Anti-human Trop-2 antibodies and their applications

Abstract

This invention provides an antibody or fragment thereof that binds to the human tumor-associated calcium signaling sensor 2 (Trop-2) protein, and the use of said antibody or fragment thereof for the prevention or treatment of diseases. The antibody or fragment thereof of this invention can effectively bind to the human Trop-2 protein and has internalization activity, which is enhanced after ADC drug labeling. Furthermore, its in vivo efficacy and safety in mouse models are no less than those of the control antibody.

Description

[0001] This patent application claims priority to Chinese Patent Application No. CN201910962965.1, filed on October 11, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biomedicine and relates to a novel anti-human Trop-2 antibody or a functional fragment thereof. This invention also relates to the application of said antibody or its functional fragment. Background Technology

[0003] Trop-2 (Tumor-associated calcium signal transducer 2), also known as tumor-associated calcium signal transducer (TACSTD2), epithelial glycoprotein-1 (EGP-1), and gastrointestinal tumor-associated antigen (GA733-1), is a cell surface glycoprotein encoded by the TACSTD2 gene. It is 323 amino acids long and its extracellular region consists of three domains. It has been shown to exist in a dimer form.

[0004] Trop-2 is a transmembrane glycoprotein. Unlike other proto-oncogenes, Trop-2 is not mutated, meaning it does not lead to genetic alterations that cause overexpression. Trop-2 stimulates cell growth through the ERK / MAPK and cyclin D1 pathways, thereby promoting tumor invasion, angiogenesis, tumor progression, and drug resistance. Trop-2 has been found to be highly expressed in various tumors, especially triple-negative breast cancer and non-small cell lung cancer, and is associated with prognosis. Conversely, Trop-2 is expressed at extremely low levels in normal tissues, making it a relatively ideal target for ADC drugs.

[0005] Antibody drugs targeting Trop-2 are currently mainly developed as antibody-drug conjugates (ADCs). Incomplete statistics indicate that more than three are in clinical trials, with small molecule conjugates primarily including irinotecan derivatives and microtubule inhibitors. Currently, the novel, low-toxicity topoisomerase inhibitor metabolite SN-38 is considered ideal. SN-38 exhibits different tumor-suppressive effects compared to existing microtubule inhibitors and DNA alkylating agents, making it particularly suitable for tumors with high heterogeneity and multiple drug resistance mechanisms, such as triple-negative breast cancer, pancreatic cancer, and gastric cancer. The most advanced clinical project is Immunomedics' IMMU-132 project, which has completed a phase III clinical trial for recurrent and metastatic triple-negative breast cancer (ASCENT-Study), a phase II clinical trial for triple-negative breast cancer (NCT02161679) as monotherapy or in combination with carboplatin, a phase II clinical trial for urothelial carcinoma (NCT03547973), and phase I / II clinical trials for solid tumors including gastric cancer, cervical cancer, and small cell lung cancer (NCT01631552). Other antibody-drug conjugate (ADC) projects with similar technologies include those from Daiichi Sankyo, Pfizer, and other pharmaceutical companies with ADC backgrounds.

[0006] Currently, there are relatively few anti-Trop-2 antibodies in clinical trials. Therefore, there is still a need in this field to find novel anti-Trop-2 antibodies that are particularly suitable for developing ADC drugs. Although Trop-2 has shown promising application prospects as a therapeutic target for tumors in previous studies, the unclear structure and biological function of the Trop-2 epitope have resulted in high-affinity monoclonal antibodies obtained in in vitro screenings often exhibiting low tumor-targeting activity and cytotoxic delivery capabilities in in vivo experiments. This has directly led to the fact that no anti-Trop-2 monoclonal antibody drug has yet completed clinical efficacy trials. Summary of the Invention

[0007] Based on the existing mechanisms of Trop-2 as a tumor therapeutic target and the progress of clinical trials of anti-Trop-2 antibody drugs, the inventors, through in-depth research and analysis, discovered that the reason why the effectiveness of anti-Trop-2 antibodies in preclinical and clinical trials did not meet theoretical expectations was not only due to insufficient affinity for Trop-2, but also because the optimal epitope for antibody binding is usually the recombinant protein of Trop-2 or its fragments, rather than the native conformation of Trop-2. Therefore, although high-affinity antibodies against Trop-2 can be screened in vitro, their effective affinity for the native conformation of the extracellular region of Trop-2 in vivo is still insufficient.

[0008] On the one hand, using recombinant proteins as immunogens and coating antigens has the advantage of easy immunogen purification, a single exogenous antigen structure, and easy screening for monoclonal antibodies against the recombinant protein. However, the modifications (e.g., glycosylation) and folding patterns of recombinant expressed proteins are often influenced by the recombinant expression system. Even when using mammalian expression systems, the purified free recombinant Trop-2 or its extracellular region differs from the native conformation of the Trop-2 extracellular region bound to the cell membrane surface. This difference in protein conformation or spatial structure leads to the specificity of antibodies produced by immunization and the effective affinity for the native conformation of the Trop-2 extracellular region in vivo.

[0009] On the other hand, using Trop-2-positive cells on the cell membrane surface as both the immunogen and the screening antigen has the advantage that the Trop-2 extracellular region has a native conformation, and monoclonal antibodies with high affinity for the Trop-2 extracellular region in vitro are expected to also have high affinity for the native conformation of the Trop-2 extracellular region in vivo. However, Trop-2-positive cells on the cell membrane surface are complex in their immunogen antigen composition, which can easily mask the immunogenicity of the Trop-2 extracellular region; furthermore, using Trop-2-positive cells on the cell membrane surface as the screening antigen for flow cytometry screening is costly and has low screening efficiency.

[0010] Based on the aforementioned previous research reports, this invention addresses the advantages and disadvantages of various technical routes for preparing monoclonal antibodies using recombinant Trop-2 protein and Trop-2 positive cells. Using recombinant Trop-2 protein as an immunogen to immunize animals to prepare hybridoma cells avoids the shielding effect on Trop-2 when using complex antigens as immunogens; using recombinant Trop-2 protein as a coating antigen for primary screening via an ELISA screening strategy ensures high throughput; and using membrane-surface Trop-2 positive cells to re-screen the initially positive hybridomas ensures that the obtained positive antibodies can bind to the native conformation of the Trop-2 extracellular region.

[0011] A further technical problem to be solved by this invention is to enable anti-Trop-2 monoclonal antibodies to more closely approximate real-world human in vivo experimental results during preclinical animal testing. To this end, based on the shared structure of the extracellular regions of human and monkey Trop-2, monoclonal antibodies capable of specifically binding to the shared structure of both extracellular regions of human and monkey Trop-2 are obtained through species cross-reactivity screening. To ensure the high specificity of the anti-Trop-2 monoclonal antibody, antibodies with specific binding ability to the extracellular region of mouse Trop-2 are excluded.

[0012] To address the aforementioned technical problems, this invention provides an anti-Trop-2 antibody through hybridoma screening and humanization technology. The antibody has a high affinity for human Trop-2 and exhibits specific killing effects on cancer cells. Simultaneously, the antibody has high internalization capacity, making it particularly suitable for developing ADC drugs.

[0013] Specifically, the present invention provides the following technical solution.

[0014] On one hand, the present invention provides a method for preparing an anti-Trop-2 monoclonal antibody, which includes the following steps:

[0015] (1) Hybridoma cells were prepared by immunizing animals with recombinant Trop-2 protein as an immunogen;

[0016] (2) Use recombinant Trop-2 protein as a coating antigen to screen positive hybridoma cells that secrete anti-Trop-2 monoclonal antibodies;

[0017] (3) Use Trop-2 positive cells on the membrane surface to rescreen the positive hybridomas in step (2);

[0018] The anti-Trop-2 monoclonal antibody specifically recognizes and binds to the natural epitope of the extracellular region of Trop-2.

[0019] Preferably, in the method for preparing anti-Trop-2 monoclonal antibody according to the present invention, the Trop-2 positive cells on the membrane surface in step (3) are recombinant animal cells, and the animal cells are from the same species as the animals immunized when preparing hybridoma cells in step (1).

[0020] More preferably, in the method for preparing anti-Trop-2 monoclonal antibody according to the present invention, hybridoma cells are prepared by immunizing mice in step (1), and Trop-2 positive cells on the membrane surface in step (3) are recombinant mouse cells expressing exogenous Trop-2 protein.

[0021] Preferably, in the method for preparing anti-Trop-2 monoclonal antibody according to the present invention, step (2) uses enzyme-linked immunosorbent assay (ELISA) to screen positive hybridoma cells that secrete anti-Trop-2 monoclonal antibody; step (3) uses flow cytometry (FACS) to rescreen hybridomas that secrete antibodies that specifically recognize and bind to the natural epitope of the extracellular region of Trop-2.

[0022] Preferably, the method for preparing the anti-Trop-2 monoclonal antibody of the present invention further includes (4) a step of identifying antibodies that specifically recognize and bind to the natural epitope of the extracellular region of Trop-2, wherein the identification includes affinity identification and specificity identification.

[0023] Preferably, in the method for preparing the anti-Trop-2 monoclonal antibody of the present invention, step (4) selects a monoclonal antibody that has specific binding ability to human Trop-2 and monkey Trop-2, but no specific binding ability to mouse.

[0024] On the other hand, the present invention provides an antibody or a fragment thereof, the 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 selected from the following (HCDR1, HCDR2, HCDR3; LCDR1, LCDR2, LCDR3):

[0025]

[0026]

[0027] The antibody or a fragment thereof is bound to human Trop-2.

[0028] Preferably, the heavy chain variable region comprises a sequence selected from:

[0029] The amino acid sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:17 or an amino acid sequence having at least 75% identity with the shown amino acid sequence; and / or

[0030] The light chain variable region comprises sequences selected from the following:

[0031] The amino acid sequence shown in any one of SEQ ID NO:18 to SEQ ID NO:36 or an amino acid sequence having at least 75% identity with the shown amino acid sequence.

[0032] According to specific embodiments of the present invention, the heavy chain variable region and light chain variable region comprising the antibody or fragment thereof may be selected from the following combinations:

[0033] (1) An amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:1; and an amino acid sequence as shown in SEQ ID NO:18 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:18.

[0034] (2) An amino acid sequence as shown in SEQ ID NO:2 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:2; and an amino acid sequence as shown in SEQ ID NO:19 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:19.

[0035] (3) An amino acid sequence as shown in SEQ ID NO:3 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:3; and an amino acid sequence as shown in SEQ ID NO:20 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:20;

[0036] (4) An amino acid sequence as shown in SEQ ID NO:4 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:4; and an amino acid sequence as shown in SEQ ID NO:20 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:20;

[0037] (5) An amino acid sequence as shown in SEQ ID NO:3 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:3; and an amino acid sequence as shown in SEQ ID NO:21 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:21.

[0038] (6) An amino acid sequence as shown in SEQ ID NO:4 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:4; and an amino acid sequence as shown in SEQ ID NO:21 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:21.

[0039] (7) An amino acid sequence as shown in SEQ ID NO:3 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:3; and an amino acid sequence as shown in SEQ ID NO:22 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:22;

[0040] (8) An amino acid sequence as shown in SEQ ID NO:3 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:3; and an amino acid sequence as shown in SEQ ID NO:23 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:23;

[0041] (9) An amino acid sequence as shown in SEQ ID NO:5 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:5; and an amino acid sequence as shown in SEQ ID NO:24 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:24;

[0042] (10) An amino acid sequence as shown in SEQ ID NO:6 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:6; and an amino acid sequence as shown in SEQ ID NO:25 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:25.

[0043] (11) An amino acid sequence as shown in SEQ ID NO:7 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:7; and an amino acid sequence as shown in SEQ ID NO:26 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:26.

[0044] (12) An amino acid sequence as shown in SEQ ID NO:7 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:7; and an amino acid sequence as shown in SEQ ID NO:27 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:27;

[0045] (13) An amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:8; and an amino acid sequence as shown in SEQ ID NO:27 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:27;

[0046] (14) An amino acid sequence as shown in SEQ ID NO:9 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:9; and an amino acid sequence as shown in SEQ ID NO:27 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:27.

[0047] (15) An amino acid sequence as shown in SEQ ID NO:10 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:10; and an amino acid sequence as shown in SEQ ID NO:27 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:27.

[0048] (16) An amino acid sequence as shown in SEQ ID NO:7 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:7; and an amino acid sequence as shown in SEQ ID NO:28 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:28.

[0049] (17) An amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:8; and an amino acid sequence as shown in SEQ ID NO:28 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:28.

[0050] (18) An amino acid sequence as shown in SEQ ID NO:9 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:9; and an amino acid sequence as shown in SEQ ID NO:28 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:28.

[0051] (19) An amino acid sequence as shown in SEQ ID NO:10 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:10; and an amino acid sequence as shown in SEQ ID NO:28 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:28.

[0052] (20) An amino acid sequence as shown in SEQ ID NO:7 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:7; and an amino acid sequence as shown in SEQ ID NO:29 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:29;

[0053] (21) An amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:8; and an amino acid sequence as shown in SEQ ID NO:29 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:29.

[0054] (22) An amino acid sequence as shown in SEQ ID NO:9 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:9; and an amino acid sequence as shown in SEQ ID NO:29 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:29;

[0055] (23) An amino acid sequence as shown in SEQ ID NO:10 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:10; and an amino acid sequence as shown in SEQ ID NO:29 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:29.

[0056] (24) An amino acid sequence as shown in SEQ ID NO:11 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:11; and an amino acid sequence as shown in SEQ ID NO:30 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:30.

[0057] (25) An amino acid sequence as shown in SEQ ID NO:12 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:12; and an amino acid sequence as shown in SEQ ID NO:31 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:31.

[0058] (26) An amino acid sequence as shown in SEQ ID NO:13 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:13; and an amino acid sequence as shown in SEQ ID NO:32 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:32;

[0059] (27) An amino acid sequence as shown in SEQ ID NO:16 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:16; and an amino acid sequence as shown in SEQ ID NO:32 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:32.

[0060] (28) An amino acid sequence as shown in SEQ ID NO:14 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:14; and an amino acid sequence as shown in SEQ ID NO:33 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:33.

[0061] (29) An amino acid sequence as shown in SEQ ID NO:16 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:16; and an amino acid sequence as shown in SEQ ID NO:33 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:33.

[0062] (30) An amino acid sequence as shown in SEQ ID NO:14 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:14; and an amino acid sequence as shown in SEQ ID NO:34 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:34.

[0063] (31) An amino acid sequence as shown in SEQ ID NO:14 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:14; and an amino acid sequence as shown in SEQ ID NO:35 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:35.

[0064] (32) An amino acid sequence as shown in SEQ ID NO:15 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:15; and an amino acid sequence as shown in SEQ ID NO:36 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:36.

[0065] (33) An amino acid sequence as shown in SEQ ID NO:14 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:14; and an amino acid sequence as shown in SEQ ID NO:36 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:36.

[0066] (34) An amino acid sequence as shown in SEQ ID NO:16 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:16; and an amino acid sequence as shown in SEQ ID NO:36 or an amino acid sequence having at least 75% identity with an amino acid sequence as shown in SEQ ID NO:36.

[0067] Generally, the antibody or fragment thereof is any form of monoclonal antibody, single-chain antibody, bifunctional antibody, single-domain antibody, nanobody, fully or partially humanized antibody, or chimeric antibody; or, the antibody or fragment thereof is a hapten or an antigen-binding fragment of a hapten, such as scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv; the antibody or fragment thereof may be from mouse, rat, human, or any other source.

[0068] Preferably, the antibody or fragment thereof further comprises a human or mouse constant region, and more preferably comprises a human or mouse light chain constant region (CL) and / or heavy chain constant region (CH);

[0069] More preferably, the antibody or fragment thereof comprises a heavy chain constant region selected from IgG, IgA, IgM, IgD or IgE and / or a κ or λ type light chain constant region.

[0070] According to a specific embodiment of the present invention, the antibody is a monoclonal antibody, preferably a mouse-derived, chimeric, or humanized monoclonal antibody; preferably, the heavy chain constant region of the monoclonal antibody is IgG1 or IgG4 subtype, and the light chain constant region is κ type.

[0071] According to a specific embodiment of the present invention, the heavy chain constant region of the monoclonal antibody contains an amino acid sequence as shown in SEQ ID NO:37 or an amino acid sequence having at least 75% identity with the amino acid sequence.

[0072] Preferably, the light chain constant region of the monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO:38 or an amino acid sequence having at least 75% identity with the amino acid sequence.

[0073] The above-described at least 75% identity refers to any percentage of identity of at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99%, etc., ≥75%.

[0074] Based on the antibody or fragment thereof of the present invention, the present invention also provides a nucleic acid molecule that encodes the heavy chain CDR, light chain CDR, heavy chain variable region, light chain variable region, heavy chain, or light chain in any antibody or fragment thereof of the present invention.

[0075] In another aspect, the present invention provides a vector comprising the nucleic acid molecule of the present invention. The vector may be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a bacteriophage vector, etc.

[0076] The vector or nucleic acid molecule of the present invention can be used to transform or transfect host cells or enter host cells in any way for purposes such as preserving or expressing antibodies.

[0077] Therefore, in another aspect, the present invention provides a host cell containing the nucleic acid molecules and / or vectors of the present invention, or the host cell being transformed or transfected by the nucleic acid molecules and / or vectors of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as bacterial or insect, fungal, plant, or animal cells.

[0078] Based on the disclosure of this invention, the antibodies or fragments thereof, nucleic acid molecules, vectors, and / or host cells provided by this invention can be obtained using any conventional techniques known in the art. The antibodies or fragments thereof, nucleic acid molecules, vectors, and / or host cells can be included in pharmaceutical compositions, and more particularly in pharmaceutical formulations, for use in various purposes as needed.

[0079] Therefore, in another aspect, the present invention also provides a pharmaceutical composition comprising the antibody or a fragment thereof described in the present invention, a nucleic acid molecule, a carrier and / or a host cell, and optionally pharmaceutically acceptable excipients.

[0080] The antibodies or fragments thereof of the present invention can be used in combination with other antibody-based drugs that have macrophage phagocytic activity. Therefore, preferably, the antibody-based drugs promote macrophage phagocytosis of cells by binding to proteins expressed on the cell surface. Therefore, the pharmaceutical compositions provided by the present invention may also contain the other antibody-based drugs, preferably macrophage-based immune checkpoint antibodies; according to a specific embodiment of the present invention, the antibody is an anti-CD47 antibody.

[0081] The present invention also provides related applications of the above-mentioned subject matter as antibodies that bind to human Trop-2 or any part thereof.

[0082] Specifically, in another aspect, the present invention provides the use of the antibody or its fragment, nucleic acid molecule, vector, host cell and / or pharmaceutical composition in the preparation of a medicament, wherein the medicament is preferably used to treat Trop-2 highly expressing cancers; preferably, the Trop-2 highly expressing cancers are gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous cell lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial carcinoma, triple-negative breast cancer or cervical cancer.

[0083] In this respect, the uses encompass the use of the antibodies or fragments thereof of the present invention in combination with other antibody-based drugs described above to prepare the drug.

[0084] The antibodies or fragments thereof provided by this invention can also be fused or conjugated with other components. For example, this invention provides a fusion protein or conjugate comprising the antibody or fragment of this invention.

[0085] Regarding fusion proteins, the fusion protein may comprise any other portion, such as an amino acid, polypeptide, or protein, that modifies the antibody or fragment thereof described in this invention.

[0086] Regarding the conjugate, the conjugate may comprise the antibody or a fragment thereof of the present invention and a drug conjugated thereto, wherein the drug is, for example, a cytotoxic agent.

[0087] Preferably, the conjugate is an antibody-drug conjugate (ADC) of the following formula: (antibody or fragment thereof of the present invention) - (linker) - (cytotoxic agent);

[0088] Preferably, the cytotoxic agent is a microtubule inhibitor (such as paclitaxel, docetaxel, etc.) or a DNA replication inhibitor (such as irinotecan or its metabolite SN-38, etc.).

[0089] According to a specific embodiment of the present invention, the conjugate is an "anti-TROP-2 antibody-linker-SN-38 antibody drug conjugate".

[0090] The present invention also provides the use of the antibody or fragment thereof, nucleic acid molecule, vector, host cell and / or pharmaceutical composition in the preparation of antibody-drug conjugates (ADCs), wherein the antibody-drug conjugates are preferably used to treat Trop-2 highly expressing cancers; preferably, the Trop-2 highly expressing cancers are gastric cancer, pancreatic cancer, colorectal cancer, ovarian cancer, squamous cell lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial carcinoma, triple-negative breast cancer or cervical cancer.

[0091] Furthermore, the present invention provides a method for preventing and / or treating a disease, the method comprising administering to a subject in need an antibody or fragment thereof, a nucleic acid molecule, a vector, a host cell, a pharmaceutical composition, a fusion protein or conjugate, and optionally other drugs or means. These optional other drugs or means refer to other drugs or means that can be administered in combination with the antibody or fragment thereof, a nucleic acid molecule, a vector, a host cell, a pharmaceutical composition, a fusion protein or conjugate, such as small molecule drugs, targeted drugs, recombinant protein drugs such as antibodies, vaccines, ADCs, oncolytic viruses, gene and nucleic acid therapeutics, and radiotherapy. The combined administration can be carried out in any form, such as simultaneously, continuously, or at intervals.

[0092] Preferably, the disease is a Trop-2 highly expressed cancer; more preferably, the Trop-2 highly expressed cancer is gastric cancer, pancreatic cancer, colorectal cancer, ovarian cancer, squamous cell lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial carcinoma, triple-negative breast cancer, or cervical cancer. The subject is a mammal, preferably a human.

[0093] Unless otherwise stated, the term "immunoglobulin sequence" is used as a general term to include full-size antibodies, their individual chains, and all their parts, domains, or fragments (including, but not limited to, antigen-binding domains or fragments such as the VHH domain or the VH / VL domain).

[0094] The term "antibody" should be understood to encompass an antibody molecule (i.e., a "complete antibody molecule") comprising two immunoglobulin heavy chains and two immunoglobulin light chains, as well as its antigen-binding fragment. As used herein, the terms "antigen-binding portion," "antigen-binding fragment," and similar terms include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the terms "antigen-binding fragment" or "antibody fragment" refer 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 a CDR, or isolated CDRs. The antigen-binding fragment of an antibody can be derived from, for example, a complete antibody molecule using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA-encoded antibody variable and (where applicable) constant domains. This type of DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or may be synthetic. DNA can be sequenced and manipulated chemically or using molecular biology techniques, such as arranging one or more variable and / or constant domains into a suitable configuration, or introducing codons; forming cysteine ​​residues; modifying, adding, or removing amino acids, etc.

[0095] 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) the smallest recognition unit consisting of amino acid residues of the hypervariable region of a mimic antibody (e.g., a separated complementarity-determining region (CDR), such as a CDR3 peptide), or a restricted FR3-CDR3-FR4 peptide. Other engineered molecules, such as those described herein as “antigen-binding fragments,” are also included: domain-specific antibodies, mono-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-grafted antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, microantibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains.

[0096] Antigen-binding fragments of antibodies typically contain at least one variable domain. Variable domains can have any size or amino acid composition and will generally contain at least one CDR adjacent to or in frame with one or more framework sequences. In antigen-binding fragments where the VH and VL domains associate, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be dimer and contain VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody can contain a monomeric VH or VL domain.

[0097] In some embodiments, the antigen-binding fragment of the antibody may contain 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 within the antigen-binding fragment of the antibody of the present invention 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 listed above, the variable and constant domains may be directly bonded to each other or may be bonded through full or partial hinge or connector regions. Hinge regions may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which create flexible or semi-flexible bonds between adjacent variable and / or constant domains within a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of the present invention may comprise any of the variable and constant domain configurations listed above that are non-covalently associated with each other and / or homodimers or heterodimers (or other multimers) with one or more monomeric VH or VL domains (e.g., via disulfide bonds).

[0098] For a complete antibody molecule, the antigen-binding fragment can be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically contain at least two distinct variable domains, each capable of specifically binding to a single antigen or different epitopes on the same antigen. Any multispecific antibody format (including the exemplary bispecific antibody formats disclosed herein) is suitable for use in the context of antigen-binding fragments of the antibodies of the present invention using conventional techniques available in the art.

[0099] The term "chimeric antibody" refers to an antibody in which (a) a constant region or a portion thereof is altered, substituted, or exchanged such that the antigen-binding site (variable region, CDR, or a portion thereof) is linked to a constant region of a different or altered type, effector function, and / or species; or (b) a variable region or a portion thereof is altered, substituted, or exchanged with a variable region of different or altered antigen specificity (e.g., a CDR and frame region from a different species). Chimeric antibodies may contain variable region fragments, for example, recombinant antibodies containing two Fab or Fv regions or scFv. As indicated above, chimeras may also contain Fc regions from a different source than the Fv region to which they are bound. In some cases, chimeric antibodies contain chimeras located within Fv regions. Examples of such chimeric antibodies are humanized antibodies in which the Fvs and CDRs are from different sources.

[0100] The term "humanized antibody" refers to an antibody in which the antigen-binding loop, or CDR, obtained from the VH and VL regions of a non-human antibody is transplanted into a human frame sequence. Humanization can be performed according to methods described in the following literature, namely, replacing the corresponding sequence of the human antibody with the non-human CDR sequence, for example, U.S. Patent Nos. 5,545,806; 5,569,825; 5,633,425; 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); Fishwild et al., Nature Biotechnology 14:845-51 (1996). Transgenic mice or other organisms, such as other mammals, can also be used to express humanized or human antibodies, as disclosed in U.S. Patent No. 6,673,986.

[0101] As used herein, the term "percentage (%) identity" refers to the percentage of amino acid (or nucleic acid) residues identical to those of the reference sequence, after alignment and the introduction of blanks, where necessary to achieve maximum percentage identity (i.e., blanks may be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences may be used for comparison purposes). The candidate sequence, such as the isolated anti-IL1-RAP antibody of the present invention, contains the percentage of amino acid (or nucleic acid) residues identical to those of the reference sequence. Alignment for determining percentage identity can be performed using BLAST 2.0 software with standard settings. Alignment can be performed to achieve maximum alignment over the full length of the comparison sequences. In some embodiments, the percentage amino acid (or nucleic acid) sequence identity of a given candidate sequence with, and or to a given reference sequence (which may optionally be expressed as a given candidate sequence having or containing a certain percentage of amino acid (or nucleic acid) sequence identity with, and or to the given reference sequence) is calculated using the following formula:

[0102] 100 × (fraction of A / B)

[0103] Where A represents the number of amino acid (or nucleic acid) residues that score identically in the alignment of the candidate and reference sequences, and B represents the total number of amino acid (or nucleic acid) residues 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 percentage amino acid (or nucleic acid) sequence identity between the candidate and reference sequences will not be equal to the percentage amino acid (or nucleic acid) sequence identity between the reference and candidate sequences.

[0104] In certain embodiments, a reference sequence aligned for comparison with a candidate sequence may exhibit 50%-100% identity across the full length of the candidate sequence or a selected portion of a consecutive amino acid (or nucleic acid) residue. The length of the candidate sequence aligned for comparison purposes is at least 30%, for example, at least 40%, for example, at least 50%, 60%, 70%, 80%, 90%, or 100% of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleic acid) residue as the corresponding position in the reference sequence, the molecules are identical at that position.

[0105] As used herein, the terms “antigen,” “immunogen,” “antibody target,” and “target analyte,” etc., refer to molecules, compounds, or complexes that can be recognized by antibodies, i.e., that can be specifically bound by antibodies. The term can refer to any molecule that can be specifically recognized by antibodies, such as peptides, polynucleotides, carbohydrates, lipids, chemical moieties, or combinations thereof (e.g., phosphorylated or glycosylated peptides). Those skilled in the art will understand that the term does not imply that the molecule is immunogenic in every case, but simply indicates that it can be targeted by antibodies.

[0106] The terms "separated" or "isolated" refer to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" product, it may be due to an alteration of its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in a living animal, and a high-purity, identical polynucleotide or polypeptide separated from this natural state is called a separated product. The terms "separated" or "isolated" do not exclude the presence of artificial or synthetic substances, nor do they exclude the presence of other impurities that do not affect the substance's activity.

[0107] The term "host cell" refers to cells that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, insect cells such as S2 Drosophila cells or Sf9 cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0108] The term "KD" refers to the dissociation equilibrium constant (KD) of a specific antibody-antigen interaction, used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. Typically, antibodies have a KD less than approximately 10-1. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 An antigen bound by a dissociation equilibrium constant of M or less, for example, as determined using surface plasmon resonance (SPR) in a BIACORE instrument. For example, the affinity of antibody-cell binding, detected on a KINEXA400 instrument using the KINEXA method.

[0109] The term "specific binding" refers to an antibody reacting with one or more antigenic determinants of an antigen without reacting with other peptides or binding to other peptides with very low affinity (Kd > 10⁻⁶). Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single-chain, Fab, Fab' and F(ab')2 fragments, Fv, scFv, and Fab expression libraries. Monoclonal antibodies (mAbs) are antibodies obtained from a single clonal cell line, which is not limited to eukaryotic, prokaryotic, or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained through recombinant techniques such as hybridoma technology, recombinant technology, phage display technology, and synthetic techniques such as CDR grafting or other existing technologies.

[0110] The term "Trop-2" refers to TROP2, a cell surface glycoprotein encoded by the TACSTD2 gene, which belongs to the TACSTD family. It is also known as tumor-associated calcium signal transducer 2 (TACSTD2), epidermal glycoprotein 1 (EGP-1), gastrointestinal tumor-associated antigen (GA733-1), and surface marker 1 (M1S1). Trop-2 is overexpressed in various malignant tumors and is an oncogene associated with the occurrence, invasion, and metastasis of malignant tumors.

[0111] The Trop-2 gene is located on the short arm of chromosome 1, specifically at 1p32.1[3]. The gene is 9072 bp long, has no introns, and has only one exon. The sequence similarity between mouse Trop-2 and human homologous gene is 87.4%. The primary structure of Trop-2 protein is a 36 kDa polypeptide composed of 323 amino acids, which is a single transmembrane surface glycoprotein. Trop-2 consists of a hydrophobic leader peptide (AA1-26), an extracellular domain (AA27-274), a transmembrane domain (AA275-297), and a cytoplasmic tail (AA298-323). The N-terminus of the TROP2 protein is the extracellular domain (ECD), which is connected to the intracellular short tail (IC) by a one-way transmembrane helix (TM), thereby fixing it to the cell membrane. Its cytoplasmic tail contains a highly conserved phosphatidylinositol 4,5-bisphosphate (PIP2) binding sequence, indicating that PIP2 plays an important role in TROP2 signal transduction. In addition to the PIP2 binding motif, it also contains conserved tyrosine and serine phosphorylation sites. A mutation at serine residue 303 eliminates the ability of Trop-2 to stimulate tumor growth. Phosphorylation of this residue is controlled by protein kinase C (PKC).

[0112] The term "antibody-drug conjugate" (ADC) refers to a small molecule drug with biological activity linked to a monoclonal antibody via a chemical link. The monoclonal antibody then acts as a carrier to target and deliver the small molecule drug to the target cells.

[0113] Antibody molecules in ADC drugs often employ humanized monoclonal antibodies, modifying the crystallizable fragment (Fc) to reduce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). First, as biological macromolecules, antibodies carry the general toxicity risks associated with other biological macromolecules, such as immunogenicity and immunotoxicity, as well as the potential ADCC, CDC, and renal basement membrane immune complex deposition associated with monoclonal antibodies. Second, the most important function of antibody molecules in ADC drugs is targeting, specifically delivering small molecule compounds to the antigen-antibody binding site. If the antibody has poor selectivity or the antigen is present in normal tissue, the cytotoxic drug may be delivered to normal cells, causing targeted toxicity. Third, in addition to targeted toxicity, the shedding of small molecules during circulation can lead to off-target toxicity. If the Fc of the antibody molecule has activity in binding to Fc receptors on immune cells, such as FcγRs / FcRNs, it readily binds to immune cells, causing them to be killed. Finally, as exogenous biological macromolecules, ADC drugs may also engulf cells in circulation and enter the cell via pinocytosis, causing cell death.

[0114] Common linkers used in ADC drugs include hydrazone bonds, disulfide bonds, and peptide bonds. Hydrazone bonds are relatively unstable and can hydrolyze under acidic conditions. Mylotarg used hydrazone bonds as a linker, which researchers believe was a major reason for its failure. Disulfide bonds can hydrolyze in high concentrations of glutathione within cells, making them less prone to extracellular detachment. Peptide bonds are the most tightly bound, breaking only under the action of lysosomal proteolytic enzymes. The stability of the linker directly affects the unintended dissociation of cytotoxic drugs; such breakage leads to the exposure of small-molecule cytotoxic drugs in vivo, i.e., off-target toxicity.

[0115] The cytotoxic drugs commonly used in ADC (anti-adrenergic cytotoxicity) therapy are routinely used chemotherapy drugs in clinical practice, which determine the main toxicity spectrum of ADC drugs. Because they are widely used clinically, their toxicity characteristics are generally well understood. The toxicity risk can be well assessed based on the drug type, such as microtubule polymerization inhibitors or DNA damage agents / DNA replication inhibitors. Microtubule inhibitors include auristatins (MMAE, MMAF, MMAD) and their derivatives, maytansinoids (DM1, DM2, DM3, DM4), paclitaxel and its derivatives (docetaxel), docetaxel, vincristine, etc. DNA damage agents / DNA replication inhibitors include irinotecan or its metabolite SN-38, etc.

[0116] "Optional," "optionally," "any," or "any" means that the event or circumstance described below may, but does not have to, occur, including the possibility or absence of such event or circumstance. For example, "optionally contains one antibody heavy chain variable region" means that the antibody heavy chain variable region of a particular sequence may, but does not have to, be present.

[0117] The term "pharmaceutical composition" refers to a mixture containing one or more of the compounds described in this invention or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, promote the absorption of the active ingredient, and thereby exert its biological activity. Therapeutic compositions should generally be sterile and stable under the conditions of manufacture and storage. Compositions can be formulated as solutions, microemulsions, dispersants, liposomes, or other ordered structures suitable for high antibody concentrations. A sterile injectable solution can be prepared by incorporating the active compound (i.e., the antibody or antibody moiety) in the required amount along with one or more of the components listed above in a suitable solvent, followed by filtration and sterilization as needed.

[0118] The methods, compositions, and combination therapies of this invention can be combined with other active agents or therapeutic modalities. The methods include administering to a subject an amount effective in treating or preventing disease (e.g., cancer) of the anti-Trop-2 antibody molecule described herein, optionally in combination with one or more inhibitors of PD-1, PD-L1, PD-L2, LAG-3, CTLA-4, Tim-3 antibodies (immunotherapy) or other tumor therapeutic antibodies, Her-2, EGFR, VEGF, VEGFR antibodies, etc., and ADCs (antibody-drug conjugates, such as T-DM1), bispecific antibodies, chemotherapeutic agents, etc., and may also include administering additional active agents or all of them in amounts or doses higher than, lower than, or equal to the amount or dose of each active agent used alone (e.g., as a monotherapy). The amount or dose of the additional active agent or all of them administered is lower than the amount or dose of each active agent used alone (e.g., as a monotherapy) (e.g., at least 20%, at least 30%, at least 40%, or at least 50%).

[0119] This invention provides a novel anti-human Trop-2 antibody with excellent biological activity: the antibody (including chimeric and humanized antibodies) provided by this invention can effectively bind to both recombinant Trop-2 protein and Trop-2 antigen expressed on the cell surface, similar to the control antibody Sacituzumab. Furthermore, the antibody provided by this invention has a high affinity for human Trop-2: compared to the control antibody Sacituzumab, the humanized antibody of this invention even has a higher specific binding ability to human Trop-2 protein, exhibiting a higher affinity than Sacituzumab. Therefore, the antibody of this invention has good therapeutic efficacy.

[0120] Experiments have demonstrated that the antibody of this invention also possesses excellent internalization ability: the internalization rate of the humanized antibody is similar to that of the control antibody Sacituzumab; and the internalization ability is significantly enhanced after being labeled as an ADC. Therefore, the antibody of this invention has the potential to be used in the development of ADC drugs. The Trop-2 antibody of this invention can also have synergistic effects with other antibodies; for example, the antibody of this invention can be used in combination with CD47 to further promote the phagocytosis of tumor cells by macrophages.

[0121] Furthermore, the antibodies of this invention have demonstrated good in vivo efficacy. ADCs prepared using the antibodies of this invention were found to have a dose-dependent inhibitory effect on tumor growth. At high doses (10 mg / kg), the efficacy of each ADC antibody was comparable to that of the control antibody Sacituzumab, and no significant toxic effects of the ADC small molecule SN38 were observed. The weight gain of animals in each experimental group was stable, with no significant difference from the control. Attached Figure Description

[0122] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0123] Figure 1 The results of screening for binding of positive hybridoma supernatant to Trop-2 on the surface of CHO cells were shown.

[0124] Figure 2 The results of screening for binding of positive hybridoma supernatant to Trop-2 on the surface of CHO cells were shown.

[0125] Figure 3 The results show the cross-reactivity of the supernatant of ELISA-positive hybridoma clones with recombinant Trop-2 from different species.

[0126] Figure 4 The results of ELISA detection of the binding activity between the anti-human Trop-2 chimeric antibody and the recombinant Trop-2 protein are shown, among which... Figure 4 A: ch3-11; Figure 4 B: ch4-3; Figure 4 C: ch23-12; Figure 4 D: ch11-4; Figure 4 E: ch17-1.

[0127] Figure 5 The results of FACS detection of the binding activity of anti-human Trop-2 chimeric antibody to recombinant Trop-2 protein on the cell surface are shown, among which... Figure 5 A: ch3-11; Figure 5 B: ch23-12; Figure 5 C: ch11-4; Figure 5 D: ch4-3; Figure 5 E: ch17-1.

[0128] Figure 6 The results of ELISA detection of species-specific binding of anti-human Trop-2 antibody to Trop-2 are shown, among which... Figure 6 A: h23-12; Figure 6 B: h4-3; Figure 6 C: Sacituzumab.

[0129] Figure 7 The results of affinity analysis of the anti-human Trop-2 antibody for the recombinant extracellular region of human Trop-2 are shown, among which... Figure 7 A: Sacituzumab; Figure 7 B: h23-12; Figure 7 C:h4-3.

[0130] Figure 8The results show the internalization of Trop-2 on the surface of N87 cells after the humanized anti-Trop-2 antibody binds to it.

[0131] Figure 9 The drug-time curves (Trop-2 assay) of a single dose of the anti-Trop-2 humanized antibody in nude mice are shown. Figure 9 A: h23-12; Figure 9 B: h4-3.

[0132] Figure 10 The inhibition rate of cell growth by the anti-Trop-2-ADC antibody was shown.

[0133] Figure 11 The curves showing the changes in body weight in Balb / C nu tumour cancer-bearing N87 mouse models are displayed. Figure 11 A: After administration of ch4-3-SN38; Figure 11 B: After administration of h23-12-SN38; Figure 11 C: After administration of isotype control antibody; Figure 11 D: High doses of ch4-3-SN38 and h23-12-SN38 were administered.

[0134] Figure 12 The curves showing tumor volume changes in the Balb / C nu tumescent gastric cancer N87 mouse model are displayed. Figure 12 A: After administration of ch4-3-SN38; Figure 12 B: After administration of h23-12-SN38; Figure 12 C: After administration of isotype control antibody; Figure 12 D: High doses of ch4-3-SN38 and h23-12-SN38 were administered.

[0135] Figure 13 The curves showing the changes in body weight in SKOV3 subcutaneous xenograft mouse models using a combination of anti-Trop2 antibody and anti-CD47 antibody are presented.

[0136] Figure 14 The tumor volume change curves of the SKOV3 subcutaneous xenograft mouse model using a combination of anti-Trop2 antibody and anti-CD47 antibody are shown.

[0137] Figure 15 The tumor volume change curve of the Balb / C nu subcutaneous xenograft mouse model of gastric cancer NCI-N87 is shown.

[0138] Figure 16 The curve showing the change in body weight of mice in the Balb / C nu subcutaneous xenograft mouse model of gastric cancer NCI-N87 is displayed.

[0139] The best way to implement an invention

[0140] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0141] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available products.

[0142] Example 1 Preparation of anti-human Trop-2 antibody hybridoma cells

[0143] Immunization: Balb / c mice were immunized with human Trop-2 recombinant protein (serum number: NP_002344.2, 1aa-274aa). Serum titers were measured by ELISA using 96-well microplates coated with human Trop-2-his recombinant protein (serum number: NP_002344.2, 1aa-274aa). Mice with serum titers meeting the fusion requirements were used for the next step of cell fusion.

[0144] Cell fusion and hybridoma preparation: Mice with the required titer were selected and subjected to a shock immunization. Three days later, the spleens of the mice were aseptically harvested, and a suspension of B lymphocytes was prepared. This suspension was mixed with SP2 / 0 myeloma cells at a ratio of 4:1, and the two cell types were fused under PEG. The fused cells were resuspended in HAT medium and aliquoted into 96-well cell culture plates. The plates were incubated at 37°C in a 5% CO2 incubator.

[0145] Example 2 Screening of anti-human Trop-2 antibody-positive hybridoma cell lines

[0146] 1. Positive hybridoma combination screening

[0147] 10-14 days after fusion, the microplates were coated with human Trop-2-his recombinant protein (sequence number: NP_002344.2, 1aa-274aa) (20ng / ml) and incubated overnight at 4°C. After washing three times with PBS, the plates were blocked with 4% skim milk powder-PBS and incubated at room temperature for 1 hour. After washing three times with PBS, the hybridoma clone culture supernatant was added and the plates were incubated at room temperature for 1 hour. The following controls were set up: (1) Positive control (PC): serum from immunized mice (diluted 1:1000 with PBS); (2) Negative control (NC): fusion wells without cell growth. Wash three times with PBST (0.05% Tween-PBS), then twice with PBS. Add HRP-goat anti-mouse IgG (Fcγ) and incubate at 37°C for 0.5 hours. Wash three more times with PBST (0.05% Tween 20-PBS), add TMB chromogenic solution, and incubate in the dark for 15-30 minutes. Add ELISA stop solution to terminate the reaction. Read the A450 value using a microplate reader.

[0148] The top 95 clones with the highest read values ​​were selected for secondary ELISA confirmation, from highest to lowest. Twenty-five antibody-secreting positive cell pools were selected for subcloning using limiting dilution. Ten days after plating, the supernatant from single-clone cells was further screened for positive clones using ELISA, following the same ELISA method. The top 21 clones with the highest read values ​​(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 further FACS-based screening, from highest to lowest read values.

[0149] 2. Screening by binding of positive hybridomas to Trop-2 on the surface of CHO cells.

[0150] The reading frame of the Trop-2 gene was cloned from a vector containing Trop-2 cDNA (Cat.:HG10428-M, Beijing Yiqiao Shenzhou) by PCR. The cloned gene was then inserted into a stable expression vector containing a glutamyl synthase (GS) selection gene via enzyme digestion. CHO-K1 cells cultured in suspension were electroporated (Nucleofector IIb, Lonza). The transfected cells were then seeded in CD CHO AGTTM medium (Cat.:12490-025, Gibco) containing 50 μM MSX (Cat.:M5379, Sigma) and seeded in 96-well cell culture plates. The cells were incubated statically at 37°C and 5% CO2 for 2-3 weeks. Pre-screening using MSX pressure was performed, and 22 cell growth wells were obtained. These were then scaled up to 24-well cell culture plates. Finally, clone 1-T-21 (CHO / Trop-2 cells) was selected by flow cytometry (FACS) for further amplification, cryopreservation, and FACS analysis.

[0151] Based on the above ELISA results, the hybridoma supernatant of the 21 clones was diluted 100-fold and incubated with the constructed CHO cell (CHO / Trop-2 cell) suspension at 37°C for 30 min. The following controls were set: (1) Positive control (PC): mouse IgG constant region form of Sacituzumab, 1 ug / ml; (2) Negative control (NC): irrelevant mouse antibody, 1 ug / ml. After washing the cells three times with PBS, goat anti-mouse IgG-FITC (Cat.:F9006, Sigma) diluted 1:200 was added and incubated for 30 min. After washing the cells three times with PBS, the mean fluorescence intensity (MFI) of the cells was detected by flow cytometry (model B49007AD, SNAW31211, BECKMAN COULTER) to verify whether the antibody secreted by the hybridoma could bind to Trop-2 on the surface of CHO cells. The results are shown in the figure. Figure 1 .

[0152] according to Figure 1Based on cell status, 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. The hybridoma supernatant was purified using a ProA affinity chromatography column, and the purified mouse antibodies were reconfirmed by binding. The antibodies were diluted to 13 nM and 0.66 nM, respectively, and then incubated with a suspension of recombinant human Trop-2-expressing CHO cells (CHO / Trop-2 cells) at 37°C for 30 min. The following controls were set up: (1) Positive control (PC): mouse IgG constant region form of Sacituzumab, 1 ug / ml; (2) Negative control (NC): irrelevant mouse antibody, 1 ug / ml. After washing cells three times with PBS, a 1:200 dilution of goat anti-mouse IgG-FITC (Cat.: F9006, Sigma) was added and incubated for 30 min. After washing cells three times with PBS, the mean fluorescence intensity (MFI) of the cells was measured by flow cytometry (model B49007AD, SNAW31211, BECKMANCOULTER) to verify whether the antibody secreted by the hybridoma could bind to Trop-2 on the surface of CHO cells. Figure 2 As shown, antibodies from the supernatants of 15 clones bound well to Trop-2 on the surface of CHO cells.

[0153] m3-11, m4-3, m11-4, m17-1, and m23-12 were selected as candidate clones for further screening.

[0154] 3. ELISA screening for species crossover of positive hybridoma clones

[0155] Human Trop-2-His recombinant protein (serial number: NP_002344.2, 1aa-274aa), cynomolgus monkey Trop-2-His recombinant protein (serial number: UniProtKB-A0A2K5UE71, 1aa-272aa), and mouse Trop-2-His recombinant protein (Cat.:50922-M08H, Beijing Yiqiao Shenzhou) were coated overnight at 4℃ with coating concentrations of 0.2 and 1 μg / mL, respectively. After washing the plate 3 times with PBS, 5% BSAPBS was added, and the plate was blocked at 37℃ for 60 min. The plate was washed 3 times with PBST. The purified mouse antibodies of the above 15 strains were diluted to 1 μg / mL with 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:39). (1) Mouse IgG constant region form of NO:40, 1ug / ml; (2) Negative control (NC): irrelevant hybridoma antibody, 1μg / mL; (3) Blank control: PBS. Incubate at 37℃ for 60min, wash 4 times with PBST; add 1:5000 diluted HRP-goat anti-mouse IgG (Fcr) (Cat:115-035-071, Jackson Immuno Research), incubate at 37℃ for 30min, wash 4 times with PBST; add TMB substrate for color development, incubate at 37℃ for 10min, then add 2M HCl to terminate the reaction; use 630nm as the reference wavelength, read and record the absorbance A450nm-630nm of the well plate at a wavelength of 450nm. Except for antibodies from clones m12-4, m17-1, m19-5, and m21-1, which showed cross-linking with mouse Trop-2, the others did not show cross-linking with mouse antibodies. However, all hybridoma antibodies could specifically bind to recombinant human and cynomolgus monkey Trop-2. Figure 3 ).

[0156] Example 3 Sequencing of murine anti-human Trop-2 antibody

[0157] Hybridoma cells m3-11, m4-3, m11-4, m17-1, and m23-12, which secrete anti-human Trop-2 antibodies, were expanded and cultured. Subtype detection was performed using the Mouse Monoclonal Antibody IgG Subclass Test Card (Cat.: A12403, VicNovo) and the Mouse Monoclonal Antibody Light / Heavy Chain Test Card (Cat.: A12401, VicNovo) according to the reagent operating procedures. The subtype identification was: heavy chain IgG1, light chain Kappa chain. This provides a basis for cloning the antibody genes of m3-11, m4-3, m11-4, m17-1, and m23-12.

[0158] Total RNA was extracted from hybridoma cells m3-11, m4-3, m11-4, m17-1, and m23-12 according to the instructions of the TRIzol kit (Cat.: 15596026, Invitrogen). The total RNA was reverse transcribed into cDNA using M-MuLV reverse transcriptase (Cat.: M0253S, NEB). Degenerate primers (refer to the book [Dong Zhiwei, Wang Yan. Antibody Engineering (Second Edition). Beijing Medical University Press, 2001, 313-314]) and the Phusion kit (Cat.: E0553L, NEB) were used to amplify the antibody light chain variable region IgVL(κ) and heavy chain variable region V. H Sequence; PCR amplification products were purified using a gel extraction kit (Cat.: AP-GX-250, Axygen); the amplified PCR products were ligated into a T vector and transformed into competent E. coli cells according to the instructions of the T vector cloning kit (Cat.: ZC205, Zhuangmeng Biotechnology); after amplification and plasmid extraction, DNA sequencing was performed to obtain the variable region sequence of the monoclonal antibody.

[0159] Sequencing results show:

[0160] The nucleotide sequence of the variable region DNA of the heavy chain of the mouse antibody cloned m3-11 is shown in SEQ ID NO:41. The amino acid sequence of the variable region of the heavy chain of the mouse antibody cloned m3-11, deduced from this DNA sequence, is shown in SEQ ID NO:1. The nucleotide sequence of the variable region DNA of the light chain of the mouse antibody cloned m3-11 is shown in SEQ ID NO:42. The amino acid sequence of the variable region of the light chain of the mouse antibody cloned m3-11, deduced from this DNA sequence, is shown in SEQ ID NO:18.

[0161] SEQ ID NO:1:

[0162] QVQLQQPGAELVKPGSSVKLSCKASGYTFT SYWMY WVKQRPGQGLEWIG EINPSNGRTNYNEKFKS KATLTVDKSSSTAYMQFSSLTSEDSAVYYCTR EGHNYDGSLGAMDH WGQGTSVTVSSSEQ ID NO:18:

[0163] DVVVTQTPLSLPVSFGDQVSISC RSSQSLTNSYGNTFLS WYLHKPGQSPQLLLY GISNRFS GVPDRFSGSGSGTDFTLKINTIKPEDLGMYYC FQSTHQPYT FGGGTKLEIK

[0164] The nucleotide sequence of the variable region DNA of the heavy chain of the mouse antibody cloned from m4-3 is shown in SEQ ID NO:43. The amino acid sequence of the variable region of the heavy chain of the mouse antibody cloned from m4-3, deduced from this DNA sequence, is shown in SEQ ID NO:2. The nucleotide sequence of the variable region DNA of the light chain of the mouse antibody cloned from m4-3 is shown in SEQ ID NO:44. The amino acid sequence of the variable region of the light chain of the mouse antibody cloned from this DNA sequence, deduced from this DNA sequence, is shown in SEQ ID NO:19.

[0165] SEQ ID NO:2:

[0166] QVQLQQSGPELVKPGASVKMSCKASGFTFT DYVIG WVKQRTGQGLEWIG EIYLGSGTIYYTEKFKG KATLTADTSSNTAYMQLSSLTSEDSAVYFCAR GSIFPFDY WGQGTTLTVSS

[0167] SEQ ID NO:19:

[0168] QIVLTQSPAIMSASPGEKVTMTC SASSSVSYMY WYQQKPGSSPRLLIY DTSTLAS GVPVRFSGSGSGTSYSLTISRMEAEDAATYYC QQWSSYPYT FGGGTKLEIK

[0169] The nucleotide sequence of the variable region DNA of the heavy chain of the mouse antibody cloned m11-4 is shown in SEQ ID NO:47. The amino acid sequence of the variable region of the heavy chain of the mouse antibody cloned m11-4, deduced from this DNA sequence, is shown in SEQ ID NO:5. The nucleotide sequence of the variable region DNA of the light chain of the mouse antibody cloned m11-4 is shown in SEQ ID NO:48. The amino acid sequence of the variable region of the light chain of the mouse antibody cloned m11-4, deduced from this DNA sequence, is shown in SEQ ID NO:24.

[0170] SEQ ID NO:5:

[0171] QVQLQQPGAELVRPGASVNLSCKASGYTFT SYWIN WVKQRPGQGLEWIG NIYPSNSYTNYNQKFKD TATLTVDKSSSTAYMQLSSPPTSEDSAVYFCSS YRSDGFAY WGQGTLVTVSA

[0172] SEQ ID NO:24:

[0173] DILLTQSPAILSVSPGEKVSFSC RASQNIGTSIH WYQQRTNGSPRLLIE FASESIS GIPSRFSGSGSGTDFTLTINSVESEDIADYYC QQSNSWPFT FGGGTKLEIK

[0174] The nucleotide sequence of the variable region DNA of the heavy chain of the mouse antibody clone m17-1 is shown in SEQ ID NO:51, and the amino acid sequence of the variable region of the heavy chain of the mouse antibody clone m17-1, deduced from this DNA sequence, is shown in SEQ ID NO:11; the nucleotide sequence of the variable region DNA of the light chain of the mouse antibody clone m17-1 is shown in SEQ ID NO:52, and the amino acid sequence of the variable region of the light chain of the mouse antibody clone m17-1, deduced from this DNA sequence, is shown in SEQ ID NO:30.

[0175] SEQ ID NO:11:

[0176] EVKLVESGGVLVKPGGSLKLSCAASGFTFS DSAMS WVRQTPEKRLEWVA SISRGDDTYYPDSVKG RITISRDFARNILYLQMTSLRSEDTAMYYCTR DRFGFAY WGQGTLVTVSA

[0177] SEQ ID NO:30:

[0178] DIVMTQSPLTLSVTIGQPASISC KSGQSLLDSDGKTYFN WLLQRPGQSPKRLIY LVSMLDS GVPDRFTGSGSGTDFTLKISRVETEDLGVYYC WQGTHFPFT FGSGTKLEIK

[0179] The nucleotide sequence of the variable region DNA of the mouse antibody heavy chain of clone m23-12 is shown in SEQ ID NO:53. The amino acid sequence of the variable region of the mouse antibody heavy chain of clone m23-12, deduced from this DNA sequence, is shown in SEQ ID NO:12. The nucleotide sequence of the variable region DNA of the mouse antibody light chain of clone m23-12 is shown in SEQ ID NO:54. The amino acid sequence of the variable region of the mouse antibody light chain of clone m23-12, deduced from this DNA sequence, is shown in SEQ ID NO:31.

[0180] SEQ ID NO:12:

[0181] QVQLQQPGAELVKPGASVKLSCKADGYIFT SYWMH WVKQRPGQGLEWIG EITPSDNYTSYNQKFKG KATLTVDKSSSTAYMQLSSLTSEDSAVYYCTR GHGNYVSFDY WGQGTTLTVSS

[0182] SEQ ID NO:31:

[0183] DIQMTQITSSLSASLGDRVTITC RASQDISNYLN WYQQKPDGTVKLLIY YTSRLHS GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC QQGYTLPPYT FGGGTKLEIK

[0184] Example 4 Preparation of anti-human Trop-2 chimeric antibody and control antibody

[0185] The light and heavy chain sequences of the control antibody (Sacituzumab) were fully synthesized. The light and heavy chain sequences were cloned separately into eukaryotic transient expression vectors to obtain light and heavy chain expression plasmids of the control antibody. These plasmids were transformed into *E. coli* for amplification, and a large number of plasmids containing the light and heavy chains of the control antibody were isolated. Using these plasmids and following the instructions of the transfection reagent 293fectin (Cat.:12347019, Gibco), the light and heavy chain plasmids of the control antibody were transformed into HEK293 cells for recombinant expression. Five to six days after cell transfection, the culture supernatant was collected and purified using 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 heavy chain amino acid sequence is shown in SEQ ID NO:39, and the light chain amino acid sequence is shown in SEQ ID NO:40.

[0186] The light chain variable region and heavy chain variable region genes of the corresponding murine antibodies 3-11, 4-3, 11-4, 17-1, and 23-12 obtained from each clone were introduced into restriction enzyme sites via PCR and cloned into eukaryotic transient expression vectors containing the coding genes for the human-kappa light chain constant region and the human IgG1 heavy chain constant region, respectively. This yielded human-mouse chimeric light chains (pKN019-ch3-11L, pKN019-ch4-3L, pKN019-ch11-4L, pKN019-ch17-1L, pKN019-ch23-12L) and human-mouse chimeric heavy chains (pKN041-ch...). Expression plasmids (pKN019-ch4-3H, pKN019-ch11-4H, pKN019-ch17-1H, pKN019-ch23-12H) were transfected into *E. coli* for amplification. A large number of plasmids containing the light and heavy chains of human-mouse chimeric antibodies were isolated. Using these plasmids and following the instructions of the transfection reagent 293fectin (Cat.: 12347019, Gibco), the light and heavy chain plasmids of chimeric antibodies ch3-11, ch4-3, ch11-4, ch17-1, and ch23-12 were transfected into HEK293 cells for recombinant expression. Five to six days after cell transfection, the culture supernatant was collected and purified using a ProA affinity chromatography column to obtain the chimeric antibodies ch3-11, ch4-3, ch17-1, ch11-4, and ch23-12.

[0187] Example 5 ELISA detection of the binding activity between anti-human Trop-2 chimeric antibody and recombinant Trop-2 protein

[0188] Human Trop-2-his recombinant protein (sequence number: NP_002344.2, 1aa-274aa) at a concentration of 0.2 μg / mL was coated overnight at 4℃ and blocked with 5% BSA at 37℃ for 60 min. Ch3-11, ch4-3, ch17-1, ch11-4, ch23-12 and control antibody Sacituzumab (initial concentration 2 μg / mL, 3-fold serial dilution, 8 gradients) were added, and the reaction was incubated at 37℃ for 60 min. The plate was washed 4 times with PBST; then, 1:5000 diluted HRP-anti-human Fc (Cat.:109-035-098, Jackson Immuno Research) was added, and the reaction was carried out for 45 min. TMB (Cat.:ME142, Beijing Taitianhe Biotechnology) substrate was added for color development for 15 min, and the reaction was stopped with 2M HCl before reading the plate. Using 630nm as the reference wavelength, the absorbance value A450nm-630nm of the well plate at a wavelength of 450nm was read and recorded.

[0189] The binding affinity of ch3-11, ch4-3, ch17-1, ch11-4, ch23-12 and the control antibody Sacituzumab to recombinant human Trop-2 protein was determined by ELISA. The half-maximal effective binding concentrations (EC50) 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 showed that the chimeric antibodies ch3-11, ch4-3, ch17-1, ch11-4, and ch23-12 had high affinity for human Trop-2 recombinant protein, and the sequences of mouse antibodies 3-11, 4-3, 11-4, 17-1, and 23-12 were correctly cloned.

[0190] Example 6 FACS detection of the binding activity of anti-human Trop-2 chimeric antibody to recombinant human Trop-2 protein on the surface of CHO cells

[0191] Recombinant human Trop-2-expressing CHO cell suspensions (CHO / Trop-2 cells) were incubated with chimeric antibodies (ch3-11, ch4-3, ch17-1, ch11-4, ch23-12) (concentrations of 30 μg / mL, 10 μg / mL, and 5 μg / mL, initially 3-fold serial dilutions, 9 gradients in total, for a total of 11 gradients) at 37°C for 30 min. The following controls were set up: (1) Positive control (PC): control antibody Sacituzumab; (2) Negative control (NC): IgG1 isotype control antibody NC-IgG1. After washing the cells 3 times with PBS, goat anti-human IgG-FITC (Cat.:F9512, Sigma) diluted 1:100 was added and incubated for 30 min. After washing the cells three times with PBS, the mean fluorescence intensity (MFI) of the cells was detected by flow cytometry (model B49007AD, SNAW31211, BECKMAN COULTER) to detect the binding ability of the chimeric antibody to human Trop-2 on the surface of CHO cells.

[0192] The binding affinity of ch3-11, ch4-3, ch17-1, ch11-4, ch23-12, and the control antibody Sacituzumab to recombinant human Trop-2 protein on the surface of CHO cells was determined by FACS. The half-maximal effective binding concentrations (EC50) 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 showed better binding activity, while ch4-3, ch17-1, and ch23-12 showed similar binding activity. The results indicate that the anti-human Trop-2 chimeric antibodies ch3-11, ch4-3, ch17-1, ch11-4, and ch23-12 can effectively bind to the recombinant human Trop-2 protein on the surface of CHO cells.

[0193] Example 7 The internalization activity of anti-human Trop-2 chimeric antibodies against cell surface Trop-2

[0194] Human pancreatic cancer cells BxPC-3, 5×10 5For each cell / tube, add chimeric antibodies ch3-11, ch4-3, ch11-4, ch23-12 diluted to 10 μg / ml, and positive control antibody Sacituzumab. Each antibody is divided into four groups (experimental group for 1h, 3h, and 5h incubation, and control group), with 2 tubes in each group. The experimental group was placed in a 37℃ thermostatic incubator and incubated for 1 h, 3 h, and 5 h, respectively, before being placed on ice. The control group was incubated on ice continuously as a negative control. After all samples were incubated, they were centrifuged at 1,500 rpm and 4℃ for 3 min, the supernatant was discarded, and the cells were washed once with ice-cold PBS. Secondary antibody, namely anti-human IgG (Fc specific)-FITC antibody (Cat.:F9512, Sigma), was added, and the cells were incubated on ice for 30 min. After centrifugation at 1,500 rpm for 3 min, the supernatant was discarded, and the cells were washed with ice-cold PBS. 200 μL of ice-cold PBS was used to resuspend the cells, and the mean fluorescence intensity (MFI) was detected by FACS. The internalization efficiency was calculated using the following formula: %MFI at time tx = MFI of samples incubated at 37℃ × 100% / MFI of control samples incubated at 4℃; Internalization percentage at time tx = 100% - %MFI at time tx.

[0195] The results are shown in Table 1. The internalization rates of ch4-3 and ch23-12 were similar to those of the control antibody Sacituzumab, while there was no significant internalization in ch3-11 and ch11-4.

[0196] Table 1. Percentage of internalization of anti-human Trop-2 chimeric antibodies mediated by Trop-2 on cell surface

[0197]

[0198] Example 8 Stability of tolerance disruption of anti-human Trop-2 chimeric antibodies

[0199] Chimeric antibodies ch3-11, ch4-3, ch11-4, and ch23-12 were placed at a concentration of 5 mg / mL in PBS, PBS containing 10% N,N-dimethylacetamide (DMA) (Cat.: ARK2190, Shanghai Feibo Chemical), and PBS containing 20% ​​DMA, respectively. After incubation at 37°C for 2 h, DMA was removed from the samples using ultrafiltration centrifuge tubes, and the replacement buffer was replaced with PBS. The purity of the samples was analyzed by high performance size exclusion chromatography (SEC-HPLC) using a G3000Wxl liquid chromatography column (Cat.: SEC-0046, Tosoh). The purity analysis results are shown in Table 2.

[0200] The results showed that all four antibodies were well tolerant to DMA. The purity decreased slightly at 10% and slightly at 20%, suggesting that the antibodies may have good tolerance to the subsequent ADC process.

[0201] Table 2. HPLC purity analysis of antibodies before and after DMA treatment

[0202] ch23-12 ch3-11 ch4-3 ch11-4 PBS 97.52％ 97.57％ 99.47％ 99.25％ 10% DMA 97.18％ 96.29％ 98.44％ 99.18％ 20% DMA 93％ 96.27％ 94％ 98.96％

[0203] Example 9 Humanization and recombinant expression of anti-human Trop-2 monoclonal antibody

[0204] 1. Humanization of mouse monoclonal antibody 23-12

[0205] (1) CDR porting

[0206] First, a comprehensive analysis of the mouse antibody heavy chain sequence was performed to identify the antigen complementarity determinant (CDR) region for antibody-antigen binding and the framework region supporting the conserved three-dimensional conformation of the antibody. Then, based on homology alignment results, the most similar human antibody template was searched in the human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX). Combining the full-sequence BLAST results and the characteristics of the heavy chain CDR3 sequence, CDR transplantation was performed, achieving full humanization of the 23-12 heavy chain variable region (VH) within the framework region. Based on homology alignment results, the most similar human antibody template was searched in the human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX). Combining the full-sequence BLAST results and the characteristics of the light chain CDR3 sequence, CDR transplantation was performed, achieving high humanization of the light chain framework region.

[0207] The nucleotide sequence of the humanized heavy chain variable region h23-12_VH1 of the 23-12 antibody CDR transplantation is shown in SEQ ID NO:55, and the amino acid sequence is shown in SEQ ID NO:13; the nucleotide sequence of the humanized light chain variable region h23-12_VL1 is shown in SEQ ID NO:56, and the amino acid sequence is shown in SEQ ID NO:32.

[0208] SEQ ID NO:13:

[0209] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQGLEWMG EITPSDNYTSYNQKFKG RVTITRDTSTSTAYMELSSLRSEDTAVYYCAR GHGNYVSFDY WGQGTLVTVSS

[0210] SEQ ID NO:32:

[0211] DIQMTQSPSSLSASVGDRVTITC RASQDISNYLN WYQQKPGKAPKLLIY YTSRLHS GVPSRFSGSGSGTDFTLTISSLQPEDFATYFC QQGYTLPPYT FGQGTKLEIKRTVAAP

[0212] (2) CDR region mutation design

[0213] Based on the sequence characteristics of the murine antibody 23-12, mutations were designed in the humanized light and heavy chain variable region CDR sequence of CDR transplantation. The mutation sites are shown in Table 3 below.

[0214] Table 3. Humanized sequence design for 23-12

[0215]

[0216] Note: Amino acid residue sites are numbered according to the Kabat numbering system.

[0217] 2. Recombinant expression of humanized monoclonal antibody 23-12

[0218] The humanized h23-12 antibody light and heavy chain variable regions (h23-12_VL1, h23-12_VH1) were totalized. The humanized h23-12_VH1 was cloned upstream of the heavy chain constant region coding gene of human IgG1 in the eukaryotic transient expression vector pKN041 via enzyme digestion. The nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO:59, and the amino acid sequence is shown in SEQ ID NO:37. Similarly, the humanized h23-12_VL1 was cloned upstream of the human light chain Cκ coding gene in the eukaryotic transient expression vector pKN019 via enzyme digestion. The nucleotide sequence of the light chain constant region is shown in SEQ ID NO:60, and the amino acid sequence is shown in SEQ ID NO:60. NO:38, construct humanized 23-12 light and heavy chain expression vectors, obtain light chain (pKN019-h23-12L1) and heavy chain (pKN041-h23-12H1) expression plasmids, transform into E. coli for amplification, and isolate the h23-12 antibody light chain and heavy chain plasmids h23-12L1 and h23-12H1.

[0219] According to the mutation design shown in Table 3, site-directed mutagenesis was performed on the light chain (pKN019-h23-12L1) and heavy chain (pKN041-h23-12H1) expression plasmids using the StarMut gene site-directed mutagenesis kit (GenStar, Cat.:T111-01). The resulting plasmids were transformed into *E. coli* for amplification, yielding h23-12 antibody light and heavy chain CDR region mutant expression plasmids (h23-12H2~h23-12H7, h23-12L2~h23-12L7), corresponding to the 23-12 humanized sequences in Table 3. Following the instructions for the transfection reagent 293fectin (Cat.:12347019, Gibco), the light and heavy chain plasmids of the 23-12 humanized antibody were combined (see Table 4) and then transformed into HEK293 cells for recombinant expression.

[0220] Table 4. Humanized 23-12 light and heavy chain sequence combinations

[0221] h23-12H1 h23-12H2 h23-12H3 h23-12H4 h23-12H5 h23-12H6 h23-12H7 h23-12L1 h23-12-1 h23-12-2 h23-12-3 h23-12-4 h23-12-5 h23-12-6 h23-12-7 h23-12L2 h23-12-8 h23-12-9 h23-12-10 h23-12-11 h23-12-12 h23-12-13 h23-12-14 h23-12L3 h23-12-15 h23-12-16 h23-12-17 h23-12-18 h23-12-19 h23-12-20 h23-12-21 h23-12L4 h23-12-22 h23-12-23 h23-12-24 h23-12-25 h23-12-26 h23-12-27 h23-12-28 h23-12L5 h23-12-29 h23-12-30 h23-12-31 h23-12-32 h23-12-33 h23-12-34 h23-12-35 h23-12L6 h23-12-36 h23-12-37 h23-12-38 h23-12-39 h23-12-40 h23-12-41 h23-12-42 h23-12L7 h23-12-43 h23-12-44 h23-12-45 h23-12-46 h23-12-47 h23-12-48 h23-12-49

[0222] Note: This table represents the sequences obtained by various combinations of the 23-12 light and heavy chains. For example, h23-12-1 indicates that the antibody is composed of the 23-12 humanized antibody light chain h23-12L1 and the humanized heavy chain h23-12H1, and so on.

[0223] Five to six days after cell transfection, the culture supernatant was collected and purified using a ProA affinity chromatography column. Different humanized antibodies against 23-12 were obtained. Antibody affinity was determined using a Fortebio Octet QKe system with an anti-human antibody Fc fragment capture antibody (AHC) bioprobe to capture the antibody Fc fragment. For the assay, the 23-12 antibody and the control antibody Sacituzumab were diluted to 4 μg / mL with PBS buffer and flowed through the AHC probe (Cat.: 18-0015, PALL) for 120 s. Human Trop-2-His recombinant protein (serial number: NP_002344.2, 1aa-274aa) was used as the mobile phase at a concentration of 60 nM. The binding time was 100 s, and the dissociation time was 300 s. After the experiment was completed, the blank control response value was subtracted, and the kinetic constant of antigen-antibody binding was calculated by fitting the 1:1 Langmuir binding mode using software.

[0224] The affinity of the h23-12 mutant combination antibody, the chimeric antibody ch23-12, and the control antibody Sacituzumab for human Trop-2-His recombinant protein was determined by ForteBio (Table 5).

[0225] Table 5. Affinity assay results of different antibodies 23-12 with recombinant human Trop-2 extracellular region protein.

[0226]

[0227]

[0228] h23-12-25 was selected. The affinity (KD) of this combination antibody is 5.02E-10M. It was named h23-12 and further functional verification was performed. The nucleotide sequence of the heavy chain variable region of this antibody is shown in SEQ ID NO:57, and the amino acid sequence is shown in SEQ ID NO:14; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO:58, and the amino acid sequence is shown in SEQ ID NO:33.

[0229] SEQ ID NO:14:

[0230] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQGLEWMG EITPSDNYGSYNQKFKG RVTITRDTSTSTAYMELSSLRSEDTAVYYCAR GHGNYVSFDY WGQGTLVTVSS

[0231] SEQ ID NO:33:

[0232] DIQMTQSPSSLSASVGDRVTITC RASQDISNYLN WYQQKPGKAPKLLIY YTSRLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYFC QQGYTLPPYT FGQGTKLEIK

[0233] 3. Humanization of mouse monoclonal antibody 4-3

[0234] (1) CDR porting

[0235] First, a comprehensive analysis of the mouse antibody heavy chain sequence was performed to identify the antigen complementarity determinant (CDR) region for antibody-antigen binding and the framework region supporting the conserved three-dimensional conformation of the antibody. Then, based on homology alignment results, the most similar human antibody template was searched in the human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX). Combining the full-sequence BLAST results and the characteristics of the heavy chain CDR3 sequence, CDR transplantation was performed, achieving full humanization of the 4-3 heavy chain variable region (VH) within the framework region. Conversely, based on homology alignment results, the most similar human antibody template was searched in the human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX). Combining the full-sequence BLAST results and the characteristics of the light chain CDR3 sequence, CDR transplantation was performed, achieving full humanization of the light chain framework region.

[0236] The nucleotide sequence of the humanized heavy chain variable region h4-3_VH1 of the 4-3 antibody CDR transplantation is shown in SEQ ID NO:45, and the amino acid sequence is shown in SEQ ID NO:3; the nucleotide sequence of the humanized light chain variable region h4-3_VL1 is shown in SEQ ID NO:46, and the amino acid sequence is shown in SEQ ID NO:20.

[0237] SEQ ID NO:3:

[0238] EVQLVQSGPVKKPGASVKVSCKASGFTFT DYVIG WVRQAPGQGLEWIG EIYLGSGTIYYTEKFKG RVTMTADTSTSTAYMELSSLRSEDTAVYYCAR GSIFPFDY WGQGTLVTVSS

[0239] SEQ ID NO:20:

[0240] DIQLTQSPSSLSASVGDRVTITC SASSSVSYMY WYQQKPGKAPKLLIY DTSTLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQWSSYPYT FGQGTKLEIK

[0241] (2) CDR region mutation design

[0242] Based on the sequence characteristics of the murine antibody 4-3, mutations were designed in the humanized light and heavy chain variable region CDR sequence of CDR transplantation. The mutation sites are shown in Table 6 below.

[0243] Table 6.4-3 Humanized Sequence Design

[0244]

[0245]

[0246] Note: Amino acid residue sites are numbered according to the Kabat numbering system.

[0247] 4. Recombinant expression of humanized monoclonal antibody 4-3

[0248] The humanized h4-3 antibody light and heavy chain variable regions (h4-3_VL1, h4-3_VH1) were fully synthesized. The humanized h4-3_VH1 was cloned upstream of the heavy chain constant region coding gene of human IgG1 in the eukaryotic transient expression vector pKN041 via enzyme digestion. The nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO:59, and the amino acid sequence is shown in SEQ ID NO:37. Similarly, the humanized h4-3_VL1 was cloned upstream of the human light chain Cκ coding gene in the eukaryotic transient expression vector pKN019 via enzyme digestion. The nucleotide sequence of the light chain constant region is shown in SEQ ID NO:60, and the amino acid sequence is shown in SEQ ID NO:60. NO:38, construct humanized 4-3 light and heavy chain expression vectors, obtain light chain (pKN019-h4-3L1) and heavy chain (pKN041-h4-3H1) expression plasmids, transform into E. coli for amplification, and isolate the h4-3 antibody light chain and heavy chain plasmids h4-3L1 and h4-3H1.

[0249] According to the mutation design shown in Table 6, site-directed mutagenesis was performed on the light chain (pKN019-h4-3L1) and heavy chain (pKN041-h4-3H1) expression plasmids using the StarMut gene site-directed mutagenesis kit (Cat.:T111-01, GenStar). The resulting plasmids were then transformed into *E. coli* for amplification, yielding h4-3 antibody light and heavy chain CDR region mutant expression plasmids (h4-3H2~h4-3H4, h4-3L2~h4-3L5), corresponding to the 4-3 humanized sequences in Table 6. Following the instructions for the 293fectin transfection reagent (Cat.:12347019, Gibco), the light and heavy chain plasmids of the 4-3 humanized antibody were combined (see Table 7) and then transformed into HEK293 cells for recombinant expression.

[0250] Table 7. Humanized 4-3 light and heavy chain sequence combinations

[0251] h4-3 H1 h4-3 H2 h4-3 H3 h4-3 H4 h4-3L1 h4-3-1 h4-3-2 h4-3-3 h4-3-4 h4-3L2 h4-3-5 h4-3-6 h4-3-7 h4-3-8 h4-3L3 h4-3-9 h4-3-10 h4-3-11 h4-3-12 h4-3L4 h4-3-13 h4-3-14 h4-3-15 h4-3-16 h4-3L5 h4-3-17 h4-3-18 h4-3-19 h4-3-20

[0252] Note: This table represents the sequences obtained by various combinations of the 4-3 light and heavy chains. For example, h4-3-1 indicates that the antibody is composed of the 4-3 humanized antibody light chain h4-3L1 and the humanized heavy chain h4-3H1, and so on.

[0253] Five to six days after cell transfection, the culture supernatant was collected and purified using a ProA affinity chromatography column. Different humanized antibodies (4-3) were obtained. Antibody affinity was determined using a Fortebio Octet QKe system instrument, employing an anti-human antibody capture antibody (AHC) bioprobe to capture the Fc fragment of the antibody. For the assay, the 4-3 antibody and the control antibody Sacituzumab were diluted to 4 μg / mL with PBS buffer and flowed through the AHC probe (Cat.: 18-0015, PALL) for 120 s. Human Trop-2-His recombinant protein (serial number: NP_002344.2, 1aa-274aa) was used as the mobile phase at a concentration of 60 nM. The binding time was 100 s, and the dissociation time was 300 s. After the experiment, the blank control response value was subtracted, and the antigen-antibody binding kinetic constant was calculated using a 1:1 Langmuir binding model fitting.

[0254] The affinity of the h4-3 mutant combination antibody, the chimeric antibody ch4-3, and the control antibody Sacituzumab for human Trop-2-His recombinant protein was determined by ForteBio (Table 8).

[0255] Table 8.4-3 Affinity assay results of different humanized antibodies with recombinant human Trop-2 extracellular region protein

[0256] Antibody combination KD value (M) Antibody combination KD value (M) Sacituzumab 7.23E-10 h4-3-10 7.63E-10 ch4-3 3.00E-10 h4-3-11 8.02E-10 h4-3-1 3.04E-10 h4-3-12 1.03E-09 h4-3-2 4.11E-10 h4-3-13 2.81E-10 h4-3-3 5.01E-10 h4-3-14 5.71E-10 h4-3-4 6.36E-10 h4-3-15 6.87E-10 h4-3-5 2.73E-10 h4-3-16 1.53E-09 h4-3-6 3.97E-10 h4-3-17 5.42E-10 h4-3-7 4.66E-10 h4-3-18 6.71E-10 h4-3-8 8.62E-10 h4-3-19 5.99E-10 h4-3-9 3.17E-10 h4-3-20 8.92E-10

[0257] h4-3-1 was selected. The affinity (KD) of this combined antibody is 3.04E-10M. It was named h4-3 and further functional verification was performed. The nucleotide sequence of the heavy chain variable region of this antibody is shown in SEQ ID NO:45, and the amino acid sequence is shown in SEQ ID NO:3; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO:46, and the amino acid sequence is shown in SEQ ID NO:20.

[0258] SEQ ID NO:3:

[0259] EVQLVQSGPVKKPGASVKVSCKASGFTFT DYVIG WVRQAPGQGLEWIG EIYLGSGTIYYTEKFKG RVTMTADTSTSTAYMELSSLRSEDTAVYYCAR GSIFPFDY WGQGTLVTVSS

[0260] SEQ ID NO:20:

[0261] DIQLTQSPSSLSASVGDRVTITC SASSSVSYMY WYQQKPGKAPKLLIY DTSTLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQWSSYPYT FGQGTKLEIK

[0262] 5. Humanization of mouse monoclonal antibody 11-4

[0263] (1) CDR porting

[0264] First, a comprehensive analysis of the mouse antibody heavy chain sequence was performed to identify the antigen complementarity determinant (CDR) region for antibody-antigen binding and the framework region supporting the conserved three-dimensional conformation of the antibody. Then, based on homology alignment results, the most similar human antibody template was searched in the human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX). Combining the full-sequence BLAST results and the characteristics of the heavy chain CDR3 sequence, CDR transplantation was performed, achieving full humanization of the 11-4 heavy chain variable region (VH) within the framework region. Conversely, based on homology alignment results, the most similar human antibody template was searched in the human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX). Combining the full-sequence BLAST results and the characteristics of the light chain CDR3 sequence, CDR transplantation was performed, achieving full humanization of the light chain framework region.

[0265] The nucleotide sequence of the humanized heavy chain variable region h11-4_VH1 of the 11-4 antibody CDR transplantation is shown in SEQ ID NO:49, and the amino acid sequence is shown in SEQ ID NO:6; the nucleotide sequence of the light chain variable region h11-4_VL1 is shown in SEQ ID NO:50, and the amino acid sequence is shown in SEQ ID NO:25.

[0266] SEQ ID NO:6

[0267] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWIN WVRQAPGQGLEWMG NIYPSNSYTNYNQKFKD RVTMTRDTTSTVYMELSSLRSEDTAVYYCAR YRSDGFAY WGQGTLVTVSS

[0268] SEQ ID NO:25EIVLTQSPATLSLSPGERATLSC RASQNIGTSIH WYQQKPGQAPRLLIY FASESIS GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQSNSWPFT FGGGTKVEIK

[0269] (2) CDR region mutation design

[0270] Based on the sequence characteristics of the murine antibody 11-4, mutations were designed in the humanized light and heavy chain variable region sequences of CDR transplantation. The mutation sites are shown in Table 9 below.

[0271] Table 9.11-4 Humanized Sequence Design

[0272]

[0273] Note: Amino acid residue sites are numbered according to the Kabat numbering system.

[0274] 6. Recombinant expression of humanized monoclonal antibody 11-4

[0275] The variable regions (h11-4_VL1 and h11-4_VH1) of the humanized h11-4 antibody light and heavy chains were fully synthesized. The humanized h11-4_VH1 was cloned upstream of the heavy chain constant region coding gene of human IgG1 in the eukaryotic transient expression vector pKN041 via enzyme digestion. The nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO:59, and the amino acid sequence is shown in SEQ ID NO:37. Similarly, the humanized h11-4_VL1 was cloned upstream of the human light chain Cκ coding gene in the eukaryotic transient expression vector pKN019 via enzyme digestion. The nucleotide sequence of the light chain constant region is shown in SEQ ID NO:60, and the amino acid sequence is shown in SEQ ID NO:60. NO:38, construct humanized 11-4 light and heavy chain expression vectors to obtain light chain (pKN019-h11-4L1) and heavy chain (pKN041-h11-4H1) expression plasmids, transform them into E. coli for amplification, and isolate the h11-4 antibody light chain and heavy chain plasmids h11-4L1 and h11-4H1.

[0276] According to the mutation design shown in Table 9, site-directed mutagenesis was performed on the light chain (pKN019-h11-4L1) and heavy chain (pKN041-h11-4H1) expression plasmids using the StarMut gene site-directed mutagenesis kit (Cat.:T111-01, GenStar). The resulting plasmids were then transformed into *E. coli* for amplification, yielding the h11-4 antibody light and heavy chain mutant expression plasmids (h11-4H2~h11-4H7, h11-4L2~h11-4L5), corresponding to the 11-4 humanized sequences in Table 9. Following the instructions for the transfection reagent 293fectin (Cat.:12347019, Gibco), the light and heavy chain plasmids of the 11-4 humanized antibody were combined (see Table 10) and then transformed into HEK293 cells for recombinant expression.

[0277] Table 10. Humanized 11-4 light and heavy chain sequence combinations

[0278]

[0279]

[0280] Note: This table represents the sequences obtained by various combinations of 11-4 light and heavy chains. For example, h11-4-1 indicates that the antibody is composed of the 11-4 humanized antibody light chain h11-4L1 and the humanized heavy chain h11-4H1, and so on.

[0281] Five to six days after cell transfection, the culture supernatant was collected and purified using a ProA affinity chromatography column. Different humanized antibodies against 11-4 were obtained. Antibody affinity was determined using a Fortebio Octet QKe system with an anti-human antibody Fc fragment capture antibody (AHC) bioprobe to capture the antibody Fc fragment. For the assay, the 11-4 antibody and the control antibody Sacituzumab were diluted to 4 μg / mL with PBS buffer and flowed through the AHC probe (Cat.: 18-0015, PALL) for 120 s. Human Trop-2-His recombinant protein (serial number: NP_002344.2, 1aa-274aa) was used as the mobile phase at a concentration of 60 nM. The binding time was 100 s, and the dissociation time was 300 s. After the experiment was completed, the blank control response value was subtracted, and the kinetic constant of antigen-antibody binding was calculated by fitting the 1:1 Langmuir binding mode using software.

[0282] The affinity of the h11-4 mutant combination antibody, the chimeric antibody ch11-4, and the control antibody Sacituzumab for human Trop-2-His recombinant protein was determined by ForteBio (Table 11).

[0283] Table 11.11-4 Affinity assay results of different humanized antibodies with recombinant human Trop-2 extracellular region protein

[0284] Antibody combination KD value (M) Antibody combination KD value (M) Sacituzumab 7.84E-10 h11-4-10 3.51E-10 ch11-4 2.64E-10 h11-4-11 3.75E-10 h11-4-1 2.16E-09 h11-4-12 2.82E-10 h11-4-2 9.91E-10 h11-4-13 2.11E-10 h11-4-3 8.68E-10 h11-4-14 3.52E-10 h11-4-4 8.11E-10 h11-4-15 3.89E-10 h11-4-5 1.08E-09 h11-4-16 2.31E-10 h11-4-6 9.52E-10 h11-4-17 2.38E-10 h11-4-7 8.03E-10 h11-4-18 3.94E-10 h11-4-8 2.22E-10 h11-4-19 2.54E-10 h11-4-9 3.01E-10 h11-4-20 2.10E-10

[0285] Example 10 ELISA detection of species specificity of anti-Trop-2 humanized antibody binding to Trop-2

[0286] Human Trop-2-his recombinant protein (serial number: NP_002344.2, 1aa-274aa), cynomolgus monkey Trop-2-His recombinant protein (serial number: UniProtKB-A0A2K5UE71, 1aa-272aa), and mouse Trop-2-His recombinant protein (Cat.:50922-M08H, Beijing Yiqiao Shenzhou) were coated overnight at 4℃ at a coating concentration of 1 μg / mL; after washing the plate three times with PBS, 5% PBS was added. Block with BSAPBS at 37°C for 60 min, wash 3 times with PBST; add different dilutions of h23-12 (starting concentration 10 μg / mL, 14 concentrations serially diluted 3-fold), h4-3 (starting concentration 3 μg / mL, 12 concentrations serially diluted 3-fold), and Sacituzumab (starting concentration 3 μg / mL, 12 concentrations serially diluted 3-fold), one parallel well per concentration, incubate at 37°C for 60 min, wash 4 times with PBST; add 1:5000 diluted HRP-anti-human Fc (Cat.:109-035-098, Jackson Immuno Research), incubate at 37°C for 30 min, wash 4 times with PBST; add TMB substrate for color development, incubate at 37°C for 10 min, then add 2M... The reaction was terminated with HCl; the absorbance A450nm-630nm of the well plate at a wavelength of 450nm was read and recorded using 630nm as the reference wavelength.

[0287] Experimental results showed that h23-12, h4-3, and the control antibody Sacituzumab could specifically bind to recombinant human and cynomolgus monkey Trop-2, but had no binding activity with recombinant mouse Trop-2. Figure 6 Table 12) provides a basis for pharmacological and toxicological experiments on humanized antibodies.

[0288] Table 12. EC50 values ​​of anti-Trop-2 humanized antibodies binding to different species of Trop-2.

[0289]

[0290] Example 11 Affinity analysis of anti-Trop-2 humanized antibody

[0291] Antibody affinity was determined using the Octet QKe system instrument from Fortebio, employing an antibody capture antibody (AHC) bioprobe to capture the Fc fragment of the anti-human antibody.

[0292] During the assay, antibodies (h23-12, h4-3, and the control antibody Sacituzumab) were diluted to 4 μg / mL with PBS buffer and flowed over the surface of an AHC probe (Cat.: 18-0015, PALL) for 120 s. Human Trop-2-his recombinant protein (serial number: NP_002344.2, 1aa-274aa) was used as the mobile phase. The Trop-2-his concentrations for each antibody were: h23-12: 23, 30, 45, 75 nM; h4-3: 23, 30, 45, 60 nM; Sacituzumab: 23, 30, 45, 75 nM. The binding time was 100 s, and the dissociation time was 300 s. After the experiment, the blank control response values ​​were subtracted, and the antigen-antibody binding kinetic constants were calculated using a 1:1 Langmuir binding model fitting software.

[0293] The reaction curves of h23-12, h4-3 and control antibody Sacituzumab with recombinant human Trop-2 protein are shown in the figure. Figure 7 As shown, the fitted curves and calculated affinities were obtained. The affinity (KD) for h23-12 was 6.40E-10M, for h4-3 it was 5.45E-10M, and for Sacituzumab it was 9.41E-10M. Detailed kinetic parameters are shown in Table 13. The results indicate that h23-12 and h4-3 have high affinity for human Trop-2, comparable to the control antibody Sacituzumab, and h23-12 has a better dissociation value than Sacituzumab.

[0294] Table 13. Affinity assay results of anti-Trop-2 humanized antibody and recombinant human Trop-2 extracellular region protein

[0295] KD value (M) kon(1 / Ms) kdis(1 / s) h23-12 6.40E-10 1.78E+05 1.14E-04 h4-3 5.45E-10 2.67E+05 1.46E-04 Sacituzumab 9.41E-10 2.08E+05 1.94E-04

[0296] Example 12 Humanized anti-human Trop-2 antibodies bind to the internalization activity of Trop-2 on the cell surface.

[0297] BXPC-3 pancreatic cancer cells naturally expressing human Trop-2 were injected at a dose of 2 × 10⁻⁶. 3 Cells were seeded at a density of 100 cells / well in 96-well cell culture plates and cultured for 24 hours. Cells were washed once with PBS, and the supernatant was discarded. Mix-n-Staining agents will be used... TM CF TM488A (Cat.: MX488AS100, Sigma) labeled h23-12 and control antibody Sacituzumab were diluted to 15 μg / mL with RPMI 1640 (containing 10% FBS) and added to BXPC-3 cells. One group was placed in a 37°C thermostatic incubator, and the other group was placed in a 4°C refrigerator as a negative control. After incubation for 30 minutes, the negative control was washed three times with PBS and observed and photographed under a fluorescence microscope. After incubation for 5 hours at 37°C, the experimental group was observed and photographed under a fluorescence microscope.

[0298] Experimental results ( Figure 8 The results showed that both humanized h23-12 and the control antibody Sacituzumab could be internalized by Trop-2 at 37°C, exhibiting a punctate distribution in the cytoplasm. This suggests that the antibodies can maintain their internalization activity after humanization.

[0299] The internalization rate on BxPC cells at 3 h was detected by FACS using the method shown in Example 7. The results are shown in Table 14, indicating that the internalization rate after humanization is comparable to that of Sac.

[0300] Table 14. Percentage of Trop-2-mediated internalization of anti-human Trop-2 antibodies on the surface of BXPC-3 cells.

[0301]

[0302]

[0303] Example 13 Pharmacokinetic study of Balb / C nude mice after single administration

[0304] Healthy female 5-week-old Balb / C nude mice were divided into groups of two and administered a single intraperitoneal injection of h23-12 (15 mg / kg). Serum was collected at 5 h, 25 h, 48 h, 96 h, 168 h, and 240 h and stored at -20 °C. A control group was set up to compare the pharmacokinetic characteristics with those of the control product Sacituzumab, which was administered intraperitoneally at the same dose as h23-12.

[0305] Healthy female 5-week-old Balb / C nude mice were divided into groups of four and administered a single intraperitoneal injection of h4-3 at 4h, 8h, 24h, 48h, 96h, 144h, 192h, and 240h. The serum was then collected and stored at -20℃ to observe its pharmacokinetic characteristics.

[0306] Serum drug concentration was detected using a coated human Trop-2-his ELISA (serum ID: NP_002344.2, 1aa-274aa), and a standard curve was simultaneously constructed. A linear curve was fitted with the standard antibody concentration as the Y-axis and the OD value as the X-axis. The antibody concentration in the serum was calculated by substituting the OD value of the detected serum into the formula, and the concentration was then determined according to the formula T. 1 / 2 =|0.693 / k|, calculate the drug half-life T 1 / 2 .

[0307] The drug-time curve results showed that h23-12, h4-3, and the control antibody Sacituzumab all had relatively long half-lives in mice, with a drug metabolism half-life T0. 1 / 2 The performance was quite good. Figure 9 A, Figure 9 B (Table 15) indicates that the antibody did not show significant inactivation in vivo and possessed good structural stability. Its metabolism conforms to the basic characteristics of monoclonal antibody drugs. 1 / 2 Approximately 170 hours.

[0308] Table 15. Pharmacokinetic parameters of anti-Trop-2 antibody after a single dose in nude mice.

[0309] <![CDATA[T 1 / 2 (Trop-2 detection) Sacituzumab(n=2) 168±14 h23-12(n=2) 174±12 h4-3(n=4) 180±57

[0310] Example 14 Affinity analysis of naked anti-Trop-2 antibody and its ADC antibody

[0311] Antibody affinity was determined using the Octet QKe system instrument from Fortebio, employing an antibody capture antibody (AHC) bioprobe to capture the Fc fragment of the anti-human antibody.

[0312] First, antibodies labeled with the ADC drug SN38 were prepared. The antibodies were reduced for 2 h in sodium phosphate buffer (pH 7.0 ± 0.5) using 20 equivalents of dithiothreitol (DTT). The reduced antibodies were then purified by ultrafiltration centrifuge to remove excess DTT, and the purified antibodies were transferred to sodium phosphate buffer (pH 7.0 ± 0.5). Using 7-15% v / v DMSO as a co-solvent, the reduced antibodies were incubated with CL2A-SN-38 at ambient temperature for 30 min. Finally, excess small molecules were removed by ultrafiltration centrifuge. The molecular weight of the antibody-drug conjugate was analyzed by mass spectrometry, and the antibody-drug conjugate ratio (DAR) was calculated. Each antibody ultimately carried an average of 7.5 SN38 molecules.

[0313] During the assay, the SN38-labeled antibodies prepared as described above—h23-12-SN38, ch4-3-SN38, ch11-4-SN38, the positive control antibody Sacituzumab-SN38, and the naked antibodies h23-12, ch4-3, ch11-4, and the positive control antibody Sacituzumab—were diluted to 4 μg / mL with PBS buffer and flowed through the surface of an AHC probe (Cat.: 18-0015, PALL) for 120 s. Human Trop-2-his recombinant protein (serial number: NP_002344.2, 1aa-274aa) was used as the mobile phase at a concentration of 60 nM. The binding time was 300 s, and the dissociation time was 300 s. After the experiment, the blank control response value was subtracted, and the antigen-antibody binding kinetic constant was calculated using 1:1 Langmuir binding mode fitting software.

[0314] As shown in Table 16, the affinity of the SN38-labeled ADC antibody for recombinant human Trop-2 protein did not change significantly compared to the naked antibody.

[0315] Table 16. Affinity assay results of anti-Trop-2 naked antibody and its ADC antibody with recombinant human Trop-2 protein

[0316]

[0317] Example 15 FACS detection of BXPC-3-mediated internalization of naked anti-Trop-2 antibody and its ADC antibody

[0318] The internalization rate on human pancreatic cancer cells BXPC-3 and human gastric cancer cells NCI-N87 was detected according to the method described in Example 7. The antibodies to be tested included antibodies labeled with the ADC drug SN38 prepared as described above: h23-12-SN38, ch4-3-SN38, ch11-4-SN38, the positive control antibody Sacituzumab-SN38, and naked antibodies h232-12, ch4-3, ch11-4, the positive control antibody Sacituzumab, and the negative isotype control antibody NC-IgG1, at 10 μg / ml.

[0319] As shown in Tables 17 and 18, the internalization rates of the naked antibody and the ADC antibody labeled with SN38 for h23-12 were similar, and the degree of internalization was similar to that of the control antibody; while the internalization rates of the ADC antibodies labeled with SN38 for ch4-3 and ch11-4 were higher than those of the naked antibody.

[0320] Table 17. Percentage of Trop-2-mediated internalization of anti-human Trop-2 antibodies on the surface of NCI-N87 cells.

[0321]

[0322] Table 18. Percentage of Trop-2-mediated internalization of anti-human Trop-2 antibodies on the surface of BXPC-3 cells.

[0323]

[0324] Example 16 Detection of the cytotoxic activity of anti-Trop-2-ADC antibody against cells

[0325] Human pancreatic cancer cells (BxPC-3) were harvested and administered at a dose of 2 × 10⁻⁶. 3 Cells were seeded per well in 96-well cell culture plates and incubated overnight at 37°C with 5% CO2. Then, according to Table 19, different concentrations of SN38-labeled anti-Trop-2 antibody samples were added (two parallel wells for each concentration, plus a blank well (untreated)). The plates were incubated at 37°C with 5% CO2 for 3 hours, followed by replacement with fresh complete culture medium. The treatment was repeated the following day for four consecutive days. The cytotoxic activity of the SN38-labeled anti-Trop-2 antibody against cells was then detected using the Cell Counting Kit-8 (CCK-8) assay.

[0326] Table 19. Anti-Trop-2-ADC antibodies and their effective concentrations

[0327]

[0328]

[0329] The results show ( Figure 10 (Table 20) All anti-Trop-2 ADC antibodies specifically killed target cells, and their killing activity was not significantly different from that of the control antibody Sacituzumab-SN38.

[0330] Table 20. Inhibitory activity of anti-Trop-2 ADC antibodies against cell growth

[0331] name <![CDATA[IC 50 (ug / ml)]]> Blank cells / h23-12-SN38 1.409 Sacituzumab-SN38 4.311 ch4-3-SN38 3.299 ch11-4-SN38 2.879

[0332] Example 17 Pharmacodynamic evaluation of anti-Trop2-ADC antibody in N87 subcutaneous xenograft model

[0333] Five-week-old female BALB / c nude mice were subcutaneously inoculated with 3×10⁻⁶ mice. 6 Personal gastric cancer cells (NCI-N87), waiting for the tumor to grow to 150mm 3The mice were randomly divided into groups of 6 each. The grouping, dosage, and frequency of administration are shown in Table 21. Each group received intravenous administration twice a week. Tumor volume and body weight were measured simultaneously with administration. A mouse was considered considered dead when its body weight decreased by more than 15% or when the tumor volume of a single animal exceeded 3000 mm. 3 Or a group of animals with an average tumor volume exceeding 2000 mm 3 The experiment was stopped and the mice were euthanized.

[0334] Table 21. Grouping, dosage, and frequency of medication in nude mice

[0335]

[0336]

[0337] like Figure 11 , Figure 12 As shown, the anti-Trop2-ADC antibody has a dose-dependent inhibitory effect on tumor growth. At high doses (10 mg / kg), no difference in efficacy was observed among the various ADC antibodies, and no significant toxicity of the ADC small molecule SN38 was observed. The weight of the animals in each experimental group increased steadily, with no significant difference from the control.

[0338] Example 18 Pharmacodynamic evaluation of the combination of anti-Trop2 antibody and anti-CD47 antibody in the SKOV3 subcutaneous xenograft model

[0339] Five-week-old female BALB / c nude mice were used, and each mouse was subcutaneously inoculated with 3 × 10⁻⁶ cells in the right flank. 6 Personal ovarian cancer cells (SKOV3), waiting for the tumor to grow to 150mm 3 Mice were randomly divided into groups of 6 (approximately 1000 mg / group). Grouping, dosage, and frequency of administration are shown in Table 22. Each group received intraperitoneal injection twice weekly for a total of 5 administrations. Tumor volume and mouse weight were measured simultaneously with administration, and the mice's condition was observed. Mice were euthanized after the last administration. The anti-CD47 antibody is described in patent application publication US20150183874A1, humanized 5F9 version 2.

[0340] Table 22. Grouping, dosage, and frequency of medication in nude mice

[0341] Group drug Dosage Dosage frequency 1 Anti-CD47 10mg / kg Biw 2 h23-12 2mg / kg Biw 3 h23-12+Anti-CD47 2mg / kg + 10mg / kg Biw 4 Negative control hIgG4 2mg / kg Biw

[0342] like Figure 13 , Figure 14As shown, compared with the negative control hIgG4, the h23-12 + Anti-CD47 combination group exhibited certain antitumor activity, while the Anti-CD47 and h23-12 single-drug groups showed no significant antitumor effect. This indicates that the Trop2 antibody and anti-CD47 antibody of the present invention can synergistically promote the phagocytosis of tumor cells by macrophages, thus having a synergistic antitumor effect.

[0343] Example 19 Pharmacodynamic evaluation of anti-Trop2-ADC antibody in N87 subcutaneous xenograft model

[0344] Five-week-old female BALB / c nude mice were subcutaneously inoculated with 3×10⁻⁶ mice. 6 Personal gastric cancer cells (NCI-N87), waiting for the tumor to grow to 100mm 3 The mice were randomly divided into groups of 6 mice each. The grouping, dosage, and frequency of administration are shown in Table 23. Each group received intravenous administration twice a week for a total of 6 weeks. Tumor volume and body weight were measured at the same time as administration. The mice were considered considered dead when their body weight decreased by more than 15% or when the tumor volume of a single animal exceeded 3000 mm. 3 Or a group of animals with an average tumor volume exceeding 2000 mm 3 The experiment was stopped and the mice were euthanized.

[0345] Table 23. Grouping, dosage, and frequency of administration in nude mice

[0346] Group drug Dosage Dosage frequency 1 ch3-11-SN38 5mg / kg Biw×6 2 ch11-4-SN38 5mg / kg Biw×6 3 Sacituzumab-SN38 5mg / kg Biw×6 4 Negative control hIgG1 5mg / kg Biw×6 5 ADC control hIgG1-SN38 5mg / kg Biw×6

[0347] like Figure 15 , Figure 16 As shown, anti-Trop2-ADC antibodies have a dose-dependent inhibitory effect on tumor growth. At a dose of 5 mg / kg, ADC antibodies ch3-11-SN38 and ch11-4-SN38 are slightly more effective than Sacituzumab-SN38, and no obvious toxic effects of the small molecule ADC SN38 were observed.

[0348] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims. sequence list <110> Maiwei (Shanghai) Biotechnology Co., Ltd. <120> Anti-human Trop-2 antibodies and their applications <130> LC20210009P-CN <140> CN202080071403.9 <141> 2020-10-12 <150> CN201910962965.1 <151> 2019-10-11 <160> 60 <170> PatentIn version 3.3 <210> 1 <211> 123 <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[[ID=3)) <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 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> twenty one 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> twenty two <211> 106 <212> PRT <213> Artificial <220> <223> Light chain variable region <400> twenty two 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> Chain link <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> Chain link <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[[ID=3⑨]] <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 gaggtgcagc tggtgcagtc tggacccgag gtgaagaagc ctggagcctc cgtgaaggtg 60 tcctgcaagg cctccggctt caccttcacc gactacgtga tcggctgggt gcgacaggct 120 tcctgcaagg cctccggctt caccttcacc gactacgtga tcggctgggt gcgacaggct 120 cctggccagg gactggagtg gatcggcgag atctacctgg gctccggcac catctactac 180 cctggccagg gactggagtg gatcggcgag atctacctgg gctccggcac catctactac 180 accgagaagt tcaagggacg ggtgaccatg acagccgaca cctccacctc caccgcctac 240 accgagaagt tcaagggacg ggtgaccatg acagccgaca cctccacctc caccgcctac 240 atggagctgt cctccctgcg gtccgaggac accgccgtgt actactgcgc tcgaggctcc 300 atggagctgt cctccctgcg gtccgaggac accgccgtgt actactgcgc tcgaggctcc 300 atcttcccct tcgactactg gggccagggc accctggtga ccgtgtcctc t 351 atcttcccct tcgactactg gggccagggc accctggtga ccgtgtcctc t 351 <210> 46<210> 46 <211> 318<211> 318 <212> DNA<212> DNA <213> Artificial<213> Artificial <220><220> <223> Light chain variable region<223> Light chain variable region <400> 46<400> 46 gacatccagc tgacccagtc tccctcctcc ctgtctgcct ccgtgggcga cagggtgacc 60 gacatccagc tgacccagtc tccctcctcc ctgtctgcct ccgtgggcga cagggtgacc 60 atcacctgct ctgcctcctc ctccgtgtcc tacatgtact ggtaccagca gaagcctggc 120 atcacctgct ctgcctcctc ctccgtgtcc tacatgtact ggtaccagca gaagcctggc 120 aaggctccca agctgctgat ctacgacacc tccaccctgg cctctggcgt gccctccagg 180 aaggctccca agctgctgat ctacgacacc tccaccctgg cctctggcgt gccctccagg 180 ttctctggct ccggatctgg caccgacttc accctgacca tctcctccct gcagcccgag 240 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 321

Claims

1. An antibody or antigen-binding fragment thereof that binds to human Trop-2, said antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein said heavy chain variable region (VH) and light chain variable region (VL) comprise a CDR combination of the following amino acid sequences: (1)HCDR1, HCDR2, HCDR3: SYWMH, EITPSDNYTSYNQKFKG, GHGNYVSFDY; LCDR1, LCDR2, LCDR3: RASQDISNYLN, YTSRLHS, QQGYTLPPYT; (2) HCDR1, HCDR2, HCDR3: SYWMH, EITPSDNYTSYNQKFKG, GEGNYVSFDY; LCDR1, LCDR2, LCDR3: RASQDISNYLN, YTSRLHS, QQGYTLPPYT; (3) HCDR1, HCDR2, HCDR3: SYWMH, EITPSDNYGSYNQKFKG, GHGNYVSFDY; LCDR1, LCDR2, LCDR3: RASQDISNYLN, YTSRLES, QQGYTLPPYT; (4) HCDR1, HCDR2, HCDR3: SYWMH, EITPSDNYTSYNQKFKG, GEGNYVSFDY; LCDR1, LCDR2, LCDR3: RASQDISNYLN, YTSRLES, QQGYTLPPYT; (5) HCDR1, HCDR2, HCDR3: SYWMH, EITPSDNYGSYNQKFKG, GHGNYVSFDY; LCDR1, LCDR2, LCDR3: RASQDISNYLN, YTSRLQS, QQGYTLPPYT; (6) HCDR1, HCDR2, HCDR3: SYWMH, EITPSDNYGSYNQKFKG, GHGNYVSFDY; LCDR1, LCDR2, LCDR3: RASQDISNYLN, YTSRLHS, QQYYTLPPYT; (7) HCDR1, HCDR2, HCDR3: SYWMH, EITPGDNYTSYNQKFKG, GHGNYVSFDY; LCDR1, LCDR2, LCDR3: RASQDISNYLN, YTSRLHS, QQGYSLPPYT; (8) HCDR1, HCDR2, HCDR3: SYWMH, EITPSDNYGSYNQKFKG, GHGNYVSFDY; LCDR1, LCDR2, LCDR3: RASQDISNYLN, YTSRLHS, QQGYSLPPYT; (9) HCDR1, HCDR2, HCDR3: SYWMH, EITPSDNYTSYNQKFKG, GEGNYVSFDY; LCDR1, LCDR2, LCDR3: RASQDISNYLN, YTSRLHS, QQGYSLPPYT; (10)HCDR1, HCDR2, HCDR3: DYVIG, EIYLGSGTIYYTEKFKG, GSIFPFDY; LCDR1, LCDR2, LCDR3: SASSVSYMY, DTSTLAS, QQWSSYPYT; or (11)HCDR1, HCDR2, HCDR3: SYWIN, NIYPSNSYTNYNQKFKD, YRSDGFAY; LCDR1, LCDR2, LCDR3: RASQNIGTSIH, FASESIS, QQSNSWPFT.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, respectively, comprising: (1) The amino acid sequence shown in SEQ ID NO:12; and the amino acid sequence shown in SEQ ID NO:31; (2) The amino acid sequence shown in SEQ ID NO:13; and the amino acid sequence shown in SEQ ID NO:32; (3) The amino acid sequence shown in SEQ ID NO:16; and the amino acid sequence shown in SEQ ID NO:32; (4) The amino acid sequence shown in SEQ ID NO:14; and the amino acid sequence shown in SEQ ID NO:33; (5) The amino acid sequence shown in SEQ ID NO:16; and the amino acid sequence shown in SEQ ID NO:33; (6) The amino acid sequence as shown in SEQ ID NO:14; and the amino acid sequence as shown in SEQ ID NO:34; (7) The amino acid sequence as shown in SEQ ID NO:14; and the amino acid sequence as shown in SEQ ID NO:35; (8) The amino acid sequence as shown in SEQ ID NO:15; and the amino acid sequence as shown in SEQ ID NO:36; (9) The amino acid sequence shown in SEQ ID NO:14; and the amino acid sequence shown in SEQ ID NO:36; (10) The amino acid sequence as shown in SEQ ID NO:16; and the amino acid sequence as shown in SEQ ID NO:36; (11) The amino acid sequence shown in SEQ ID NO:2; and the amino acid sequence shown in SEQ ID NO:19; (12) The amino acid sequence as shown in SEQ ID NO:3; and, the amino acid sequence as shown in SEQ ID NO:20; or (13) The amino acid sequence as shown in SEQ ID NO:5; and the amino acid sequence as shown in SEQ ID NO:

24.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment comprises heavy chain variable regions and light chain variable regions selected from the following combinations: (1) An amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:12; and an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:31; (2) An amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:13; and an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:32; (3) An amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:16; and an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:32; (4) An amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:14; and an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:33; (5) An amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:16; and an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:33; (6) An amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:14; and an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:34; (7) An amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:14; and an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:35; (8) An amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:15; and an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:36; (9) An amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:14; and an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:36; (10) An amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:16; and an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:36; (11) An amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:2; and an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:19; (12) An amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:3; and an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:20; or (13) An amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:5; and an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:

24.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody or its antigen-binding fragment is a monoclonal antibody, single-chain antibody, bifunctional antibody, or partially humanized antibody, chimeric antibody, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv.

5. The antibody or its antigen-binding fragment according to claim 4, characterized in that, The bifunctional antibody is BsFv.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody or its antigen-binding fragment also contains a human or mouse constant region.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody or its antigen-binding fragment also contains a human or mouse light chain constant region (CL) or heavy chain constant region (CH).

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody or its antigen-binding fragment contains a heavy chain constant region selected from IgG, IgA, IgM, IgD or IgE, or a κ or λ type light chain constant region.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody is a monoclonal antibody.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody is a mouse-derived, chimeric, or humanized monoclonal antibody.

11. The antibody or its antigen-binding fragment according to claim 9, characterized in that, The heavy chain constant region of the monoclonal antibody is IgG1 or IgG4 subtype, and the light chain constant region is κ type.

12. The antibody or its antigen-binding fragment according to claim 9, characterized in that, The heavy chain constant region of the monoclonal antibody contains an amino acid sequence as shown in SEQ ID NO:

37.

13. The antibody or its antigen-binding fragment according to claim 9, characterized in that, The light chain constant region of the monoclonal antibody contains an amino acid sequence as shown in SEQ ID NO:

38.

14. The antibody or its antigen-binding fragment according to claim 12, characterized in that, The light chain constant region of the monoclonal antibody contains an amino acid sequence as shown in SEQ ID NO:

38.

15. The antibody or its antigen-binding fragment according to claim 9, characterized in that, The heavy chain constant region of the monoclonal antibody contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO:

37.

16. The antibody or its antigen-binding fragment according to claim 9, characterized in that, The light chain constant region of the monoclonal antibody contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO:

38.

17. The antibody or antigen-binding fragment thereof according to claim 15, characterized in that, The light chain constant region of the monoclonal antibody contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO:

38.

18. A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 17.

19. A carrier comprising the nucleic acid molecule of claim 18.

20. A host cell comprising the nucleic acid molecule of claim 18 or the vector of claim 19, or the host cell being transformed or transfected by the nucleic acid molecule of claim 18 or the vector of claim 19; and the host cell is not a plant cell.

21. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 17, a nucleic acid molecule as described in claim 18, a carrier as described in claim 19 or a host cell as described in claim 20, and optionally pharmaceutically acceptable excipients.

22. The pharmaceutical composition according to claim 21, characterized in that, The pharmaceutical composition also contains other antibody drugs.

23. The pharmaceutical composition according to claim 22, characterized in that, The antibody drug in question is a macrophage-based immune checkpoint antibody.

24. The pharmaceutical composition according to claim 22, characterized in that, The antibody drug in question is an anti-CD47 antibody.

25. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 17, the nucleic acid molecule of claim 18, the vector of claim 19, the host cell of claim 20, or the pharmaceutical composition of any one of claims 21 to 24 in the preparation of a medicament for the treatment of gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous cell lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial carcinoma, triple-negative breast cancer, or cervical cancer.

26. A kit comprising an antibody molecule or antigen-binding fragment thereof as claimed in any one of claims 1 to 17, a nucleic acid molecule as claimed in claim 18, a vector as claimed in claim 19, a host cell as claimed in claim 20, or a pharmaceutical composition as claimed in any one of claims 21 to 24.

27. A conjugate comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 17 and a drug conjugated thereto, wherein the drug is a cytotoxic agent.

28. The conjugate according to claim 27, wherein the conjugate is an antibody-drug conjugate (ADC) of the following formula: (antibody or antigen-binding fragment thereof according to any one of claims 1 to 17) - (linker) - (cytotoxic agent).

29. The conjugate according to claim 27 or 28, characterized in that, The cytotoxic agent is a microtubule inhibitor or a DNA replication inhibitor.

30. The conjugate according to claim 29, characterized in that, The microtubule inhibitor is paclitaxel or docetaxel.

31. The conjugate according to claim 29, characterized in that, The DNA replication inhibitor is irinotecan or its metabolite SN-38.

32. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 17, the nucleic acid molecule of claim 18, the vector of claim 19, or the host cell of claim 20 in the preparation of an antibody-drug conjugate (ADC) for the treatment of gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous cell lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial carcinoma, triple-negative breast cancer, or cervical cancer.

33. Use of the conjugate of any one of claims 27 to 31 in the preparation of a medicament for the treatment of gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous cell lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial carcinoma, triple-negative breast cancer, or cervical cancer.

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