatography column to obtain the control antibody. The amino acid sequence of the control antibody sacituzumab was obtained from WHO Drug Information (Vol. 31, No. 1, 2017). The amino acid sequence of the heavy chain is as set forth in SEQ ID NO:39, and the amino acid sequence of the light chain is as set forth in SEQ ID NO:40.

[0096] The light-chain variable region genes and heavy-chain variable region genes of mouse antibodies 3-11, 4-3, 11-4, 17-1, and 23-12 obtained from the above clones, into which restriction enzyme cleavage sites had been introduced by PCR, were cloned upstream of the gene encoding the human κ light chain constant region and upstream of the gene encoding the human IgG1 heavy chain constant region, respectively, contained in a eukaryotic transient expression vector. For amplification, E. coli cells were transformed with the expression plasmids expressing the resulting human-mouse chimeric light chains (pKN019-ch3-11L, pKN019-ch4-3L, pKN019-ch11-4L, pKN019-ch17-1L, and pKN019-ch23-12L) and the expression plasmids expressing the human-mouse chimeric heavy chains (pKN041-ch3-11H, pKN019-ch4-3H, pKN019-ch11-4H, pKN019-ch17-1H, and pKN019-ch23-12H). Plasmid recovery yielded numerous plasmids containing the human-mouse chimeric light chains and the human-mouse chimeric heavy chains, respectively. For recombinant expression, the plasmids containing the light and heavy chains of chimeric antibodies ch3-11, ch4-3, ch11-4, ch17-1, and ch23-12 were transfected into HEK293 cells using 293fectin (catalog number 12347019, Gibco) transfection reagent according to the manufacturer's instructions. Five to six days after cell transfection, the culture supernatants were collected and purified through a ProA affinity chromatography column to obtain chimeric antibodies ch3-11, ch4-3, ch11-4, ch17-1, and ch23-12.

[0097] Example 5 Detection of binding activity of anti-human Trop-2 chimeric antibodies to recombinant Trop-2 protein by ELISA Plates were coated with recombinant human Trop-2-his protein (accession number: NP_002344.2, AA 1-274) at a concentration of 0.2 μg / ml overnight at 4°C and then blocked with 5% BSA in a 37°C incubator for 60 minutes. Chimeric antibodies ch3-11, ch4-3, ch17-1, ch11-4, and ch23-12 and control antibody sacituzumab (a total of eight serial dilutions obtained by three-fold serial dilutions of an initial solution of 2 μg / ml) were added to the plates and then incubated in a 37°C incubator for 60 minutes. The plates were washed four times with PBST, and a 1:5000 dilution of HRP-anti-human Fc (catalog number: 109-035-098, Jackson ImmunoResearch) was added to the plates and allowed to react for 45 minutes. TMB (catalog number ME142, GalaxyBio, Beijing) substrate was added and allowed to develop for 15 minutes, after which the reaction was stopped by adding 2 M HCl. The absorbance was read at 450 nm and 630 nm (as the reference wavelength), and the A450 nm–630 nm values ​​of each well in the plate were recorded.

[0098] The binding affinities of ch3-11, ch4-3, ch17-1, ch11-4, and ch23-12, as well as the control antibody sacituzumab, to recombinant human Trop-2 protein were determined by ELISA. The 50% effective concentration (EC50) values ​​for antibody binding were 0.3147 nM, 0.3195 nM, 0.3278 nM, 0.2366 nM, 0.4581 nM, and 0.271 nM, respectively (Figure 4). The results indicated that chimeric antibodies ch3-11, ch4-3, ch17-1, ch11-4, and ch23-12 had high affinity for recombinant human Trop-2 protein and that the sequences of murine antibodies 3-11, 4-3, 11-4, 17-1, and 23-12 were accurately cloned.

[0099] Example 6 Detection of binding activity of anti-human Trop-2 chimeric antibodies to recombinant human Trop-2 protein on the surface of CHO cells by FACS A suspension of CHO cells recombinantly expressing human Trop-2 (CHO / Trop-2 cells) was incubated with chimeric antibodies ch3-11, ch4-3, ch17-1, ch11-4, and ch23-12 (dilutions at 30 μg / ml and 10 μg / ml, as well as 9 serial dilutions obtained by 3-fold serial dilutions of an initial solution of 5 μg / ml, for a total of 11 concentrations) at 37°C for 30 minutes. The following controls were set: (1) positive control (PC): control antibody sacituzumab; (2) negative control (NC): IgG1 isotype control antibody NC-IgG1. The cells were washed three times with PBS, and a 1:100 dilution of goat anti-human IgG-FITC (catalog: F9512, Sigma) was added to the cells and then incubated for 30 minutes. The cells were then washed again three times with PBS, and the mean fluorescence intensity (MFI) of the cells was measured using a flow cytometer (model B49007AD, SNAW31211, BECKMAN COULTER) to detect the binding ability of the chimeric antibody to human Trop-2 on the surface of CHO cells.

[0100] The binding affinities of ch3-11, ch4-3, ch17-1, ch11-4, and ch23-12, as well as the control antibody sacituzumab, to recombinant human Trop-2 protein on the surface of CHO cells were determined by FACS. The 50% effective concentration (EC50) values ​​for antibody binding were 0.993 nM, 3.326 nM, 2.918 nM, 1.154 nM, 2.748 nM, and 2.316 nM, respectively (Figure 5). Compared with the control antibody sacituzumab, ch3-11 and ch11-4 had better binding activity, while ch4-3, ch17-1, and ch23-12 had similar binding activity. The results showed that the anti-human Trop-2 chimeric antibodies ch3-11, ch4-3, ch17-1, ch11-4 and ch23-12 could effectively bind to recombinant human Trop-2 protein on the surface of CHO cells.

[0101] Example 7 Internalization activity of anti-human Trop-2 chimeric antibodies against Trop-2 on the cell surface 5 × 10 BxPC-3 human pancreatic cancer cells were added to each tube containing chimeric antibodies ch3-11, ch4-3, ch11-4, ch23-12, and the positive control antibody sacituzumab. 5 The cells were added in the amount of cells / tube, and each antibody was diluted to 10 μg / ml. Four groups were set up for each antibody (experimental groups incubated for 1, 3, and 5 hours, respectively, and a control group), each containing two tubes. The experimental groups were placed in an electrically heated incubator at 37°C and incubated for 1, 3, and 5 hours, respectively, and then placed on ice. The control group served as a negative control and was always incubated on ice. After incubation of all samples was completed, the samples were centrifuged at 1,500 rpm for 3 minutes at 4°C, and the supernatant was discarded. The cell pellets were washed once with ice-cold PBS, and the secondary antibody, anti-human IgG (Fc-specific)-FITC antibody (catalog: F9512, Sigma), was added to the cells and then incubated on ice for 30 minutes. The cells were then centrifuged at 1,500 rpm for 3 minutes, and the supernatant was discarded. The cell pellet was washed with ice-cold PBS and resuspended in 200 μl of ice-cold PBS, followed by FACS to detect the mean fluorescence intensity (MFI). The internalization efficiency was calculated by the following formula: %MFI tx = MFI of sample incubated at 37°C / MFI of control sample incubated at 4°C × 100; and Internalization rate (%tx) = 100 - %MFI tx.

[0102] The results are shown in Table 1, and as shown, ch4-3 and ch23-12 had similar internalization rates to the control antibody sacituzumab, while no obvious internalization was observed for ch3-11 and ch11-4.

[0103] Table 1. Internalization rate of anti-human Trop-2 chimeric antibodies mediated by Trop-2 on the cell surface. TIFF2025163135000008.tif56128

[0104] Example 8 Stability of anti-human Trop-2 chimeric antibodies intolerant to destruction Chimeric antibodies ch3-11, ch4-3, ch11-4, and ch23-12 were placed in PBS, PBS containing 10% N,N-dimethylacetamide (DMA) (catalog: ARK2190, Shanghai Feibo Chemical Technology Co., Ltd.), and PBS containing 20% ​​DMA at a concentration of 5 mg / ml each for 2 hours at 37°C. DMA was then removed from the samples using ultrafiltration centrifuge tubes containing the PBS used for buffer exchange. The samples were analyzed for purity by size-exclusion high-performance liquid chromatography (SEC-HPLC) using a G3000WXL liquid chromatography column (catalog: SEC-0046, Tosoh Corporation). The purity analysis results are shown in Table 2.

[0105] The results showed that all four antibodies could tolerate DMA well, with less obvious reduction in purity at 10% DMA and only a slight decrease at 20% DMA, suggesting that the antibodies would likely tolerate the subsequent ADC conjugation process well.

[0106] Table 2. Purity of antibody analyzed by HPLC before and after DMA treatment TIFF2025163135000009.tif40166

[0107] Example 9 Humanization and recombinant expression of an anti-human Trop-2 monoclonal antibody 1. Humanization of mouse monoclonal antibody 23-12 (1) CDR grafting First, we comprehensively analyzed the heavy chain sequence of the mouse antibody to determine the complementarity-determining regions (CDRs) responsible for antigen binding and the framework regions that support the antibody's conserved three-dimensional structure. Based on the results of homology alignment, we then searched for the most similar human template in a human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX). Based on the results of full-sequence BLAST analysis, we performed CDR grafting to fully humanize the heavy chain variable region (VH) of the mouse antibody 23-12 within the framework regions. Based on the results of the homology alignment, the most similar human template was searched for in the human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX), and CDR grafting was performed according to the full-sequence BLAST results in combination with the sequence characteristics of the light chain CDR3 to highly humanize the light chain variable region (VL) of mouse antibody 23-12 within the framework regions.

[0108] The nucleotide and amino acid sequences of the humanized heavy chain variable region (h23-12_VH1) of CDR-grafted antibody 23-12 are set forth in SEQ ID NO: 55 and SEQ ID NO: 13, respectively. The nucleotide and amino acid sequences of the humanized light chain variable region (h23-12_VL1) of CDR-grafted antibody 23-12 are set forth in SEQ ID NO: 56 and SEQ ID NO: 32, respectively. TIFF2025163135000010.tif58162

[0109] (2) Mutation design in CDR Mutations in the CDR sequences within the CDR-grafted humanized light chain variable region and the CDR-grafted humanized heavy chain variable region were designed according to the sequence characteristics of the murine antibody 23-12. The mutation sites are shown in Table 3.

[0110] Table 3: Design of humanized sequence of mouse antibody 23-12 TIFF2025163135000011.tif61128Note: Amino acid residue positions were numbered according to the Kabat numbering system.

[0111] 2. Recombinant expression of humanized monoclonal antibody 23-12 The light and heavy chain variable region genes (h23-12_VL1 and h23-12_VH1) of the CDR-grafted antibody 23-12 were fully synthesized. The humanized h23-12_VH1 gene was cloned into the eukaryotic transient expression vector pKN041 by enzymatic digestion upstream of the gene encoding the heavy chain constant region of human IgG1. The nucleotide and amino acid sequences of the heavy chain constant region are shown in SEQ ID NO:59 and SEQ ID NO:37, respectively. The humanized h23-12_VL1 gene was cloned into the eukaryotic transient expression vector pKN019 by enzymatic digestion upstream of the gene encoding the human Cκ light chain. The nucleotide and amino acid sequences of the light chain constant region are shown in SEQ ID NO:60 and SEQ ID NO:38, respectively. Thus, expression plasmids containing the light and heavy chains of CDR-grafted antibody 23-12 were constructed, and for amplification, E. coli cells were transformed with the resulting expression plasmids expressing the light chain (pKN019-h23-12L1) and heavy chain (pKN019-h23-12H1), and plasmids expressing the light chain h23-12L1 and heavy chain h23-12H1 of CDR-grafted antibody 23-12 were isolated and obtained.

[0112] Site-directed mutagenesis was performed on expression plasmids expressing the light chain (pKN019-h23-12L1) and heavy chain (pKN019-h23-12H1) using the StarMut gene Site-directed Mutagenesis Kit (catalog: T111-01, GenStar) according to the mutation design shown in Table 3. The mutated plasmids were transformed into E. coli cells for amplification, resulting in expression plasmids expressing the light and heavy chains of a humanized monoclonal antibody with CDR mutations (h23-12H2...h23-12H7; h23-12L2...h23-12L7), which correspond to the humanized sequences of mouse antibody 23-12 shown in Table 3. All the plasmids containing various humanized heavy and light chain sequences of mouse antibody 23-12 were combined as shown in Table 4 and transfected into HEK293 cells for recombinant expression using 293fectin (catalog: 12347019, Gibco) transfection reagent according to the manufacturer's instructions.

[0113] Table 4. Combinations of humanized heavy and light chain sequences of mouse antibody 23-12 TIFF2025163135000012.tif84167Note: Table 4 shows antibodies derived from various heavy and light chain combinations derived from murine antibody 23-12. For example, h23-12-1 refers to an antibody composed of the humanized light chain h23-12L1 and humanized heavy chain h23-12H1 of murine antibody 23-12.

[0114] Five to six days after cell transfection, the culture supernatant was purified through a ProA affinity chromatography column to obtain various humanized antibodies. The affinity of the resulting antibodies was determined by an assay involving capturing the Fc fragment of the antibody with an anti-human IgG Fc capture (AHC) biosensor using a Fortebio Octet QKe system. For the assay, each humanized antibody and the control antibody sacituzumab were diluted to 4 μg / ml in PBS and flowed over the surface of an AHC biosensor (catalog number: 18-0015, PALL) for 120 seconds. Recombinant human Trop-2-his protein (accession number: NP_002344.2, AA 1-274) was used as the mobile phase at a concentration of 60 nM. The association time was 100 seconds, and the dissociation time was 300 seconds. Once the assay was completed, the software was used to fit the data, minus the response value of the blank control, to a 1:1 Langmuir binding model, and then calculate the kinetic constants of antigen-antibody binding.

[0115] The affinity of antibodies resulting from combinations of variants of murine antibody 23-12, chimeric antibody ch23-12 and control antibody sacituzumab to recombinant human Trop-2-his protein was determined by ForteBio (Table 5).

[0116] Table 5. Results of antibody affinity detection for recombinant human Trop-2 extracellular domain TIFF2025163135000013.tif220167

[0117] Antibody h23-12-25, with a measured affinity of 5.02E-10M, was selected and designated h23-12 for further functional validation. The nucleotide and amino acid sequences of the heavy chain variable region of the antibody are set forth in SEQ ID NO:57 and SEQ ID NO:14, respectively. The nucleotide and amino acid sequences of the light chain variable region of the antibody are set forth in SEQ ID NO:58 and SEQ ID NO:33, respectively. TIFF2025163135000014.tif58161

[0118] 3. Humanization of mouse monoclonal antibody 4-3 (1) CDR grafting First, we comprehensively analyzed the heavy chain sequence of the mouse antibody to determine the complementarity-determining regions (CDRs) responsible for antigen binding and the framework regions supporting the antibody's conserved three-dimensional structure. Based on the results of homology alignment, we then searched for the most similar human template in a human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX). Based on the sequence characteristics of the heavy chain CDR3 and the results of full-sequence BLAST, we performed CDR grafting to fully humanize the heavy chain variable region (VH) of the mouse antibody 4-3 within the framework regions. Based on the results of the homology alignment, the most similar human template was searched for in the human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX), and CDR grafting was performed according to the full-sequence BLAST results in combination with the sequence characteristics of the light chain CDR3 to fully humanize the light chain variable region (VL) of mouse antibody 4-3 within the framework regions.

[0119] The nucleotide and amino acid sequences of the humanized heavy chain variable region (h4-3_VH1) of CDR-grafted antibody 4-3 are set forth in SEQ ID NO: 45 and SEQ ID NO: 3, respectively. The nucleotide and amino acid sequences of the humanized light chain variable region (h4-3_VL1) of CDR-grafted antibody 4-3 are set forth in SEQ ID NO: 46 and SEQ ID NO: 20, respectively. TIFF2025163135000015.tif58162

[0120] (2) Mutation design in CDR According to the sequence characteristics of mouse antibody 4-3, mutations in the CDR sequences in the CDR-grafted humanized light chain variable region and the CDR-grafted humanized heavy chain variable region were designed. The mutation sites are shown in Table 6.

[0121] Table 6: Design of humanized sequence of mouse antibody 4-3 TIFF2025163135000016.tif45128Note: Amino acid residue positions were numbered according to the Kabat numbering system.

[0122] 4. Recombinant expression of humanized monoclonal antibody 4-3 The light and heavy chain variable region genes (h4-3_VL1 and h4-3_VH1) of the CDR-grafted antibody 4-3 were fully synthesized. The humanized h4-3_VH1 gene was cloned into the eukaryotic transient expression vector pKN041 by enzymatic digestion upstream of the gene encoding the heavy chain constant region of human IgG1. The nucleotide and amino acid sequences of the heavy chain constant region are shown in SEQ ID NO:59 and SEQ ID NO:37, respectively. The humanized h4-3_VL1 gene was cloned into the eukaryotic transient expression vector pKN019 by enzymatic digestion upstream of the gene encoding the human Cκ light chain. The nucleotide and amino acid sequences of the light chain constant region are shown in SEQ ID NO:60 and SEQ ID NO:38, respectively. Thus, expression plasmids containing the light and heavy chains of CDR-grafted antibody 4-3 were constructed, and the resulting expression plasmids expressing the light chain (pKN019-h24-3L1) and heavy chain (pKN019-h4-3H1) were transformed into E. coli cells for amplification, and plasmids expressing the light chain h4-3L1 and heavy chain h4-3H1 of CDR-grafted antibody 4-3 were isolated and obtained.

[0123] Site-directed mutagenesis was performed on expression plasmids expressing the light chain (pKN019-h4-3L1) and heavy chain (pKN019-h4-3H1) using the StarMut gene Site-directed Mutagenesis Kit (catalog: T111-01, GenStar) according to the mutation design shown in Table 6. The mutated plasmids were transformed into E. coli cells for amplification, resulting in expression plasmids expressing the light and heavy chains (h4-3H2...h4-3H4; h4-3L2...h4-3L5) of humanized monoclonal antibodies with CDR mutations corresponding to the humanized sequences of mouse antibody 4-3 shown in Table 6. All plasmids containing the various humanized heavy and light chain sequences of mouse antibody 4-3 were combined as shown in Table 7 and transfected into HEK293 cells using 293fectin (catalog: 12347019, Gibco) transfection reagent according to the manufacturer's instructions for recombinant expression.

[0124] Table 7. Combinations of humanized heavy and light chain sequences of mouse antibody 4-3 TIFF2025163135000017.tif49161Note: Table 7 shows antibodies derived from various heavy and light chain combinations derived from mouse antibody 4-3. For example, h4-3-1 refers to an antibody composed of the humanized light chain h4-3L1 and humanized heavy chain h4-3H1 of mouse antibody 4-3.

[0125] Five to six days after cell transfection, the culture supernatant was purified through a ProA affinity chromatography column to obtain various humanized antibodies. The affinity of the resulting antibodies was determined by an assay involving capturing the Fc fragment of the antibody with an anti-human IgG Fc capture (AHC) biosensor using a Fortebio Octet QKe system. For the assay, each humanized antibody and the control antibody sacituzumab were diluted to 4 μg / ml in PBS and flowed over the surface of an AHC biosensor (catalog number: 18-0015, PALL) for 120 seconds. Recombinant human Trop-2-his protein (accession number: NP_002344.2, AA 1-274) was used as the mobile phase at a concentration of 60 nM. The association time was 100 seconds, and the dissociation time was 300 seconds. Once the assay was completed, the software was used to fit the data, minus the response value of the blank control, to a 1:1 Langmuir binding model, and then calculate the kinetic constants of antigen-antibody binding.

[0126] The affinity of antibodies resulting from combinations of variants of murine antibody 23-12, chimeric antibody ch4-3 and control antibody sacituzumab to recombinant human Trop-2-his protein was determined by ForteBio (Table 8).

[0127] Table 8: Results of antibody affinity detection for recombinant human Trop-2 extracellular domain TIFF2025163135000018.tif98167

[0128] Antibody h4-3-1, with a measured affinity of 3.04E-10M, was selected and designated h4-3, which was used for subsequent functional validation. The nucleotide and amino acid sequences of the heavy chain variable region of the antibody are set forth in SEQ ID NO:45 and SEQ ID NO:3, respectively. The nucleotide and amino acid sequences of the light chain variable region of the antibody are set forth in SEQ ID NO:46 and SEQ ID NO:20, respectively. TIFF2025163135000019.tif58162

[0129] 5. Humanization of Mouse Monoclonal Antibody 11-4 (1) CDR grafting First, we comprehensively analyzed the heavy chain sequence of the mouse antibody to determine the complementarity-determining regions (CDRs) responsible for antigen binding and the framework regions supporting the antibody's conserved three-dimensional structure. Based on the results of homology alignment, we then searched for the most similar human template in a human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX). Based on the sequence characteristics of the heavy chain CDR3 and the results of full-sequence BLAST, we performed CDR grafting to fully humanize the heavy chain variable region (VH) of the mouse antibody 11-4 within the framework regions. Based on the results of the homology alignment, the most similar human template was searched for in the human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX), and CDR grafting was performed according to the full-sequence BLAST results in combination with the sequence characteristics of the light chain CDR3, to fully humanize the light chain variable region (VL) of mouse antibody 11-4 within the framework regions.

[0130] The nucleotide and amino acid sequences of the humanized heavy chain variable region (h11-4_VH1) of CDR-grafted antibody 11-4 are set forth in SEQ ID NO: 49 and SEQ ID NO: 50, respectively. The nucleotide and amino acid sequences of the humanized light chain variable region (h11-4_VL1) of CDR-grafted antibody 4-3 are set forth in SEQ ID NO: 50 and SEQ ID NO: 25, respectively. TIFF2025163135000020.tif58162

[0131] (2) Mutation design in CDR According to the sequence characteristics of mouse antibody 11-4, mutations in the CDR sequences in the CDR-grafted humanized light chain variable region and the CDR-grafted humanized heavy chain variable region were designed. The mutation sites are shown in Table 9.

[0132] Table 9: Design of humanized sequence of mouse antibody 11-4 TIFF2025163135000021.tif77147Note: Amino acid residue positions were numbered according to the Kabat numbering system.

[0133] 6. Recombinant expression of humanized monoclonal antibody 11-4 The light and heavy chain variable region genes (h11-4_VL1 and h11-4_VH1) of the CDR-grafted antibody 11-4 were fully synthesized. The humanized h11-4_VH1 gene was cloned into the eukaryotic transient expression vector pKN041 by enzymatic digestion upstream of the gene encoding the heavy chain constant region of human IgG1. The nucleotide and amino acid sequences of the heavy chain constant region are shown in SEQ ID NO:59 and SEQ ID NO:37, respectively. The humanized h11-4_VL1 gene was cloned into the eukaryotic transient expression vector pKN019 by enzymatic digestion upstream of the gene encoding the human Cκ light chain. The nucleotide and amino acid sequences of the light chain constant region are shown in SEQ ID NO:60 and SEQ ID NO:38, respectively. Thus, expression plasmids containing the light and heavy chains of CDR-grafted antibody 11-4 were constructed, and the resulting expression plasmids expressing the light chain (pKN019-h11-4L1) and heavy chain (pKN019-h11-4H1) were transformed into E. coli cells for amplification, and plasmids expressing the light chain h11-4L1 and heavy chain h11-4H1 of CDR-grafted antibody 11-4 were isolated and obtained.

[0134] Site-directed mutagenesis was performed on expression plasmids expressing the light chain (pKN019-h11-4L1) and heavy chain (pKN019-h11-4H1) using the StarMut gene Site-directed Mutagenesis Kit (catalog: T111-01, GenStar) according to the mutation design shown in Table 9. The mutated plasmids were transformed into E. coli cells for amplification, and expression plasmids expressing the light and heavy chains of humanized monoclonal antibodies with CDR mutations (h11-4H2...h11-4H7; h11-4L2...h11-4L5) corresponding to the humanized sequence of mouse antibody 4-3 shown in Table 9. All the plasmids containing various humanized heavy and light chain sequences of mouse antibody 11-4 were combined as shown in Table 10 and transfected into HEK293 cells for recombinant expression using 293fectin (catalog: 12347019, Gibco) transfection reagent according to the manufacturer's instructions.

[0135] Table 10. Combinations of humanized heavy and light chain sequences of mouse antibody 11-4 TIFF2025163135000022.tif50167Note: Table 10 shows antibodies derived from various heavy and light chain combinations derived from murine antibody 11-4. For example, h11-4-1 refers to an antibody composed of the humanized light chain h11-4L1 and humanized heavy chain h11-4H1 of murine antibody 11-4.

[0136] Five to six days after cell transfection, the culture supernatant was purified through a ProA affinity chromatography column to obtain various humanized antibodies. The affinity of the resulting antibodies was determined by an assay involving capturing the Fc fragment of the antibody with an anti-human IgG Fc capture (AHC) biosensor using a Fortebio Octet QKe system. For the assay, each humanized antibody and the control antibody sacituzumab were diluted to 4 μg / ml in PBS and flowed over the surface of an AHC biosensor (catalog number: 18-0015, PALL) for 120 seconds. Recombinant human Trop-2-his protein (accession number: NP_002344.2, AA 1-274) was used as the mobile phase at a concentration of 60 nM. The association time was 100 seconds, and the dissociation time was 300 seconds. Once the assay was completed, the software was used to fit the data, minus the response value of the blank control, to a 1:1 Langmuir binding model, and then calculate the kinetic constants of antigen-antibody binding.

[0137] The affinity of antibodies resulting from combinations of variants of murine antibody 11-4, chimeric antibody ch11-4 and control antibody sacituzumab to recombinant human Trop-2-his protein was determined by ForteBio (Table 11).

[0138] Table 11: Results of antibody affinity detection for recombinant human Trop-2 extracellular domain TIFF2025163135000023.tif96163

[0139] Example 10 Detection of species-specific binding of anti-Trop-2 humanized antibodies to Trop-2 by ELISA Plates were coated overnight at 4°C with recombinant human Trop-2-his protein (accession number: NP_002344.2, AA 1-274), recombinant cynomolgus monkey Trop-2-his protein (accession number: UniProtKB-A0A2K5UE71, AA 1-272), and recombinant mouse Trop-2-his protein (catalog number: 50922-M08H, Beijing Yiqiao Shenzhou Science and Technology Co., Ltd.) at a coating concentration of 1 μg / ml each. After washing three times with PBS, the plates were blocked with 5% BSA PBS at 37°C for 60 minutes and then washed three times with PBST. Different concentrations of h23-12 (14 serial dilutions obtained by 3-fold serial dilutions from a solution with an initial concentration of 10 μg / ml), h4-3 (12 serial dilutions obtained by 3-fold serial dilutions from a solution with an initial concentration of 3 μg / ml), and sacituzumab (12 serial dilutions obtained by 3-fold serial dilutions from a solution with an initial concentration of 3 μg / ml) were added to the plate, with each concentration provided in a parallel well. The plate was incubated at 37°C for 60 minutes and then washed four times with PBST. A 1:5000 dilution of HRP anti-human Fc (catalog: 109-035-098, Jackson ImmunoResearch) was added to the plate and incubated at 37°C for 30 minutes. The plate was then washed four times with PBST, and TMB substrate was added and developed at 37°C for 10 minutes. The reaction was then stopped by adding 2M HCl. The absorbance was read at 450 nm and 630 nm (as the reference wavelength), and the A450 nm-630 nm values ​​of the wells in the plate were recorded.

[0140] The experimental results showed that h23-12, h4-3, and the control antibody sacituzumab could specifically bind to recombinant human Trop-2 and cynomolgus monkey Trop-2, but had no binding activity to mouse Trop-2 (Figure 6 and Table 12), providing a basis for pharmacological and toxicological experiments of the humanized antibodies.

[0141] Table 12. EC50 of binding of anti-Trop-2 humanized antibodies to Trop-2 in different species TIFF2025163135000024.tif40128

[0142] Example 11 Affinity analysis of anti-human Trop-2 humanized antibodies Antibody affinity was determined by an assay involving capturing the Fc fragment of the antibody with an anti-human IgG Fc capture (AHC) biosensor using an Octet QKe system instrument from Fortebio.

[0143] For the assay, each antibody (h23-12, h4-3, and the control antibody sacituzumab) was diluted to 4 μg / ml in PBS and flowed over the surface of an AHC biosensor (catalog number: 18-0015, PALL) for 120 seconds. Recombinant human Trop-2-his protein (accession number: NP_002344.2, AA 1-274) was used as the mobile phase. Trop-2-his protein was used at the following concentrations corresponding to the various antibodies: for h23-12, Trop-2 was used at concentrations of 23, 30, 45, and 75 nM; for h4-3, Trop-2 was used at concentrations of 23, 30, 45, and 60 nM; and for sacituzumab, Trop-2 was used at concentrations of 23, 30, 45, and 75 nM. The association time was 100 seconds, and the dissociation time was 300 seconds. Once the assay was completed, the software was used to fit the data, minus the response value of the blank control, to a 1:1 Langmuir binding model, and then calculate the kinetic constants of antigen-antibody binding.

[0144] The reaction curves of h23-12, h4-3, and the control antibody sacituzumab with recombinant human Trop-2 protein are shown in Figure 7. Curve fitting and affinity calculations indicated that h23-12 had an affinity measured by a KD value of 6.40E-10 M, h4-3 had an affinity measured by a KD value of 5.45E-10 M, and sacituzumab had an affinity measured by a KD value of 9.41E-10 M. Kinetic parameters are detailed in Table 13. The results showed that h23-12 and h4-3 had high affinity for human Trop-2 comparable to that of the control antibody sacituzumab, and h23-12 had a better dissociation value than sacituzumab.

[0145] Table 13: Results of affinity detection of anti-human Trop-2 humanized antibodies to recombinant human Trop-2 extracellular domain TIFF2025163135000025.tif44151

[0146] Example 12 Internalization activity of a humanized anti-human Trop-2 antibody that binds to Trop-2 on the cell surface 2 × 10 NCI-N87 human gastric cancer cells that naturally express human Trop-2 3 Cells were seeded into 96-well cell culture plates at a density of 100 cells / well and cultured for 24 hours. The cells were washed once with PBS, and the supernatant was discarded. h23-12 labeled with Mix-n-Stain™ CF™ 488A (catalog: MX488AS100, Sigma) and the control antibody sacituzumab were diluted to 15 μg / ml in RPMI 1640 (containing 10% FBS) and added to NCI-N87 cells. The plates were divided into two groups: one group was placed in an electrically heated incubator at 37°C, and the other group, serving as a negative control, was placed in a refrigerator at 4°C. The negative control plate was incubated for 30 minutes, washed three times with PBS, and observed and photographed using a fluorescent microscope. The experimental group plates were incubated at 37°C for 5 hours, then observed and photographed using a fluorescent microscope.

[0147] The experimental results (Figure 8) showed that both humanized h23-12 and the control antibody sacituzumab could be endocytosed by Trop-2-mediated endocytosis and distributed as spots in the cytoplasm, suggesting that the internalization activity was maintained after antibody humanization.

[0148] The internalization rate on BxPC cells at 3 hours was measured by FACS using the procedure described in Example 7. The results, which suggest that the internalization rate after humanization is similar to that of sacituzumab, are shown in Table 14.

[0149] Table 14. Internalization rate of anti-human Trop-2 antibody mediated by Trop-2 on the surface of BxPC cells. TIFF2025163135000026.tif40128

[0150] Example 13 Pharmacokinetic study in Balb / C nude mice administered a single dose Healthy female 5-week-old Balb / C nude mice were divided into groups of two. A single dose of h23-12 (15 mg / kg) was intraperitoneally injected into the mice. Serum samples were collected at 5, 25, 48, 96, 168, and 240 hours post-transplant and stored at -20°C. A control group was also established, and mice in the control group were intraperitoneally injected with the control antibody sacituzumab at the same dose as h23-12 for comparison. The pharmacokinetic properties of the antibody were observed.

[0151] Healthy female 5-week-old Balb / C nude mice were divided into groups of four. Mice were intraperitoneally injected with a single dose of h4-3 (20 mg / kg). Serum samples were collected at 4, 8, 24, 48, 96, 144, 192, and 240 hours, respectively, and stored at -20°C. The pharmacokinetic properties of the antibody were observed.

[0152] The drug concentration in serum was measured by ELISA using coated human Trop-2-his (accession number: NP_002344.2, AA 1-274). A standard curve was then established. A linear curve was fitted by plotting the concentration of the standard antibody (Y-axis) against the OD value (X-axis). The antibody content in serum was calculated by substituting the OD value of the detected serum into the formula: T 1 / 2 = |0.693 / k| according to the antibody drug half-life T 1 / 2 was calculated.

[0153] The drug concentration versus time curve results showed that h23-12, h4-3, and the control antibody sacituzumab all had relatively long half-lives and showed comparable drug metabolic half-lives (Panel 9A, Panel 9B, and Table 15), suggesting that the antibodies had good structural stability in vivo without obvious inactivation phenomena. The antibody metabolism met the basic characteristics of monoclonal antibody drugs and was consistent with T 1 / 2 is about 170 hours.

[0154] Table 15. Pharmacokinetic parameters of anti-Trop-2 antibodies in nude mice administered a single dose. TIFF2025163135000027.tif43128

[0155] Example 14 Affinity analysis of anti-Trop-2 naked antibodies and their ADCs Antibody affinity was determined by an assay involving capturing the Fc fragment of the antibody with an anti-human IgG Fc capture (AHC) biosensor using an Octet QKe system instrument from Fortebio.

[0156] First, we prepared an antibody labeled with SN38 for use in the ADC drug. The antibody was reduced using 20 equivalents of dithiothreitol (DTT) in sodium phosphate buffer, pH 7.0 ± 0.5, for 2 hours. The reduced antibody was purified using an ultrafiltration centrifuge to remove excess DTT and exchanged into sodium phosphate buffer, pH 7.0 ± 0.5. The reduced antibody was then incubated with CL2A-SN-38 for 30 minutes at ambient temperature using 7-15% (v / v) DMSO as a cosolvent. Finally, excess small molecules were removed through an ultrafiltration centrifuge. The molecular weight of the resulting antibody-drug conjugate was analyzed by mass spectrometry, and the antibody-drug ratio (DAR) was calculated. Finally, we confirmed that 7.5 SN38 molecules were linked to one antibody.

[0157] For the assay, the SN38-labeled antibodies (h23-12-SN38, ch4-3-SN38, ch11-4-SN38, and the positive control antibody sacituzumab-SN38) prepared as described above, as well as the naked antibodies h23-12, ch4-3, ch11-4, and the positive control antibody sacituzumab, were diluted to 4 μg / ml in PBS and flowed over the surface of an AHC biosensor (catalog number: 18-0015, PALL) for 120 seconds. Recombinant human Trop-2-his protein (accession number: NP_002344.2, AA 1-274) was used as the mobile phase at a concentration of 60 nM. The association time was 300 seconds, and the dissociation time was 300 seconds. After the assay was completed, the data, minus the response value of the blank control, were fitted to a 1:1 Langmuir binding model using software, and the kinetic constants of antigen-antibody binding were then calculated.

[0158] As shown in Table 16, when antibodies were labeled with SN38, thereby forming ADCs, no significant change in the affinity of the antibodies was observed compared to the affinity when the antibodies were naked.

[0159] Table 16. Results of affinity detection of anti-Trop-2 naked antibodies and their ADCs TIFF2025163135000028.tif50128

[0160] Example 15 FACS detection of BXPC-3 cell-mediated internalization of anti-Trop-2 naked antibody and its ADC. The internalization rates of BXPC-3 human pancreatic cancer cells and NCI-N87 human gastric cancer cells were detected according to the procedure described in Example 7. The antibodies used for detection included the ADC drugs, i.e., the SN38-labeled antibodies prepared as described above: h23-12-SN38, ch4-3-SN38, ch11-4-SN38, the positive control antibody sacituzumab-SN38, as well as naked antibodies h32-12, ch4-3, ch11-4, the positive control antibody sacituzumab, and the negative isotype control antibody NC-IgG1, each at 10 μg / ml.

[0161] As shown in Tables 17 and 18, naked h23-12 and h23-12 labeled with SN38 to form ADCs had similar internalization rates, which were similar to the internalization rate of the control antibody. ch4-3 and ch11-4 had higher internalization rates when labeled with SN38 to form ADCs than when naked.

[0162] Table 17. Internalization rate of anti-human Trop-2 antibodies mediated by Trop-2 on the surface of NCI-N87 cells TIFF2025163135000029.tif50128

[0163] Table 18. Internalization rate of anti-human Trop-2 antibody mediated by Trop-2 on the surface of BXPC-3 cells TIFF2025163135000030.tif58128

[0164] Example 16 Detection of cell-killing activity of anti-Trop-2-ADC BxPC-3 human pancreatic cancer cells were plated in 96-well cell culture plates at 2 × 10 3The cells were seeded at 1000 x g / well and cultured overnight in an incubator at 37°C and 5% CO2. Then, different concentrations of SN38-labeled anti-Trop-2 antibody samples (each concentration was provided in two parallel wells) were added to the plate according to Table 19, and blank wells containing cells only (without any treatment) were set up. The cells were incubated in an incubator at 37°C and 5% CO2 for 3 hours, and then the culture medium was replaced with fresh complete medium. The next day, the treatment was repeated as the previous day. After four consecutive days of treatment, the cell-killing activity of the SN38-labeled anti-Trop-2 antibody was detected using Cell Counting Kit-8 (CCK-8).

[0165] Table 19. Anti-Trop-2 ADCs and their actual concentrations TIFF2025163135000031.tif72163

[0166] Results showed that each anti-Trop-2 ADC specifically killed target cells, with no significant difference in killing activity from the control antibody sacituzumab-SN38 (Figure 10 and Table 20).

[0167] Table 20. Inhibitory activity of anti-Trop-2 ADCs on cell proliferation TIFF2025163135000032.tif51128

[0168] Example 17 Pharmacodynamic evaluation of anti-Trop-2 ADCs in the N87 subcutaneous xenograft tumor model Five-week-old female BALB / c nude mice were treated with 3 × 10 6 Human gastric cancer cells (NCI-N87) were subcutaneously inoculated, and the tumor grew to approximately 150 mm 3 When the tumors grew to 1000 mm3, the mice were randomly assigned to groups of 6 mice per group. The group assignments, dosages, and administration frequency for each group are shown in Table 21. Each group received intravenous injections twice a week, and the tumor volume and body weight of each mouse were measured at the same time. The mice were discontinued if their body weights decreased by more than 15% or if their tumor volume reached 3000 mm3. 3 or when the mean tumor volume for the entire group of animals exceeds 2000 mm 3If the serotonin concentration exceeded 100mg / kg, the experiment was stopped and the mice were euthanized.

[0169] Table 21: Grouping of nude mice and dosage and frequency of administration TIFF2025163135000033.tif82128

[0170] As shown in Figures 11 and 12, the anti-Trop-2 ADCs had a dose-dependent inhibitory effect on tumor growth, and no difference in efficacy was observed among the ADCs at high doses (10 mg / kg). No significant toxic effects of the small molecule SN38 in the ADCs were observed, and the body weight of animals in each experimental group steadily increased without significant differences from controls.

[0171] Example 18 Pharmacodynamic evaluation of coadministration of anti-Trop-2 and anti-CD47 antibodies in a SKOV3 subcutaneous xenograft tumor model Five-week-old female BALB / c nude mice were each given 3 × 10 6 SKOV3 human ovarian cancer cells were subcutaneously inoculated, and tumors grew to approximately 150 mm 3 When the tumors grew to 1000 mcg, the mice were randomly assigned to groups of 6 mice per group. The group assignments, dosages, and administration frequency are shown in Table 22. Each group received intravenous injections twice a week for a total of 5 times, and tumor volume and body weight of each mouse were measured at the same time. The condition of the mice was observed, and after the last administration, the mice were euthanized. The anti-CD47 antibody used was described in Patent Application Publication US2015 / 0183874, i.e., humanized 5F9, version 2.

[0172] Table 22: Grouping of nude mice and dosage and frequency of administration TIFF2025163135000034.tif40128

[0173] As shown in Figures 13 and 14, the group co-administered with h23-12 and anti-CD47 antibody showed some inhibitory activity against tumors compared to the negative control hIgG4, while the anti-CD47 antibody group and h23-12 group showed no significant inhibitory effect against tumors. This indicates that the anti-Trop-2 antibody and anti-CD47 antibody of the present disclosure synergistically promote phagocytosis of tumor cells by macrophages and have a synergistic inhibitory effect against tumors.

[0174] Example 19 Pharmacodynamic evaluation of anti-Trop-2 ADCs in the N87 subcutaneous xenograft tumor model Five-week-old female BALB / c nude mice were treated with 3 × 10 6 Human gastric cancer cells (NCI-N87) were subcutaneously inoculated, and the tumors grew to approximately 100 mm 3 When the tumors grew to 1000 mm3, the mice were randomly assigned to groups of 6 mice per group. The group assignments, dosages, and administration frequency for each group are shown in Table 23. Each group received intravenous injections twice a week, and the tumor volume and body weight of each mouse were measured at the same time. The mice were discontinued if their body weights decreased by more than 15% or if their tumor volume reached 3000 mm3. 3 or when the mean tumor volume for the entire group of animals exceeds 2000 mm 3 If the serotonin concentration exceeded 100mg / kg, the experiment was stopped and the mice were euthanized.

[0175] Table 23: Grouping of nude mice and dosage and frequency of administration TIFF2025163135000035.tif48128

[0176] As shown in Figures 15 and 16, anti-Trop-2 ADCs had a dose-dependent inhibitory effect on tumor growth, with ADCs ch3-11-SN38 and ch11-4-SN38 at a dose of 5 mg / kg having slightly better effects than sacituzumab-SN38. No significant toxic effects of the small molecule SN38 in the ADCs were observed.

[0177] The above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications to the present invention without departing from the spirit of the present invention, which should be included within the scope of the appended claims.

[0178] Sequence information Sequence Listing <110> MABWELL (SHANGHAI) BIOSCIENCE CO., LTD. <120> ANTI-HUMAN TROP-2 ANTIBODY AND APPLICATION THEREOF <150> CN 201910962965.1 <151> 2019-10-11 <160> 60 <170> PatentIn version 3.3 <210> 1 <211> one two three <212> PRT <213> Artificial <220> <223> heavy chain variable region <400> 1 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Tyr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Phe Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly His Asn Tyr Asp Gly Ser Leu Gly Ala Met Asp His 100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 2 <211> 117 <212> PRT <213> Artificial <220> <223> heavy chain variable region <400> 2 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Val Ile Gly Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Leu Gly Ser Gly Thr Ile Tyr Tyr Thr Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Ser Ile Phe Pro Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 3 <211> 117 <212> PRT <213> Artificial <220> <223> heavy chain variable region <400> 3 Glu Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Val Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Leu Gly Ser Gly Thr Ile Tyr Tyr Thr Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Ile Phe Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 4 <211> 117 <212> PRT <213> Artificial <220> <223> heavy chain variable region <400> 4 Glu Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Val Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Leu Gly Ser Gly Thr Ile Tyr Tyr Ala Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Ile Phe Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 5 <211> 117 <212> PRT <213> Artificial <220> <223> heavy chain variable region <400> 5 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Asn Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asn Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Thr Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ser Ser Tyr Arg Ser Asp Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210> 6 <211> 117 <212> PRT <213> Artificial <220> <223> heavy chain variable region <400> 6 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Tyr Pro Ser Asn Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Arg Ser Asp Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 7 <211> 117 <212> PRT <213> Artificial <220> <223> heavy chain variable region <400> 7 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Tyr Pro Ser Asn Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Ser Tyr Arg Ser Asp Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 8 <211> 117 <212> PRT <213> Artificial <220> <223> heavy chain variable region <400> 8 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Tyr Pro Ser Asn Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Ser Tyr Arg Ser Glu Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 9 <211> 117 <212> PRT <213> Artificial <220> <223> heavy chain variable region <400> 9 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Tyr Pro Ser Asn Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Ser Tyr Arg Ser Gly Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 10 <211> 117 <212> PRT <213> Artificial <220> <223> heavy chain variable region <400> 10 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Tyr Pro Ser Asn Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Ser Tyr Arg Ser Asp Ala Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 11 <211> 115 <212> PRT <213> Artificial <220> <223> heavy chain variable region <400> 11 Glu Val Lys Leu Val Glu Ser Gly Gly Val Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30 Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Arg Gly Asp Asp Thr Tyr Tyr Pro Asp Ser Val Lys 50 55 60 Gly Arg Ile Thr Ile Ser Arg Asp Phe Ala Arg Asn Ile Leu Tyr Leu 65 70 75 80 Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Thr 85 90 95 Arg Asp Arg Phe Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ala 115 <210> 12 <211> 119 <212> PRT <213> Artificial <220> <223> heavy chain variable region <400> 12 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Asp Gly Tyr Ile Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Thr Pro Ser Asp Asn Tyr Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly His Gly Asn Tyr Val Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 13 <211> 119 <212> PRT <213> Artificial <220> <223> heavy chain variable region <400> 13 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Thr Pro Ser Asp Asn Tyr Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly His Gly Asn Tyr Val Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 14 <211> 119 <212> PRT <213> Artificial <220> <223> heavy chain variable region <400> 14 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Thr Pro Ser Asp Asn Tyr Gly Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly His Gly Asn Tyr Val Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 15 <211> 119 <212> PRT <213> Artificial <220> <223> heavy chain variable region <400> 15 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Thr Pro Gly Asp Asn Tyr Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly His Gly Asn Tyr Val Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 16 <211> 119 <212> PRT <213> Artificial <220> <223> heavy chain variable region <400> 16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Thr Pro Ser Asp Asn Tyr Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Glu Gly Asn Tyr Val Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 17 <211> 119 <212> PRT <213> Artificial <220> <223> heavy chain variable region <400> 17 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Thr Pro Ser Asp Asn Tyr Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gln Gly Asn Tyr Val Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 18 <211> 112 <212> PRT <213> Artificial <220> <223> light chain variable region <400> 18 Asp Val Val Val Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Phe Gly 1 5 10 15 Asp Gln Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Thr Asn Ser 20 25 30 Tyr Gly Asn Thr Phe Leu Ser Trp Tyr Leu His Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Leu Tyr Gly Ile Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Asn Thr Ile Lys Pro Glu Asp Leu Gly Met Tyr Tyr Cys Phe Gln Ser 85 90 95 Thr His Gln Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 19 <211> 106 <212> PRT <213> Artificial <220> <223> light chain variable region <400> 19 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Thr Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Tyr Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 20 <211> 106 <212> PRT <213> Artificial <220> <223> light chain variable region <400> 20 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 21 <211> 106 <212> PRT <213> Artificial <220> <223> light chain variable region <400> 21 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 22 <211> 106 <212> PRT <213> Artificial <220> <223> light chain variable region <400> 22 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 23 <211> 106 <212> PRT <213> Artificial <220> <223> light chain variable region <400> 23 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 24 <211> 107 <212> PRT <213> Artificial <220> <223> light chain variable region <400> 24 Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Lys Val Ser Phe Ser Cys Arg Ala Ser Gln Asn Ile Gly Thr Ser 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Glu Phe Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 25 <211> 107 <212> PRT <213> Artificial <220> <223> light chain variable region <400> 25 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Ile Gly Thr Ser 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Phe Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 26 <211> 107 <212> PRT <213> Artificial <220> <223> light chain variable region <400> 26 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Ile Gly Thr Ser 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Glu Phe Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 27 <211> 107 <212> PRT <213> Artificial <220> <223> light chain variable region <400> 27 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Ile Gly Thr Ser 20 25 30 Ile Glu Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Glu Phe Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 28 <211> 107 <212> PRT <213> Artificial <220> <223> light chain variable region <400> 28 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Ile Gly Thr Ser 20 25 30 Ile Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Glu Phe Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 29 <211> 107 <212> PRT <213> Artificial <220> <223> light chain variable region <400> 29 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Ile Gly Thr Ser 20 25 30 Ile Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Glu Phe Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 30 <211> 112 <212> PRT <213> Artificial <220> <223> light chain variable region <400> 30 Asp Ile Val Met Thr Gln Ser Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Gly Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Phe Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Met Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Thr Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 31 <211> 108 <212> PRT <213> Artificial <220> <223> light chain variable region <400> 31 Asp Ile Gln Met Thr Gln Ile Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Pro 85 90 95 Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 32 <211> 114 <212> PRT <213> Artificial <220> <223> light chain variable region <400> 32 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser 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 Phe Cys Gln Gln Gly Tyr Thr Leu Pro Pro 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro <210> 33 <211> 108 <212> PRT <213> Artificial <220> <223> light chain variable region <400> 33 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Glu Ser 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 Phe Cys Gln Gln Gly Tyr Thr Leu Pro Pro 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 34 <211> 114 <212> PRT <213> Artificial <220> <223> light chain variable region <400> 34 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Gln Ser 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 Phe Cys Gln Gln Gly Tyr Thr Leu Pro Pro 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro <210> 35 <211> 114 <212> PRT <213> Artificial <220> <223> light chain variable region <400> 35 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser 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 Phe Cys Gln Gln Tyr Tyr Thr Leu Pro Pro 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro <210> 36 <211> 114 <212> PRT <213> Artificial <220> <223> light chain variable region <400> 36 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser 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 Phe Cys Gln Gln Gly Tyr Ser Leu Pro Pro 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro <210> 37 <211> 330 <212> PRT <213> Artificial <220> <223> heavy chain constant region <400> 37 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 Arg 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 Glu Glu 225 230 235 240 Met 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> 38 <211> 107 <212> PRT <213> Artificial <220> <223> light chain constant region <400> 38 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> 39 <211> 451 <212> PRT <213> Artificial <220> <223> Sacituzumab, heavy chain <400> 39 Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Ser Leu 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 Arg 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 Glu Glu Met 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> 40 <211> 214 <212> PRT <213> Artificial <220> <223> Sacituzumab, light chain <400> 40 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp 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 Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Val 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> 41 <211> 369 <212> DNA <213> Artificial <220> <223> heavy chain variable region <400> 41 caggtccaac tgcagcagcc tggggctgaa ctggtgaagc ctgggtcttc agtgaagctg 60 tcctgcaagg cttctggcta caccttcact agttactgga tgtactgggt gaagcagagg 120 cctggacagg gccttgagtg gattggagag attaatccta gtaacggtcg tactaattac 180 aatgagaagt tcaagagcaa ggccacactg actgtagaca aatcgtccag cacagcctac 240 atgcaattca gcagcctgac atctgaggac tctgcggtct attactgtac aagagaaggc 300 cataattacg atggttccct cggggctatg gaccactggg gtcaaggaac ctcagtcacc 360 gtctcctca 369 <210> 42 <211> 336 <212> DNA <213> Artificial <220> <223> light chain variable region <400> 42 gatgttgtgg tgactcaaac tccactctcc ctgcctgtca gctttggaga tcaggtttct 60 atctcttgca ggtctagtca gagtcttaca aacagttatg ggaacacctt tttgtcttgg 120 tacctgcaca agcctggcca gtctccacag ctcctcctct atgggatttc caacagattt 180 tctggggtgc cagacaggtt cagtggcagt ggttcaggga cagatttcac actcaagatc 240 aacacaataa agcctgagga cctgggaatg tattactgct ttcaaagtac acatcagccg 300 tacacgttcg gaggggggac caagctggaa ataaaa 336 <210> 43 <211> 351 <212> DNA <213> Artificial <220> <223> heavy chain variable region <400> 43 caggttcagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaagatg 60 tcctgcaagg cttctggatt cacattcact gactatgtta taggctgggt gaagcagaga 120 actggacagg gccttgagtg gattggagag atttatcttg gaagtggtac tatttactac 180 actgagaagt tcaagggcaa ggccacactg actgcagaca catcctccaa cacagcctac 240 atgcagctca gcagcctgac gtctgaagac tctgcggtct atttctgtgc aaggggatct 300 attttcccct ttgactactg gggccaaggc accactctca cagtctcctc a 351 <210> 44 <211> 318 <212> DNA <213> Artificial <220> <223> light chain variable region <400> 44 caaattgttc tcacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60 atgacctgca gtgccagctc aagtgtaagt tacatgtact ggtaccagca gaagccagga 120 tcctccccca gactcctgat ttatgacaca tccaccctgg cttctggagt ccctgttcgc 180 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagccgaat ggaggctgaa 240 gatgctgcca cttactactg ccagcagtgg agtagttacc cttacacgtt cggagggggg 300 accaagctgg aaataaaa 318 <210> 45 <211> 351 <212> DNA <213> Artificial <220> <223> heavy chain variable region <400> 45 gaggtgcagc tggtgcagtc tggacccgag gtgaagaagc ctggagcctc cgtgaaggtg 60 tcctgcaagg cctccggctt caccttcacc gactacgtga tcggctgggt gcgacaggct 120 cctggccagg gactggagtg gatcggcgag atctacctgg gctccggcac catctactac 180 accgagaagt tcaagggacg ggtgaccatg acagccgaca cctccacctc caccgcctac 240 atggagctgt cctccctgcg gtccgaggac accgccgtgt actactgcgc tcgaggctcc 300 atcttcccct tcgactactg gggccagggc accctggtga ccgtgtcctc t 351 <210> 46 <211> 318 <212> DNA <213> Artificial <220> <223> light chain variable region <400> 46 gacatccagc tgacccagtc tccctcctcc ctgtctgcct ccgtgggcga cagggtgacc 60 atcacctgct ctgcctcctc ctccgtgtcc tacatgtact ggtaccagca gaagcctggc 120 aaggctccca agctgctgat ctacgacacc tccaccctgg cctctggcgt gccctccagg 180 ttctctggct ccggatctgg caccgacttc accctgacca tctcctccct gcagcccgag 240 gacttcgcca cctactactg ccagcagtgg tcctcctacc cctacacctt cggacagggc 300 accaagctgg agatcaag 318 <210> 47 <211> 351 <212> DNA <213> Artificial <220> <223> heavy chain variable region <400> 47 caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggggcttc agtgaacctg 60 tcctgcaagg cttctggcta caccttcacc agctactgga taaactgggt gaagcagagg 120 cctggacaag gccttgagtg gatcggaaat atttatcctt ctaatagtta tactaactac 180 aatcaaaagt tcaaggacac ggccacattg actgtagaca aatcctccag cacagcctac 240 atgcagctca gcagcccgac atctgaggac tctgcggtct atttctgttc aagttatagg 300 tccgacgggt ttgcttactg gggccaaggg actcttgtca ctgtctctgc a 351 <210> 48 <211> 321 <212> DNA <213> Artificial <220> <223> light chain variable region <400> 48 gacatcttgc tgactcagtc tccagccatc ctgtctgtga gtccaggaga aaaagtcagt 60 ttctcctgca gggccagtca gaacattggc acaagcatac actggtatca gcaaagaaca 120 aatggttctc caaggcttct catagaattt gcttctgagt ctatctctgg gatcccttcc 180 aggtttagtg gcagtggatc agggacagat tttactctta ccatcaacag tgtggagtct 240 gaagatattg cagattatta ctgtcaacaa agtaatagct ggccgttcac gttcggaggg 300 gggaccaagc tggaaataaa a 321 <210> 49 <211> 351 <212> DNA <213> Artificial <220> <223> heavy chain variable region <400> 49 caggtgcagc tggtgcagtc tggagccgag gtgaagaagc ctggagcctc cgtgaaggtg 60 tcctgcaagg cctccggcta caccttcacc tcctactgga tcaactgggt gcggcaggct 120 cctggccagg gactggagtg gatgggcaac atctacccat ccaactccta caccaactac 180 aaccagaagt tcaaggacag ggtgaccatg accagagaca cctccacctc caccgtgtac 240 atggagctgt cctccctgcg gtccgaggac acagccgtgt actactgcgc tcggtaccgg 300 tctgacggct tcgcctactg gggacagggc accctggtga ccgtgtcctc c 351 <210> 50 <211> 321 <212> DNA <213> Artificial <220> <223> light chain variable region <400> 50 gagatcgtgc tgacccagtc tcctgccacc ctgtccctgt ctcctggcga gagagccacc 60 ctgtcctgca gagcctccca gaacatcggc acctccatcc actggtacca gcagaagcct 120 ggccaggctc ctcggctgct gatctacttc gcctccgagt ccatctctgg catccctgct 180 cggttctctg gctccggatc tggcaccgac ttcaccctga ccatctcctc cctggagcct 240 gaggacttcg ccgtgtacta ctgccagcag tccaactcct ggcccttcac cttcggaggt 300 ggcaccaagg tggagatcaa g 321 <210> 51 <211> 345 <212> DNA <213> Artificial <220> <223> heavy chain variable region <400> 51 gaggtgaagc tggtggagtc tgggggagtc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt gactctgcca tgtcttgggt tcgccagact 120 ccagagaaga ggctggagtg ggtcgcatcc attagtcgtg gtgatgacac atattatcca 180 gacagtgtga agggccgaat caccatttcc agagattttg ccagaaacat cctgtatttg 240 caaatgacca gtctgaggtc tgaggacacg gccatgtatt actgtacaag agatcggttc 300 gggtttgctt actggggcca agggactctg gtcactgtct ctgca 345 <210> 52 <211> 336 <212> DNA <213> Artificial <220> <223> light chain variable region <400> 52 gacattgtga tgacccagtc tccactcact ttgtcggtta ccattggaca acctgcctcc 60 atctcttgca agtcaggtca gagcctctta gatagtgatg gaaagacata ttttaattgg 120 ttgttacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc tatgctggac 180 tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240 agcagagtgg agactgagga tttgggagtt tattattgct ggcaaggtac acattttcca 300 ttcacgttcg gctcggggac aaagttggaa ataaag 336 <210> 53 <211> 357 <212> DNA <213> Artificial <220> <223> heavy chain variable region <400> 53 caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60 tcctgtaagg ctgatggcta catcttcacc agttactgga tgcactgggt gaaacagagg 120 cctggacaag gccttgagtg gatcggagag attactcctt ctgataatta tacttcctac 180 aatcaaaagt tcaagggcaa ggccacattg actgtagaca aatcctccag cacagcctac 240 atgcagctca gcagcctgac gtctgaggac tctgcggtct attactgtac aagaggccac 300 ggtaactacg tcagctttga ctactggggc caaggcacca ctctcacagt ctcctca 357 <210> 54 <211> 324 <212> DNA <213> Artificial <220> <223> light chain variable region <400> 54 gacatccaga tgacacagat tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60 atcacttgca gggcaagtca ggacattagc aattatttaa actggtatca gcagaaacca 120 gatggaactg ttaaactcct gatctactac acatcaagat tacactcagg agtcccctca 180 aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240 gaagatattg ccacttactt ttgccaacag ggttatacgc ttcctccgta cacgttcgga 300 ggggggacca agctggaaat aaaa 324 <210> 55 <211> 357 <212> DNA <213> Artificial <220> <223> heavy chain variable region <400> 55 caggtgcagc tggtgcagtc cggagccgag gtgaagaagc ctggagcctc cgtgaaggtg 60 tcctgcaagg cctccggcta caccttcacc tcctactgga tgcactgggt gcggcaggct 120 cctggccagg gactggagtg gatgggcgag atcacaccct ccgacaacta cacctcctac 180 aaccagaagt tcaagggacg ggtgaccatc accagggaca cctccacctc caccgcctac 240 atggagctgt cctccctgcg gtccgaggac accgccgtgt actactgcgc tcgaggccac 300 ggcaactacg tgtccttcga ctactgggga cagggcaccc tggtgaccgt gtcctcc 357 <210> 56 <211> 324 <212> DNA <213> Artificial <220> <223> light chain variable region <400> 56 gacatccaga tgacccagtc tccctcctcc ctgtctgcct ccgtgggaga ccgggtgacc 60 atcacctgca gagcctccca ggacatctcc aactacctga actggtacca gcagaagcct 120 ggcaaggctc ccaagctgct gatctactac acctccaggc tgcactccgg agtgccctcc 180 cggttctccg gctctggctc cggaaccgac ttcaccctga ccatctcctc cctgcagccc 240 gaggacttcg ccacctactt ctgccagcag ggctacaccc tgcctcccta caccttcggc 300 cagggcacca agctggagat caag 324 <210> 57 <211> 357 <212> DNA <213> Artificial <220> <223> heavy chain variable region <400> 57 caggtgcagc tggtgcagtc cggagccgag gtgaagaagc ctggagcctc cgtgaaggtg 60 tcctgcaagg cctccggcta caccttcacc tcctactgga tgcactgggt gcggcaggct 120 cctggccagg gactggagtg gatgggcgag atcacaccct ccgacaacta cggctcctac 180 aaccagaagt tcaagggacg ggtgaccatc accagggaca cctccacctc caccgcctac 240 atggagctgt cctccctgcg gtccgaggac accgccgtgt actactgcgc tcgaggccac 300 ggcaactacg tgtccttcga ctactgggga cagggcaccc tggtgaccgt gtcctcc 357 <210> 58 <211> 324 <212> DNA <213> Artificial <220> <223> light chain variable region <400> 58 gacatccaga tgacccagtc tccctcctcc ctgtctgcct ccgtgggaga ccgggtgacc 60 atcacctgca gagcctccca ggacatctcc aactacctga actggtacca gcagaagcct 120 ggcaaggctc ccaagctgct gatctactac acctccaggc tggagtccgg agtgccctcc 180 cggttctccg gctctggctc cggaaccgac ttcaccctga ccatctcctc cctgcagccc 240 gaggacttcg ccacctactt ctgccagcag ggctacaccc tgcctcccta caccttcggc 300 cagggcacca agctggagat caag 324 <210> 59 <211> 990 <212> DNA <213> Artificial <220> <223> heavy chain constant region <400> 59 gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960 cagaagagcc tctccctgtc cccgggtaaa 990 <210> 60 <211> 321 <212> DNA <213> Artificial <220> <223> light chain constant region <400> 60 agaaccgtgg cggcgccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggtaccgcta gcgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg t 321

Claims

1. (1) preparing hybridoma cells by immunizing an animal with a recombinant Trop-2 protein as an immunogen; (2) screening for positive hybridoma cells secreting anti-Trop-2 monoclonal antibodies using recombinant Trop-2 protein as a coating antigen; (3) rescreening the positive hybridoma cells obtained in step (2) using cells positive for Trop-2 on the cell membrane surface.

1. A method for preparing an anti-Trop-2 monoclonal antibody, comprising: A method in which an anti-Trop-2 monoclonal antibody specifically recognizes and binds to a native epitope in the Trop-2 extracellular domain.

2. 2. The method for preparing an anti-Trop-2 monoclonal antibody according to claim 1, wherein the cells positive for Trop-2 on the cell membrane surface in step (3) are recombinant animal cells derived from the same species as the animal immunized in preparing the hybridoma cells in step (1).

3. 3. The method for preparing an anti-Trop-2 monoclonal antibody according to claim 2, wherein in step (1), hybridoma cells are prepared by immunizing a mouse, and in step (3), the cells positive for Trop-2 on the cell membrane surface are recombinant mouse cells expressing exogenous Trop-2 protein.

4. 2. The method for preparing an anti-Trop-2 monoclonal antibody according to claim 1, wherein in step (2), positive hybridoma cells secreting anti-Trop-2 monoclonal antibodies are screened using enzyme-linked immunosorbent assay (ELISA); and in step (3), rescreening is performed by flow cytometry (FACS) analysis to obtain hybridomas secreting antibodies that specifically recognize and bind to native epitopes of the Trop-2 extracellular domain.

5. Process (4): Identifying antibodies that specifically recognize and bind to native epitopes of the Trop-2 extracellular domain.

2. The method for preparing an anti-Trop-2 monoclonal antibody according to claim 1, further comprising:

6. 6. The method for preparing an anti-Trop-2 monoclonal antibody according to claim 5, wherein in step (4), a monoclonal antibody is selected that has specific binding ability to human Trop-2 and cynomolgus monkey Trop-2 but not to mouse Trop-2.

7. An anti-Trop-2 monoclonal antibody or a fragment thereof obtained by the method for preparing an anti-Trop-2 monoclonal antibody according to any one of claims 1 to 6.

8. The monoclonal antibody is 1×10 -8 Less than M, 5 x 10 -9 Less than M, 1 x 10 -10 Less than M or 5 x 10 -10 8. The anti-Trop-2 monoclonal antibody or fragment thereof of claim 7, which binds to the recombinant human Trop-2 extracellular domain with an affinity measured by a KD value of less than M.

9. An antibody or fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL) comprising a combination of CDRs (HCDR1, HCDR2, HCDR3; and LCDR1, LCDR2, LCDR3) selected from the group consisting of: 。

10. 10. The antibody or fragment thereof according to claim 9, wherein the heavy chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 17, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth, and / or the light chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 18 to 36, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth.

11. 11. The antibody or fragment thereof of claim 9 or 10, wherein the heavy chain variable region and the light chain variable region comprised by the antibody or fragment thereof comprise one of the following: (1) an amino acid sequence set forth in SEQ ID NO:1, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:1; and an amino acid sequence set forth in SEQ ID NO:18, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:18; (2) an amino acid sequence set forth in SEQ ID NO:2, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:2; and an amino acid sequence set forth in SEQ ID NO:19, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:19; (3) an amino acid sequence set forth in SEQ ID NO:3, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:3; and an amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:20; (4) an amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:4; and an amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:20; (5) an amino acid sequence set forth in SEQ ID NO:3, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:3; and an amino acid sequence set forth in SEQ ID NO:21, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:21; (6) an amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:4; and an amino acid sequence set forth in SEQ ID NO:21, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:21; (7) an amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 3; and an amino acid sequence set forth in SEQ ID NO: 22, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 22; (8) an amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 3; and an amino acid sequence set forth in SEQ ID NO: 23, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 23; (9) an amino acid sequence set forth in SEQ ID NO: 5, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 5; and an amino acid sequence set forth in SEQ ID NO: 24, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 24; (10) The amino acid sequence set forth in SEQ ID NO: 6, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 6; and the amino acid sequence set forth in SEQ ID NO: 25, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 25; (11) The amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 7; and the amino acid sequence set forth in SEQ ID NO: 26, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 26; (12) The amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 7; and the amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 27; (13) The amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 8; and the amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 27; (14) The amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 9; and the amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 27; (15) The amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 10; and the amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 27; (16) The amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 7; and the amino acid sequence set forth in SEQ ID NO: 28, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 28; (17) The amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 8; and the amino acid sequence set forth in SEQ ID NO: 28, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 28; (18) The amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 9; and the amino acid sequence set forth in SEQ ID NO: 28, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 28; (19) The amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 10; and the amino acid sequence set forth in SEQ ID NO: 28, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 28; (20) The amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 7; and the amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 29; (21) The amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 8; and the amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 29; (22) The amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 9; and the amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 29; (23) The amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 10; and the amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 29; (24) The amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 11; and the amino acid sequence set forth in SEQ ID NO: 30, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 30; (25) The amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 12; and the amino acid sequence set forth in SEQ ID NO: 31, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 31; (26) The amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 13; and the amino acid sequence set forth in SEQ ID NO: 32, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 32; (27) The amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 16; and the amino acid sequence set forth in SEQ ID NO: 32, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 32; (28) The amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 14; and the amino acid sequence set forth in SEQ ID NO: 33, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 33; (29) The amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 16; and the amino acid sequence set forth in SEQ ID NO: 33, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 33; (30) The amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 14; and the amino acid sequence set forth in SEQ ID NO: 34, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 34; (31) The amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 14; and the amino acid sequence set forth in SEQ ID NO: 35, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 35; (32) The amino acid sequence set forth in SEQ ID NO: 15, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 15; and the amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 36; (33) The amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 14; and the amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 36; and (34) An amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 16; and an amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:

36.

12. It may be in any form, such as a monoclonal antibody, a single chain antibody, a diabody, a single domain antibody, a nanobody, a fully or partially humanized antibody, or a chimeric antibody, or a half antibody or an antigen-binding fragment of a half antibody, such as a scFv, BsFv, dsFv, (dsFv) 2 , Fab, Fab', F(ab') 2 or Fv, Preferably, it further comprises a human or mouse constant region, preferably a human or mouse light chain constant region (CL) and / or a heavy chain constant region (CH), More preferably, the antibody comprises a heavy chain constant region selected from the group consisting of IgG, IgA, IgM, IgD and IgE, and / or a kappa or lambda light chain constant region. An antibody or fragment thereof according to any one of claims 1 to 11.

13. the antibody is a monoclonal antibody, preferably a mouse monoclonal antibody, a chimeric monoclonal antibody or a humanized monoclonal antibody, and preferably the heavy chain constant region of the monoclonal antibody is of the IgG1 or IgG4 subtype, and the light chain constant region of the monoclonal antibody is of the κ type; Preferably, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 37, or an amino acid sequence having at least 75% identity to the amino acid sequence shown, and preferably, the light chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 38, or an amino acid sequence having at least 75% identity to the amino acid sequence shown. An antibody or fragment thereof according to any one of claims 1 to 12.

14. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or fragment thereof according to any one of claims 1 to 13, or encoding the heavy chain CDR, light chain CDR, heavy chain variable region, light chain variable region, heavy chain or light chain contained in said antibody or fragment thereof.

15. 15. A vector comprising the nucleic acid molecule of claim 14.

16. A host cell comprising the nucleic acid molecule of claim 14 and / or the vector of claim 15 or transformed or transfected with the nucleic acid molecule of claim 14 and / or the vector of claim 15.

17. A pharmaceutical composition comprising the antibody or fragment thereof of any one of claims 1 to 13, the nucleic acid molecule of claim 14, the vector of claim 15, or the host cell of claim 16, and optionally a pharmaceutically acceptable excipient.

18. the pharmaceutical composition comprises an additional antibody-based drug; Preferably, the antibody-based further drug is an antibody against a macrophage-associated immune checkpoint, more preferably an anti-CD47 antibody.

18. The pharmaceutical composition of claim 17.

19. Use of an antibody or fragment thereof according to any one of claims 1 to 13, a nucleic acid molecule according to claim 14, a vector according to claim 15, a host cell according to claim 16, and / or a pharmaceutical composition according to claim 17 or 18 in the manufacture of a medicament, comprising: Preferably, the medicament is for treating a cancer with high Trop-2 expression, Preferably, the Trop-2 highly expressing cancer is gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous cell lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial cancer, triple-negative breast cancer or cervical cancer. use.

20. A kit comprising the antibody or fragment thereof of any one of claims 1 to 13, the nucleic acid molecule of claim 14, the vector of claim 15, the host cell of claim 16, or the pharmaceutical composition of claim 17 or 18.

21. A fusion protein comprising the antibody or fragment thereof according to any one of claims 1 to 13.

22. A conjugate comprising the antibody or fragment thereof according to any one of claims 1 to 13 and a drug conjugated thereto, wherein the drug is a cytotoxic agent.

23. The conjugate has the formula: (An antibody or fragment thereof according to any one of claims 1 to 13)-(linker)-(cytotoxic agent) is an antibody drug conjugate (ADC) represented by Preferably, the cytotoxic agent is a tubulin inhibitor (e.g., paclitaxel, docetaxel, etc.) or a DNA replication inhibitor (e.g., irinotecan or its active metabolite SN-38, etc.).

23. The conjugate of claim 22.

24. Use of the antibody or fragment thereof of any one of claims 1 to 13, the nucleic acid molecule of claim 14, the vector of claim 15, or the host cell of claim 16 in the manufacture of an antibody drug conjugate (ADC), comprising: Preferably, the ADC is for treating a cancer with high Trop-2 expression, Preferably, the Trop-2 highly expressing cancer is gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous cell lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial cancer, triple-negative breast cancer or cervical cancer. use.

25. 10. A method for preventing and / or treating a disease, comprising administering to a subject in need thereof the antibody or fragment thereof of any one of claims 1 to 13, the nucleic acid molecule of claim 14, the vector of claim 15, the host cell of claim 16, the pharmaceutical composition of claim 17 or 18, the fusion protein of claim 21, or the conjugate of claim 22 or 23, and optionally other drugs or measures, Preferably, the disease is a cancer with high Trop-2 expression, Preferably, the Trop-2 highly expressing cancer is gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous cell lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial cancer, triple-negative breast cancer or cervical cancer. The method.

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