Anti-human Trop-2 antibody and its applications

Abstract

The present invention provides an antibody or a fragment thereof that binds to human tumor-associated calcium signal transducer 2 (Trop-2) protein, and the use of the antibody or the fragment thereof for preventing or treating diseases. The antibody or the fragment thereof of the present invention can effectively bind to human Trop-2 protein, has internalization activity, and the internalization activity is enhanced after being labeled with an ADC drug, and the in vivo efficacy and safety in a mouse model are not lower than those of a control antibody.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a novel anti-human Trop-2 antibody or a functional fragment thereof. The present invention also relates to the application of the antibody or the functional fragment thereof. Background Art

[0002] Trop-2 (Tumor-associated calcium signal transducer 2), also known as tumor-associated calcium signal transducer (TACSTD2), epithelial glycoprotein-1 antigen (EGP-1), and gastrointestinal tumor-associated antigen 1 (GA733-1), is a cell surface glycoprotein encoded by the TACSTD2 gene, with a full length of 323 amino acids. The extracellular region consists of three domains and has been confirmed to exist in the form of a dimer.

[0003] Trop-2 is a transmembrane glycoprotein. Different from other proto-oncogenes, Trop2 has no mutation, which means it has no genetic composition change leading to overexpression. Trop-2 stimulates cell growth through the ERK / MAPK and cyclin D1 pathways, thereby promoting mechanisms such as 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, non-small cell lung cancer, etc., and is related to prognosis. Relatively, Trop-2 is extremely lowly expressed in normal tissues, making it an ideal target for ADC drugs.

[0004] Antibody drugs targeting Trop-2 are currently mainly developed as ADC drugs. Incomplete statistics show that there are more than 3 clinical research varieties, and the small molecule conjugates mainly include derivatives of irinotecan and tubulin inhibitors. Currently, it is considered ideal to select the novel and low-toxic metabolite SN-38 of topoisomerase inhibitor. The tumor suppression of SN-38 is different from that of existing microtubule inhibitors and DNA alkylating agents, and it is particularly suitable for tumors with high heterogeneity and multiple drug resistance mechanisms, such as triple-negative breast cancer, pancreatic cancer, gastric cancer and other tumors. The project with the fastest clinical progress is the IMMU-132 project of Immunomedics, which has conducted phase III clinical trials for recurrent and metastatic triple-negative breast cancer (ASCENT-Study), phase II clinical trials for treating triple-negative breast cancer alone or in combination with carboplatin (NCT02161679), phase II clinical trials for treating urothelial carcinoma (NCT03547973), phase I / II clinical trials for treating solid tumors including gastric cancer, cervical cancer, small cell lung cancer (NCT01631552), etc. Antibody-drug conjugate projects with similar technologies also include Daiichi Sankyo, Pfizer, and major drug companies with an ADC foundation, etc.

[0005] Currently, there are few clinical research varieties of antibodies against Trop-2. Therefore, there is still a need in this field to find novel anti-Trop-2 antibodies that are particularly suitable for developing ADC drugs. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an anti-Trop-2 antibody through hybridoma screening and humanization technology, which has high affinity for human Trop-2 and has a specific killing effect on cancer cells; at the same time, the antibody has high internalization ability and is particularly suitable for developing ADC drugs.

[0007] In view of the above technical problems, the present invention provides the following technical solutions.

[0008] On the one hand, the present invention provides an antibody or a fragment thereof, which comprises 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 CDR combination selected from the following (HCDR1, HCDR2, HCDR3; LCDR1, LCDR2, LCDR3):

[0009]

[0010]

[0011] The antibody or its fragment binds to human Trop-2.

[0012] Preferably, the heavy chain variable region comprises a sequence selected from the following:

[0013] The amino acid sequence shown by 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

[0014] The light chain variable region contains a sequence selected from the following:

[0015] The amino acid sequence shown by 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.

[0016] According to a specific embodiment of the present invention, the heavy chain variable region and the light chain variable region contained in the antibody or its fragment of the present invention can be selected from the following combinations:

[0017] (1) The amino acid sequence shown by SEQ ID NO: 1, or an amino acid sequence having at least 75% identity with the amino acid sequence shown by SEQ ID NO: 1; and, the amino acid sequence shown by SEQ ID NO: 18, or an amino acid sequence having at least 75% identity with the amino acid sequence shown by SEQ ID NO: 18;

[0018] (2) The amino acid sequence shown by SEQ ID NO: 2, or an amino acid sequence having at least 75% identity with the amino acid sequence shown by SEQ ID NO: 2; and, the amino acid sequence shown by SEQ ID NO: 19, or an amino acid sequence having at least 75% identity with the amino acid sequence shown by SEQ ID NO: 19;

[0019] (3) The amino acid sequence shown by SEQ ID NO: 3, or an amino acid sequence having at least 75% identity with the amino acid sequence shown by SEQ ID NO: 3; and, the amino acid sequence shown by SEQ ID NO: 20, or an amino acid sequence having at least 75% identity with the amino acid sequence shown by SEQ ID NO: 20;

[0020] (4) The amino acid sequence shown by SEQ ID NO: 4, or an amino acid sequence having at least 75% identity with the amino acid sequence shown by SEQ ID NO: 4; and, the amino acid sequence shown by SEQ ID NO: 20, or an amino acid sequence having at least 75% identity with the amino acid sequence shown by SEQ ID NO: 20;

[0021] (5) The amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 3; and, the amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 21;

[0022] (6) The amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 4; and, the amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 21;

[0023] (7) The amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 3; and, the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 22;

[0024] (8) The amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 3; and, the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 23;

[0025] (9) The amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 5; and, the amino acid sequence shown in SEQ ID NO: 24 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 24;

[0026] (10) The amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 6; and, the amino acid sequence shown in SEQ ID NO: 25 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 25;

[0027] (11) The amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:7; and, the amino acid sequence shown in SEQ ID NO:26 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:26;

[0028] (12) The amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:7; and, the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:27;

[0029] (13) The amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:8; and, the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:27;

[0030] (14) The amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:9; and, the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:27;

[0031] (15) The amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:10; and, the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:27;

[0032] (16) The amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:7; and, the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:28;

[0033] (17) The amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 8; and, the amino acid sequence shown in SEQ ID NO: 28 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 28;

[0034] (18) The amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 9; and, the amino acid sequence shown in SEQ ID NO: 28 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 28;

[0035] (19) The amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 10; and, the amino acid sequence shown in SEQ ID NO: 28 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 28;

[0036] (20) The amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 7; and, the amino acid sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 29;

[0037] (21) The amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 8; and, the amino acid sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 29;

[0038] (22) The amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 9; and, the amino acid sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 29;

[0039] (23) The amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 10; and, the amino acid sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 29;

[0040] (24) The amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 11; and, the amino acid sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 30;

[0041] (25) The amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 12; and, the amino acid sequence shown in SEQ ID NO: 31 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 31;

[0042] (26) The amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 13; and, the amino acid sequence shown in SEQ ID NO: 32 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 32;

[0043] (27) The amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 16; and, the amino acid sequence shown in SEQ ID NO: 32 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 32;

[0044] (28) The amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 14; and, the amino acid sequence shown in SEQ ID NO: 33 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 33;

[0045] (29) The amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:16; and, the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:33;

[0046] (30) The amino acid sequence shown in SEQ ID NO:14 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:14; and, the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:34;

[0047] (31) The amino acid sequence shown in SEQ ID NO:14 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:14; and, the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:35;

[0048] (32) The amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:15; and, the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:36;

[0049] (33) The amino acid sequence shown in SEQ ID NO:14 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:14; and, the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:36;

[0050] (34) The amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:16; and, the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:36.

[0051] Generally, the antibody or its fragment can be in any form such as monoclonal antibody, single-chain antibody, bispecific antibody, single-domain antibody, nanobody, fully or partially humanized antibody or chimeric antibody, or the antibody or its fragment 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 its fragment can be from mouse, rat, human or any other source;

[0052] Preferably, the antibody or its fragment further comprises a human or murine constant region, preferably comprising a human or murine light chain constant region (CL) and / or heavy chain constant region (CH);

[0053] More preferably, the antibody or its fragment comprises a heavy chain constant region selected from IgG, IgA, IgM, IgD or IgE and / or a light chain constant region of κ or λ type.

[0054] According to a specific embodiment of the present invention, the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; preferably, the heavy chain constant region of the monoclonal antibody is of IgG1 or IgG4 subtype, and the light chain constant region is of κ type;

[0055] According to a specific embodiment of the present invention, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having at least 75% identity with the amino acid sequence;

[0056] Preferably, the light chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:38 or an amino acid sequence having at least 75% identity with the amino acid sequence.

[0057] The at least 75% identity described above in the present invention is any percentage identity of ≥75%, such as at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% identity.

[0058] Based on the antibody or its fragment of the present invention, on the other hand, the present invention also provides a nucleic acid molecule encoding the heavy chain CDR, light chain CDR, heavy chain variable region, light chain variable region, heavy chain or light chain in any antibody or its fragment of the present invention.

[0059] On yet another aspect, the present invention provides a vector comprising the nucleic acid molecule of the present invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome and a phage vector, etc.

[0060] The vector or nucleic acid molecule of the present invention can be used to transform or transfect a host cell or enter the host cell in any way for purposes such as preservation or expression of antibodies.

[0061] Therefore, on the other hand, the present invention provides a host cell, which comprises the nucleic acid molecule and / or vector of the present invention, or the host cell is transformed or transfected by the nucleic acid molecule and / or vector of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.

[0062] Based on the disclosure of the present invention, the antibody or its fragment, nucleic acid molecule, vector and / or host cell provided by the present invention can be obtained by using any conventional technical method known in the art. The antibody or its fragment, nucleic acid molecule, vector and / or host cell can be included in a pharmaceutical composition, and more particularly included in a pharmaceutical preparation, so as to be used for various purposes according to actual needs.

[0063] Therefore, in yet another aspect, the present invention further provides a pharmaceutical composition, which comprises the antibody or its fragment, nucleic acid molecule, vector and / or host cell of the present invention, and optionally a pharmaceutically acceptable excipient.

[0064] The antibody or its fragment of the present invention can be used in combination with other antibody drugs having macrophage phagocytosis. Therefore, preferably, the antibody drug promotes the phagocytosis of the cell by macrophages through binding to the cell surface-expressed protein. Therefore, the pharmaceutical composition provided by the present invention may further comprise the other antibody drug, preferably a macrophage class immune checkpoint antibody; according to the specific embodiment of the present invention, the antibody is an anti-CD47 antibody.

[0065] As an antibody that binds to human Trop-2 or any part thereof, the present invention also provides related applications of the above subject matter.

[0066] Specifically, in yet 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 drug for treating Trop-2 highly expressed cancer; preferably, the Trop-2 highly expressed cancer is gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial cancer, triple-negative breast cancer or cervical cancer.

[0067] In this aspect, the use covers the use of the antibody or its fragment of the present invention in combination with the other antibody drugs described above in the preparation of the drug.

[0068] The antibody or its fragment provided by the present invention can also be fused or conjugated with other parts. For example, the present invention provides a fusion protein or conjugate, which comprises the antibody or its fragment of the present invention.

[0069] Regarding the fusion protein, the fusion protein may comprise any other part that modifies the antibody or its fragment described in the present invention, such as an amino acid, a polypeptide or a protein.

[0070] Regarding the conjugate, the conjugate may comprise the antibody or its fragment of the present invention and a drug conjugated thereto, wherein the drug is, for example, a cytotoxic agent.

[0071] Preferably, the conjugate is an antibody-drug conjugate (ADC) represented by the following formula: (antibody or its fragment of the present invention)-(linker)-(cytotoxic agent);

[0072] Preferably, the cytotoxic agent is a tubulin inhibitor (such as paclitaxel, docetaxel, etc.) or a DNA replication inhibitor (such as irinotecan or its metabolically active substance SN-38, etc.).

[0073] According to a specific embodiment of the present invention, the conjugate is an "anti-TROP-2 antibody-linker-SN-38 antibody-drug conjugate".

[0074] The present invention also provides the use of the antibody or its fragment, nucleic acid molecule, vector, host cell and / or pharmaceutical composition in the preparation of an antibody-drug conjugate (ADC) for treating Trop-2 overexpressing cancers; preferably, the Trop-2 overexpressing cancers are gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial cancer, triple-negative breast cancer or cervical cancer.

[0075] The present invention provides a novel anti-human Trop-2 antibody, which has good biological activity: whether it is Trop-2 recombinant protein or Trop-2 antigen expressed on the cell surface, the antibody provided by the present invention (including chimeric antibody and humanized antibody) can effectively bind, similar to the control antibody Sacituzumab. At the same time, the antibody provided by the present invention has high affinity for human Trop-2: compared with the control antibody Sacituzumab, the humanized antibody of the present invention even has higher specific binding ability to human Trop-2 protein, and the affinity is higher than that of Sacituzumab. Therefore, the antibody of the present invention has good drug efficacy.

[0076] Experiments have proved that the antibody of the present invention also has good internalization ability: the internalization rate of the humanized antibody is similar to that of the control antibody Sacituzumab; the internalization ability is significantly enhanced after being labeled as ADC. Therefore, the antibody of the present invention has the potential for developing ADC drugs. The Trop-2 antibody of the present invention can also have a synergistic effect with other antibodies. For example, the antibody of the present invention can be used in combination with CD47 to further promote the phagocytosis of tumor cells by macrophages.

[0077] In addition, the antibody of the present invention has also been proven to have good in vivo efficacy. When the ADC is prepared with the antibody of the present invention, it is found that the anti-Trop2-ADC antibody has a dose-dependent inhibitory effect on tumor growth. At high doses (10 mg / kg), the efficacy of each ADC antibody is equivalent to that of the control antibody Sacituzumab, and no obvious toxic effect of the ADC small molecule SN38 is observed. The body weights of the animals in each experimental group increase steadily, showing no significant difference from the control group. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Hereinafter, the embodiments of the present invention will be described in detail with reference to the accompanying drawings, where:

[0079] Figure 1 Shows the screening results of the binding of the positive hybridoma supernatant to Trop-2 on the surface of CHO cells.

[0080] Figure 2 Shows the screening results of the binding of the positive hybridoma supernatant to Trop-2 on the surface of CHO cells.

[0081] Figure 3 Shows the cross-reactivity results of the ELISA detection of the positive hybridoma clone supernatant with recombinant Trop-2 of different species.

[0082] Figure 4 Shows the binding activity results of the ELISA detection of the anti-human Trop-2 chimeric antibody with the Trop-2 recombinant protein, where 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.

[0083] Figure 5 Shows the binding activity results of the FACS detection of the anti-human Trop-2 chimeric antibody with the Trop-2 recombinant protein on the cell surface, where 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.

[0084] Figure 6 Shows the species-specific results of the ELISA detection of the anti-human Trop-2 antibody binding to Trop-2, where Figure 6 A: h23-12; Figure 6 B: h4-3; Figure 6 C: Sacituzumab.

[0085] Figure 7 Shows the results of the affinity analysis of anti-human Trop-2 antibody against the recombinant protein of the extracellular region of human Trop-2, where Figure 7 A: Sacituzumab; Figure 7 B: h23-12; Figure 7 C: h4-3.

[0086] Figure 8 Shows the results of the internalization observation after the anti-Trop-2 humanized antibody binds to Trop-2 on the surface of N87 cells.

[0087] Figure 9 Shows the plasma concentration-time curve (for Trop-2 detection) of the anti-Trop-2 humanized antibody after a single-dose administration in nude mice, where Figure 9 A: h23-12; Figure 9 B: h4-3.

[0088] Figure 10 Shows the inhibition rate of the anti-Trop-2-ADC antibody on cell growth.

[0089] Figure 11 Shows the change curve of the body weight of the mice in the Balb / C nu nude mice model bearing gastric cancer N87 tumors, where Figure 11 A: After administration of ch4-3-SN38; Figure 11 B: After administration of h23-12-SN38; Figure 11 C: After administration of the isotype control antibody; Figure 11 D: After high-dose administration of ch4-3-SN38 and h23-12-SN38.

[0090] Figure 12 Shows the change curve of the tumor volume of the mice in the Balb / C nu nude mice model bearing gastric cancer N87 tumors, where Figure 12 A: After administration of ch4-3-SN38; Figure 12 B: After administration of h23-12-SN38; Figure 12 C: After administration of the isotype control antibody; Figure 12 D: After high-dose administration of ch4-3-SN38 and h23-12-SN38.

[0091] Figure 13 Shows the change curve of the body weight of the mice in the SKOV3 subcutaneous xenograft tumor mouse model after the combination of anti-Trop2 antibody and anti-CD47 antibody.

[0092] Figure 14 Shows the change curve of the tumor volume of the mice in the SKOV3 subcutaneous xenograft tumor mouse model after the combination of anti-Trop2 antibody and anti-CD47 antibody.

[0093] Figure 15 Shows the tumor volume change curve of a subcutaneous transplanted tumor mouse model of Balb / C nu nude mice bearing gastric cancer NCI-N87.

[0094] Figure 16 Shows the change curve of the body weight of mice in a subcutaneous transplanted tumor mouse model of Balb / C nu nude mice bearing gastric cancer NCI-N87. Specific implementation manners

[0095] The present invention will be described below with reference to specific embodiments. Those skilled in the art can understand that these embodiments are only used to illustrate the present invention and do not limit the scope of the present invention in any way.

[0096] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The medicinal material raw materials, reagent materials, etc. used in the following embodiments are all commercially available products unless otherwise specified.

[0097] Example 1 Preparation of anti-human Trop-2 antibody hybridoma cells

[0098] Immunization: Balb / c mice were immunized with human Trop-2 recombinant protein (sequence number: NP_002344.2, 1aa-274aa), and the serum titer was detected by ELISA using a 96-well enzyme-linked immunosorbent assay plate coated with human Trop-2-his recombinant protein (sequence number: NP_002344.2, 1aa-274aa); mice with a serum titer meeting the fusion requirements were used for the next cell fusion.

[0099] Cell fusion and hybridoma preparation: Mice with a titer meeting the requirements were selected for booster immunization. Three days later, the spleens of the mice were aseptically removed to prepare a B lymphocyte suspension, which was mixed with SP2 / 0 myeloma cells at a ratio of 4:1, and the two types of cells were fused under the action of PEG. The fused cells were resuspended with HAT medium and then aliquoted into 96-well cell culture plates. Incubate in a 37°C, 5% CO 2 incubator.

[0100] Example 2 Screening of anti-human Trop-2 antibody positive hybridoma cell lines

[0101] 1. Screening of positive hybridoma binding

[0102] 10 - 14 days after fusion, coat an ELISA plate with human Trop-2-his recombinant protein (sequence number: NP_002344.2, 1aa - 274aa) (20 ng / ml) at 4°C overnight; after washing three times with PBS, block with 4% skim milk-PBS at room temperature for 1 hr; wash three times with PBS, add the hybridoma clone culture supernatant at room temperature for 1 hr. Set the following controls: (1) Positive control (PC): serum from immunized mice (diluted 1:1000 with PBS); (2) Negative control (NC): fusion wells without cell growth. After washing three times with PBST (0.05% Tween-PBS) and twice with PBS, add HRP-goat anti-mouse IgG (Fcγ) at 37°C for 0.5 hr; then wash 3 times with PBST (0.05% Tween 20-PBS), add TMB chromogenic solution, develop color in the dark for 15 - 30 min, add ELISA stop solution to terminate the reaction; read the A450 value with an ELISA reader.

[0103] Select the top 95 clones with high readings in descending order for secondary ELISA confirmation. Select 25 antibody-secreting positive cell pools and perform subcloning using the limiting dilution method. 10 days after plating, pick the supernatants of monoclonal cells and further screen for positive clones by ELISA. The ELISA method is the same as above. Select the top 21 clones with high readings in descending order, namely m1-1, m3-11, m4-3, m5-5, m6-6, m7-13, m11-4, m12-2, m12-4, m13-2, m14-2, m15-3, m16-7, m17-1, m18-4, m19-5, m20-4, m21-1, m22-1, m23-12, m24-3 for the next step of FACS combined screening.

[0104] 2. Screening for binding of positive hybridomas to Trop-2 on the surface of CHO cells

[0105] Clone the open reading frame of the Trop-2 gene from the vector containing Trop-2 cDNA (Cat.: HG10428-M, Sino Biological Inc., Beijing) by PCR and clone it into a stable expression vector containing the glutamine synthetase (GS) selection gene by restriction enzyme digestion. Electroporate suspension-cultured CHO-K1 cells (Nucleofector IIb, Lonza). Place the transfected cells in CD CHO AGTTM medium (Cat.: 12490-025, Gibco) containing 50 μM MSX (Cat.: M5379, Sigma), inoculate into a 96-well cell culture plate, and culture at 37°C in 5% CO 2Static culture was carried out for 2 - 3 weeks. Through MSX pressure screening, 22 cell growth wells obtained from the pre - screening were amplified to a 24 - well cell culture plate. Finally, clone No. 1 - T - 21 (CHO / Trop - 2 cells) was selected by flow cytometry (FACS) for scale - up culture, cryopreservation, and used for FACS detection.

[0106] According to the above ELISA results, the hybridoma supernatants of these 21 clones were 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): murine IgG constant region form of Sacituzumab, 1 μg / ml; (2) Negative control (NC): irrelevant murine antibody, 1 μg / ml. After washing the cells 3 times with PBS, 1:200 - diluted goat anti - murine IgG - FITC (Cat.: F9006, Sigma) was added and incubated for 30 min. After washing the cells 3 times with PBS, the mean fluorescence intensity (MFI) of the cells was detected by a flow cytometer (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 Figure 1 .

[0107] According to Figure 1 and the 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, m23 - 12 were selected. The hybridoma supernatants were purified using a ProA affinity chromatography column, and the purified murine antibodies were confirmed for binding again. The antibodies were diluted to 13 nM and 0.66 nM respectively, and then incubated with the suspension of CHO cells (CHO / Trop - 2 cells) recombinantly expressing human Trop - 2 at 37 °C for 30 min. The following controls were set: (1) Positive control (PC): murine IgG constant region form of Sacituzumab, 1 μg / ml; (2) Negative control (NC): irrelevant murine antibody, 1 μg / ml. After washing the cells 3 times with PBS, 1:200 - diluted goat anti - murine IgG - FITC (Cat.: F9006, Sigma) was added and incubated for 30 min. After washing the cells 3 times with PBS, the mean fluorescence intensity (MFI) of the cells was detected by a flow cytometer (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. As Figure 2 shown, the antibodies from the supernatants of 15 clones had good binding to Trop - 2 on the surface of CHO cells.

[0108] Select m3-11, m4-3, m11-4, m17-1, m23-12 as candidate clones for further screening.

[0109] 3. Species-cross ELISA screening of positive hybridoma clones

[0110] Coat human Trop-2-His recombinant protein (sequence number: NP_002344.2, 1aa-274aa), cynomolgus monkey Trop-2-His recombinant protein (sequence number: UniProtKB-A0A2K5UE71, 1aa-272aa), and mouse Trop-2-His recombinant protein (Cat.: 50922-M08H, Sino Biological Inc.) overnight at 4°C, with coating concentrations of 0.2 and 1 μg / mL respectively; after washing the plate 3 times with PBS, add 5% BSA in PBS and incubate at 37°C for 60 min, then wash the plate 3 times with PBST; dilute the above 15 purified mouse antibodies to 1 μg / mL with PBS, and set the following controls: (1) Positive control (PC): Mouse IgG constant region form of Sacituzumab (WHO Drug Information (Vol.31, No.1, 2017), SEQ ID NO:39 and SEQ ID NO:40), 1 μg / ml; (2) Negative control (NC): Irrelevant hybridoma antibody, 1 μg / mL; (3) Blank control: PBS. Incubate at 37°C for 60 min, then wash the plate 4 times with PBST; add HRP-goat anti-mouse IgG (Fcr) (Cat: 115-035-071, Jackson Immuno Research) diluted 1:5000, incubate at 37°C for 30 min, and wash the plate 4 times with PBST; add TMB substrate for color development, incubate at 37°C for 10 min, then add 2M HCl to terminate the reaction; read and record the absorbance A450nm-630nm of the microplate at a wavelength of 450 nm with 630 nm as the reference wavelength. Except for the antibodies of clones m12-4, m17-1, m19-5, and m21-1 that cross-react with mouse Trop-2, the others do not cross-react with mice, but all hybridoma antibodies can specifically bind to recombinant human and cynomolgus monkey Trop-2 ( Figure 3 ).

[0111] Example 3 Sequence determination of mouse anti-human Trop-2 antibody

[0112] After the hybridoma cells m3-11, m4-3, m11-4, m17-1, and m23-12 that secrete anti-human Trop-2 antibodies were expanded in culture, subtype detection was performed using a Mouse Monoclonal Antibody IgG Subclass Test Card (Cat.: A12403, VicNovo) and a Mouse Monoclonal Antibody Light / Heavy Chain Test Card (Cat.: A12401, VicNovo) according to the reagent operation procedures. The subtype identification was: the heavy chain was IgG1 and the light chain was the Kappa chain, providing a basis for the cloning of the antibody genes of m3-11, m4-3, m11-4, m17-1, and m23-12.

[0113] Total cellular RNA was extracted from the hybridoma cells m3-11, m4-3, m11-4, m17-1, and m23-12 according to the steps described in the TRIzol kit (Cat.: 15596026, Invitrogen) manual; the total RNA of the hybridoma cells 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). Peking University Medical Press, 2001, 313-314]) and the Phusion kit (Cat.: E0553L, NEB) were used to amplify the variable region of the antibody light chain IgVL(κ) and the variable region V H sequence; the PCR amplification products were purified using a gel extraction kit (Cat.: AP-GX-250, Axygen); the amplified PCR products were ligated to the T vector and transformed into competent Escherichia coli cells according to the instructions of the T vector cloning kit (Cat.: ZC205, Zhuangmeng Biotech). After strain amplification and plasmid extraction, DNA sequencing was performed to obtain the variable region sequences of the monoclonal antibodies.

[0114] The sequencing results showed:

[0115] The nucleotide sequence of the variable region of the heavy chain of the mouse antibody cloned from m3-11 is shown in SEQ ID NO: 41, and the amino acid sequence of the variable region of the heavy chain of the mouse antibody cloned from m3-11 deduced from this DNA sequence is shown in SEQ ID NO: 1; the nucleotide sequence of the variable region of the light chain of the mouse antibody cloned from m3-11 is shown in SEQ ID NO: 42, and the amino acid sequence of the variable region of the light chain of the mouse antibody cloned from m3-11 deduced from this DNA sequence is shown in SEQ ID NO: 18.

[0116] SEQ ID NO: 1:

[0117] QVQLQQPGAELVKPGSSVKLSCKASGYTFT SYWMY WVKQRPGQGLEWI

[0118] G EINPSNGRTNYNEKFKS KATLTVDKSSSTAYMQFSSLTSEDSAVYYCTR E

[0119] GHNYDGSLGAMDH WGQGTSVTVSS

[0120] SEQ ID NO:18:

[0121] DVVVTQTPLSLPVSFGDQVSISC RSSQSLTNSYGNTFLS WYLHKPGQSPQL

[0122] LLY GISNRFS GVPDRFSGSGSGTDFTLKINTIKPEDLGMYYC FQSTHQPYT

[0123] FGGGTKLEIK

[0124] The nucleotide sequence of the murine antibody heavy chain variable region DNA of clone m4-3 is shown in SEQ ID NO:43, and the amino acid sequence of the murine antibody heavy chain variable region deduced from this DNA sequence is shown in SEQ ID NO:2; the nucleotide sequence of the murine antibody light chain variable region DNA of clone m4-3 is shown in SEQ ID NO:44, and the amino acid sequence of the murine antibody light chain variable region deduced from this DNA sequence is shown in SEQ ID NO:19.

[0125] SEQ ID NO:2:

[0126] QVQLQQSGPELVKPGASVKMSCKASGFTFT DYVIG WVKQRTGQGLEWIG

[0127] EIYLGSGTIYYTEKFKG KATLTADTSSNTAYMQLSSLTSEDSAVYFCAR GSI

[0128] FPFDY WGQGTTLTVSS

[0129] SEQ ID NO:19:

[0130] QIVLTQSPAIMSASPGEKVTMTC SASSSVSYMY WYQQKPGSSPRLLIY DTS

[0131] TLAS GVPVRFSGSGSGTSYSLTISRMEAEDAATYYC QQWSSYPYT FGGGT

[0132] KLEIK

[0133] The nucleotide sequence of the heavy chain variable region DNA of the mouse antibody cloned m11-4 is shown in SEQ ID NO:47, and the amino acid sequence of the heavy chain variable region of the mouse antibody cloned m11-4 deduced from this DNA sequence is shown in SEQ ID NO:5; the nucleotide sequence of the light chain variable region DNA of the mouse antibody cloned m11-4 is shown in SEQ ID NO:48, and the amino acid sequence of the light chain variable region of the mouse antibody cloned m11-4 deduced from this DNA sequence is shown in SEQ ID NO:24.

[0134] SEQ ID NO:5:

[0135] QVQLQQPGAELVRPGASVNLSCKASGYTFT SYWIN WVKQRPGQGLEWIG

[0136] NIYPSNSYTNYNQKFKD TATLTVDKSSSTAYMQLSSPTSEDSAVYFCSS YRSDGFAY WGQGTLVTVSA

[0137] SEQ ID NO:24:

[0138] DILLTQSPAILSVSPGEKVSFSC RASQNIGTSIH WYQQRTNGSPRLLIE FASESIS GIPSRFSGSGSGTDFTLTINSVESEDIADYYC QQSNSWPFT FGGGTKLEIK

[0139] The nucleotide sequence of the heavy chain variable region DNA of the mouse antibody cloned m17-1 is shown in SEQ ID NO:51, and the amino acid sequence of the heavy chain variable region of the mouse antibody cloned m17-1 deduced from this DNA sequence is shown in SEQID NO:11; the nucleotide sequence of the light chain variable region DNA of the mouse antibody cloned m17-1 is shown in SEQ ID NO:52, and the amino acid sequence of the light chain variable region of the mouse antibody cloned m17-1 deduced from this DNA sequence is shown in SEQ ID NO:30.

[0140] SEQ ID NO:11:

[0141] EVKLVESGGVLVKPGGSLKLSCAASGFTFS DSAMS WVRQTPEKRLEWVA SISRGDDTYYPDSVKG RITISRDFARNILYLQMTSLRSEDTAMYYCTR DRFGFAY WGQGTLVTVSA

[0142] SEQ ID NO:30:

[0143] DIVMTQSPLTLSVTIGQPASISC KSGQSLLDSDGKTYFN WLLQRPGQSPKRLIY LVSMLDS GVPDRFTGSGSGTDFTLKISRVETEDLGVYYC WQGTHFPFT FGSGTKLEIK

[0144] The nucleotide sequence of the mouse antibody heavy chain variable region DNA of clone m23-12 is shown in SEQ ID NO:53, and the amino acid sequence of the mouse antibody heavy chain variable region deduced from this DNA sequence is shown in SEQ ID NO:12; the nucleotide sequence of the mouse antibody light chain variable region DNA of clone m23-12 is shown in SEQ ID NO:54, and the amino acid sequence of the mouse antibody light chain variable region deduced from this DNA sequence is shown in SEQ ID NO:31.

[0145] SEQ ID NO:12:

[0146] QVQLQQPGAELVKPGASVKLSCKADGYIFT SYWMH WVKQRPGQGLEWIG EITPSDNYTSYNQKFKG KATLTVDKSSSTAYMQLSSLTSEDSAVYYCTR GHGNYVSFDY WGQGTTLTVSS

[0147] SEQ ID NO:31:

[0148] DIQMTQITSSLSASLGDRVTITC RASQDISNYLN WYQQKPDGTVKLLIY YTSRLHS GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC QQGYTLPPYT FGGGTKLEIK

[0149] Example 4 Preparation of anti-human Trop-2 chimeric antibody and control antibody

[0150] The light and heavy chain sequences of the control antibody (Sacituzumab) were fully synthesized, and the light and heavy chain sequences were respectively cloned into eukaryotic transient expression vectors to obtain the light and heavy chain expression plasmids of the control antibody. These plasmids were transferred into Escherichia 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 according to the operating instructions of the transfection reagent 293fectin (Cat.: 12347019, Gibco), the light and heavy chain plasmids of the control antibody were respectively transferred into HEK293 cells for recombinant expression. Five to six days after cell transfection, the culture supernatant was taken, and the expression supernatant was purified using a ProA affinity chromatography column to obtain the control antibody. Among them, the amino acid sequence of the control antibody Sacituzumab was derived from WHO Drug Information (Vol. 31, No. 1, 2017), the amino acid sequence of the heavy chain was shown in SEQ ID NO: 39, and the amino acid sequence of the light chain was shown in SEQ ID NO: 40.

[0151] The light and heavy chain variable region genes of the corresponding murine antibodies 3-11, 4-3, 11-4, 17-1, 23-12 obtained from each clone were introduced with restriction enzyme sites by PCR and respectively cloned into eukaryotic transient expression vectors upstream of the genes encoding the human-kappa light chain constant region and the human IgG1 heavy chain constant region to obtain 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-ch3-11H, pKN019-ch4-3H, pKN019-ch11-4H, pKN019-ch17-1H, pKN019-ch23-12H) expression plasmids. These plasmids were transferred into Escherichia coli for amplification, and a large number of plasmids containing the light and heavy chains of the human-mouse chimeric antibody were isolated. Using this plasmid and according to the operating instructions of the transfection reagent 293fectin (Cat.: 12347019, Gibco), the light and heavy chain plasmids of the chimeric antibodies ch3-11, ch4-3, ch11-4, ch17-1, ch23-12 were respectively transferred into HEK293 cells for recombinant expression. Five to six days after cell transfection, the culture supernatant was taken, and the expression supernatant was purified using a ProA affinity chromatography column to obtain the chimeric antibodies ch3-11, ch4-3, ch17-1, ch11-4, ch23-12.

[0152] Example 5 ELISA detection of the binding activity of anti-human Trop-2 chimeric antibody to Trop-2 recombinant protein

[0153] Human Trop-2-his recombinant protein (sequence number: NP_002344.2, 1aa-274aa), with a concentration of 0.2 μg / mL, was coated overnight at 4°C and blocked with 5% BSA in a 37°C incubator for 60 min. ch3-11, ch4-3, ch17-1, ch11-4, ch23-12 and the control antibody Sacituzumab (starting concentration of 2 μg / mL, 3-fold serial dilution, 8 gradients) were added respectively. After reacting in a 37°C incubator 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 reacted for 45 min, and TMB (Cat.: ME142, Beijing TaiTianHe Bio) substrate was added for color development for 15 min. After termination with 2M HCl, the plate was read. With 630 nm as the reference wavelength, the absorbance value A450nm-630nm of the well plate at a wavelength of 450 nm was read and recorded.

[0154] The binding ability of ch3-11, ch4-3, ch17-1, ch11-4, ch23-12 and the control antibody Sacituzumab to human Trop-2 recombinant protein was determined by ELISA, and their half-maximal effective binding concentration (EC50) values were 0.3147 nM, 0.3195 nM, 0.3278 nM, 0.2366 nM, 0.4581 nM and 0.271 nM ( Figure 4 ), which were basically equivalent. The results showed that ch3-11, ch4-3, ch17-1, ch11-4, ch23-12 chimeric antibodies had high affinity for human Trop-2 recombinant protein, and the sequences of murine antibodies 3-11, 4-3, 11-4, 17-1, 23-12 were cloned correctly.

[0155] Example 6 FACS was used to detect the binding activity of anti-human Trop-2 chimeric antibodies to human Trop-2 recombinant protein on the surface of CHO cells

[0156] Suspensions of CHO cells recombinantly expressing human Trop-2 (CHO / Trop-2 cells) were incubated with chimeric antibodies (ch3-11, ch4-3, ch17-1, ch11-4, ch23-12) (at concentrations of 30 μg / mL, 10 μg / mL, 5 μg / mL, starting with 3-fold serial dilutions for 9 gradients, a total of 11 gradients) at 37 °C for 30 min. The following controls were set: (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, 1:100 diluted goat anti-human IgG-FITC (Cat.: F9512, Sigma) was added and incubated for 30 min. After washing the cells 3 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 antibodies to human Trop-2 on the surface of CHO cells.

[0157] The binding abilities of ch3-11, ch4-3, ch17-1, ch11-4, ch23-12 and the control antibody Sacituzumab to the recombinant human Trop-2 protein on the surface of CHO cells were determined by FACS, and their half-maximal effective binding concentration (EC50) values were 0.993 nM, 3.326 nM, 2.918 nM, 1.154 nM, 2.748 nM and 2.316 nM ( Figure 5 ). Compared with the control antibody Sacituzumab, ch3-11 and ch11-4 had better binding activities, and ch4-3, ch17-1 and ch23-12 had similar binding activities. The results showed that the anti-human Trop-2 chimeric antibodies ch3-11, ch4-3, ch17-1, ch11-4, ch23-12 could effectively bind to the recombinant human Trop-2 protein on the surface of CHO cells.

[0158] Example 7 Internalization activity of anti-human Trop-2 chimeric antibodies binding to Trop-2 on the cell surface

[0159] Human pancreatic cancer cells BxPC-3 were taken, 5×10 5Cells / tube were added with chimeric antibodies ch3-11, ch4-3, ch11-4, ch23-12 diluted to 10 μg / ml and the positive control antibody Sacituzumab. Each antibody was divided into four groups (experimental groups incubated for 1 h, 3 h, 5 h and the control group), with 2 tubes in each group. The experimental groups were placed in an electrothermal constant temperature incubator at 37°C, incubated for 1 h, 3 h, and 5 h respectively, and then placed on ice. The control group was incubated on ice all the time as the negative control. After all samples were incubated, they were centrifuged at 1,500 rpm at 4°C for 3 min, the supernatant was discarded, washed once with ice-cold PBS, the secondary antibody, namely anti-human IgG (Fc specific)-FITC antibody (Cat.: F9512, Sigma), was added, incubated on ice for 30 minutes, then centrifuged at 1,500 rpm for 3 min, the supernatant was discarded, washed with ice-cold PBS, 200 μl of ice-cold PBS was taken to resuspend the cells, and the mean fluorescence intensity MFI was detected by FACS. The internalization efficiency was calculated by the formula: %MFI at the tx time point = MFI of the sample incubated at 37°C × 100% / MFI of the control sample incubated at 4°C; the internalization percentage at the tx time point = 100% - %MFI at the tx time point.

[0160] The results are shown in Table 1. The internalization ratios of ch4-3 and ch23-12 were similar to that of the control antibody Sacituzumab, while there was no obvious internalization of ch3-11 and ch11-4.

[0161] Table 1. Internalization percentages of anti-human Trop-2 chimeric antibodies mediated by cell surface Trop-2

[0162]

[0163] Example 8 Stability of anti-human Trop-2 chimeric antibodies against tolerance disruption

[0164] The chimeric antibodies ch3-11, ch4-3, ch11-4, and ch23-12 were placed in PBS, PBS containing 10% N,N-dimethylacetamide (DMA) (Cat.: ARK2190, Shanghai Feibo Chemical Industry), and PBS containing 20% DMA at a concentration of 5 mg / mL respectively. After being placed at 37°C for 2 h, the DMA in the samples was removed using an ultrafiltration centrifugal tube, and the replacement buffer was PBS. The sample purity was analyzed by high performance size exclusion chromatography (SEC-HPLC) using a G3000Wxl liquid chromatography analysis column (Cat.: SEC-0046, Tosoh). The purity analysis results are shown in Table 2.

[0165] The results showed that all 4 antibodies could tolerate DMA well. The purity decreased slightly when the concentration of DMA was 10%, and decreased slightly more when the concentration was 20%, indicating that the antibodies may have good tolerance to the subsequent ADC process.

[0166] Table 2. HPLC Purity Analysis of Antibody before and after DMA Processing

[0167]

[0168]

[0169] Example 9 Humanization and Recombinant Expression of Anti-Human Trop-2 Monoclonal Antibody

[0170] 1. Humanization of Murine Monoclonal Antibody 23-12

[0171] (1) CDR Transplantation

[0172] First, comprehensively analyze the heavy chain sequence of the murine antibody to determine the antigen complementary determining region (CDR) where the antibody binds to the antigen and the framework region that supports the conserved three-dimensional conformation of the antibody. Subsequently, according to the homology alignment results, search for the most similar human antibody template in the human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX). Combining the results of the full-sequence blast and the characteristics of the heavy chain CDR3 sequence, perform CDR transplantation to achieve the full humanization of the variable region of the heavy chain (VH) of 23-12 in the framework region. According to the homology alignment results, search for the most similar human antibody template in the human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX). Combining the results of the full-sequence blast and the characteristics of the light chain CDR3 sequence, perform CDR transplantation to achieve a high degree of humanization of the light chain framework region.

[0173] 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. SEQ ID NO:13:

[0174] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQGLEWMG EITPSDNYTSYNQKFKG RVTITRDTSTSTAYMELSSLRSEDTAVYYCAR GHGNYVSFDY WGQGTLVTVSS

[0175] SEQ ID NO:32:

[0176] DIQMTQSPSSLSASVGDRVTITC RASQDISNYLN WYQQKPGKAPKLLIY YTSRLHS GVPSRFSGSGSGTDFTLTISSLQPEDFATYFC QQGYTLPPYT FGQGTKLEIKRTVAAP

[0177] (2) CDR region mutation design

[0178] According to the sequence characteristics of murine antibody 23-12, the CDR region sequences of the humanized heavy and light chain variable regions for CDR transplantation were subjected to mutation design, and the mutation sites are shown in Table 3 below.

[0179] Table 3. Design of 23-12 humanized sequence

[0180]

[0181]

[0182] Note: The amino acid residue site numbers follow the Kabat numbering system.

[0183] 2. Recombinant expression of humanized monoclonal antibody 23-12

[0184] The sequences of the humanized heavy and light chain variable regions of the h23-12 antibody (h23-12_VL1, h23-12_VH1) designed for humanization were fully synthesized. The humanized h23-12_VH1 was cloned by restriction enzyme digestion into the upstream of the heavy chain constant region encoding gene of the eukaryotic transient expression vector pKN041. 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. The humanized h23-12_VL1 was cloned by restriction enzyme digestion into the upstream of the light chain Cκ encoding gene of the eukaryotic transient expression vector pKN019. 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:38. Humanized 23-12 light and heavy chain expression vectors were constructed to obtain light chain (pKN019-h23-12L1) and heavy chain (pKN041-h23-12H1) expression plasmids, which were transferred into Escherichia coli for amplification, and the plasmids h23-12L1 and h23-12H1 of the h23-12 antibody light and heavy chains were isolated.

[0185] According to the mutation design shown in Table 3, using the StarMut Site-Directed Mutagenesis Kit (GenStar, Cat.: T111-01), site-directed mutagenesis was performed on the light chain (pKN019-h23-12L1) and heavy chain (pKN041-h23-12H1) expression plasmids respectively, and then transferred into Escherichia coli for amplification to obtain the mutant expression plasmids of the CDR regions of the light and heavy chains of the h23-12 antibody (h23-12H2~h23-12H7, h23-12L2~h23-12L7), which corresponded to the 23-12 humanized sequences in Table 3 respectively; according to the operation instructions of the transfection reagent 293fectin (Cat.: 12347019, Gibco), the light and heavy chain plasmids of the 23-12 humanized antibody were combined as shown in Table 4 and then transferred into HEK293 cells for recombinant expression.

[0186] Table 4. Combinations of Humanized 23-12 Light and Heavy Chain Sequences

[0187]

[0188]

[0189] Note: This table shows the sequences obtained from various 23-12 light and heavy chain combinations. For example, h23-12-1 indicates that this antibody is composed of the 23-12 humanized antibody light chain h23-12L1 and the humanized heavy chain h23-12H1, and so on.

[0190] At 5 - 6 days after cell transfection, the culture supernatant was taken, and the expression supernatant was purified using a ProA affinity chromatography column. For the obtained different humanized antibodies of 23-12, the antibody affinity was measured using the Octet QKe system instrument of Fortebio company by the method of capturing the antibody Fc segment with the capture antibody (AHC) bioprobe against the human antibody Fc segment. During the measurement, the 23-12 antibody and the control antibody Sacituzumab were diluted to 4 μg / mL with PBS buffer and flowed through the surface of the AHC probe (Cat.: 18-0015, PALL) for 120 s. The human Trop-2-His recombinant protein (sequence number: NP_002344.2, 1aa - 274aa) was used as the mobile phase, and the concentration of the Trop-2-His recombinant protein was 60 nM. The binding time was 100 s, and the dissociation time was 300 s. After the experiment, the blank control response value was deducted, and the software was used for 1:1 Langmuir binding mode fitting to calculate the kinetic constants of antigen-antibody binding.

[0191] The affinities of the h23-12 mutant combination antibodies, the chimeric antibody ch23-12, and the control antibody Sacituzumab for the human Trop-2-His recombinant protein were measured by ForteBio (Table 5).

[0192] Table 5. Affinity determination results of different antibodies and recombinant human Trop-2 extracellular domain protein

[0193]

[0194]

[0195] Select h23-12-25. The affinity (KD) of this combined antibody is 5.02E-10 M, named h23-12, for further functional verification. 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.

[0196] SEQ ID NO:14:

[0197] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQGLEW

[0198] MG EITPSDNYGSYNQKFKG RVTITRDTSTSTAYMELSSLRSEDTAVYYCA

[0199] R GHGNYVSFDY WGQGTLVTVSS

[0200] SEQ ID NO:33:

[0201] DIQMTQSPSSLSASVGDRVTITC RASQDISNYLN WYQQKPGKAPKLLIY Y

[0202] TSRLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYFC QQGYTLPPYT FGQG

[0203] TKLEIK

[0204] 3. Humanization of murine monoclonal antibody 4-3

[0205] (1) CDR grafting

[0206] First, the heavy chain sequence of the murine antibody was comprehensively analyzed to determine the antigen - complementary determining region (CDR) where the antibody binds to the antigen and the framework region that supports the conserved three - dimensional conformation of the antibody. Subsequently, based on the results of homology alignment, 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 results of the full - sequence blast and the characteristics of the heavy - chain CDR3 sequence, CDR transplantation was performed to achieve full humanization of the framework region of the 4 - 3 heavy - chain variable region (VH). According to the results of homology alignment, 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 results of the full - sequence blast and the characteristics of the light - chain CDR3 sequence, CDR transplantation was performed to achieve full humanization of the framework region of the light chain.

[0207] 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.

[0208] SEQ ID NO:3:

[0209] EVQLVQSGPEVKKPGASVKVSCKASGFTFT DYVIG WVRQAPGQGLEWIG

[0210] EIYLGSGTIYYTEKFKG RVTMTADTSTSTAYMELSSLRSEDTAVYYCAR GS

[0211] IFPFDY WGQGTLVTVSS

[0212] SEQ ID NO:20:

[0213] DIQLTQSPSSLSASVGDRVTITC SASSSVSYMY WYQQKPGKAPKLLIY DTS

[0214] TLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQWSSYPYT FGQGTK

[0215] LEIK

[0216] (2) CDR region mutation design

[0217] Based on the sequence characteristics of the murine antibody 4-3, the CDR region sequences of the humanized light and heavy chain variable regions for CDR transplantation were subjected to mutation design, and the mutation sites are shown in Table 6 below.

[0218] Table 6. Design of 4-3 humanized sequences

[0219]

[0220] Note: The amino acid residue site numbers follow the Kabat numbering system.

[0221] 4. Recombinant expression of humanized monoclonal antibody 4-3

[0222] The sequences of the humanized light and heavy chain variable regions (h4-3_VL1, h4-3_VH1) of the h4-3 antibody were fully synthesized. The humanized h4-3_VH1 was cloned by restriction enzyme digestion into the upstream of the heavy chain constant region coding gene of the eukaryotic transient expression vector pKN041. 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; the humanized h4-3_VL1 was cloned by restriction enzyme digestion into the upstream of the coding gene of the human light chain Cκ of the eukaryotic transient expression vector pKN019. 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:38. Humanized 4-3 light and heavy chain expression vectors were constructed to obtain light chain (pKN019-h4-3L1) and heavy chain (pKN041-h4-3H1) expression plasmids, which were transferred into Escherichia coli for amplification, and the plasmids h4-3L1 and h4-3H1 of the h4-3 antibody light and heavy chains were isolated.

[0223] According to the mutation design shown in Table 6, using the StarMut site-directed mutagenesis kit (Cat.:T111-01, GenStar), site-directed mutagenesis was performed on the light chain (pKN019-h4-3L1) and heavy chain (pKN041-h4-3H1) expression plasmids respectively, and then transferred into Escherichia coli for amplification to obtain h4-3 antibody light and heavy chain CDR region mutant expression plasmids (h4-3H2~h4-3H4, h4-3L2~h4-3L5), which correspond to the 4-3 humanized sequences in Table 6 respectively; according to the operation instructions of the transfection reagent 293fectin (Cat.:12347019, Gibco), the light and heavy chain plasmids of the 4-3 humanized antibody were combined as shown in Table 7 and transferred into HEK293 cells for recombinant expression.

[0224] Table 7. Combination of humanized 4-3 light and heavy chain sequences

[0225] 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

[0226] Note: This table shows the sequences obtained from various 4-3 light and heavy chain combinations. 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.

[0227] Five to six days after cell transfection, the culture supernatant was taken, and the expression supernatant was purified using a ProA affinity chromatography column to obtain different humanized antibodies of 4-3. The antibody affinity was measured using the Octet QKe system instrument from Fortebio and the method of capturing the antibody Fc segment with the capture antibody (AHC) bioprobe against the human antibody Fc segment. When measuring, the 4-3 antibody and the control antibody Sacituzumab were diluted to 4 μg / mL with PBS buffer and flowed through the surface of the AHC probe (Cat.: 18-0015, PALL) for 120 s. The human Trop-2-His recombinant protein (sequence number: NP_002344.2, 1aa-274aa) was used as the mobile phase, and the concentration of the Trop-2-His recombinant protein was 60 nM. The binding time was 100 s, and the dissociation time was 300 s. After the experiment, the blank control response value was deducted, and the software was used for 1:1 Langmuir binding mode fitting to calculate the kinetic constant of antigen-antibody binding.

[0228] The affinity of the h4-3 mutant combination antibody, the chimeric antibody ch4-3, and the control antibody Sacituzumab for the human Trop-2-His recombinant protein was measured by ForteBio (Table 8).

[0229] Table 8. Results of the affinity measurement of different humanized antibodies of 4-3 for the recombinant protein of the extracellular region of human Trop-2

[0230] 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

[0231] h4-3-1 was selected. The affinity (KD) of this combination antibody was 3.04E-10 M, named h4-3, and further functional verification was carried out. 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.

[0232] SEQ ID NO:3:

[0233] EVQLVQSGPEVKKPGASVKVSCKASGFTFT DYVIG WVRQAPGQGLEWIG

[0234] EIYLGSGTIYYTEKFKG RVTMTADTSTSTAYMELSSLRSEDTAVYYCAR GS

[0235] IFPFDY WGQGTLVTVSS

[0236] SEQ ID NO:20:

[0237] DIQLTQSPSSLSASVGDRVTITC SASSSVSYMY WYQQKPGKAPKLLIY DTS

[0238] TLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQWSSYPYT FGQGTK

[0239] LEIK

[0240] 5. Humanization of Murine Monoclonal Antibody 11-4

[0241] (1) CDR Grafting

[0242] First, a comprehensive analysis was performed on the heavy chain sequence of the murine antibody to determine the antigen - complementary determining region (CDR) where the antibody binds to the antigen and the framework region that supports the conserved three - dimensional conformation of the antibody. Subsequently, based on the results of homology alignment, 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 results of the full - sequence blast and the characteristics of the heavy - chain CDR3 sequence, CDR grafting was carried out to achieve full humanization of the variable region (VH) of the 11 - 4 heavy chain in the framework region. According to the results of homology alignment, 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 results of the full - sequence blast and the characteristics of the light - chain CDR3 sequence, CDR grafting was carried out to achieve full humanization of the light - chain framework region.

[0243] The nucleotide sequence of the humanized heavy - chain variable region h11 - 4_VH1 of the 11 - 4 antibody CDR grafting 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.

[0244] SEQ ID NO:6

[0245] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWIN WVRQAPGQGLEW

[0246] MG NIYPSNSYTNYNQKFKD RVTMTRDTSTSTVYMELSSLRSEDTAVYYC

[0247] AR YRSDGFAY WGQGTLVTVSS

[0248] SEQ ID NO:25

[0249] EIVLTQSPATLSLSPGERATLSC RASQNIGTSIH WYQQKPGQAPRLLIY FAS

[0250] ESIS GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQSNSWPFT FGGGTKVE

[0251] IK

[0252] (2) CDR region mutation design

[0253] Based on the sequence characteristics of murine antibody 11-4, mutation design was carried out on the humanized heavy and light chain variable region sequences for CDR transplantation. The mutation sites are shown in Table 9 below.

[0254] Table 9. Design of 11-4 humanized sequence

[0255]

[0256]

[0257] Note: The amino acid residue site numbers follow the Kabat numbering system.

[0258] 6. Recombinant expression of humanized monoclonal antibody 11-4

[0259] The sequences of the variable regions of the light and heavy chains (h11-4_VL1, h11-4_VH1) of the humanized h11-4 antibody were fully synthesized. The humanized h11-4_VH1 was cloned by enzymatic digestion into the upstream of the heavy chain constant region encoding gene of human IgG1 in the eukaryotic transient expression vector pKN041. 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. The humanized h11-4_VL1 was cloned by enzymatic digestion into the upstream of the light chain Cκ encoding gene in the eukaryotic transient expression vector pKN019. 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:38. Humanized 11-4 light and heavy chain expression vectors were constructed to obtain light chain (pKN019-h11-4L1) and heavy chain (pKN041-h11-4H1) expression plasmids, which were transferred into Escherichia coli for amplification, and the plasmids h11-4L1 and h11-4H1 of the light and heavy chains of the h11-4 antibody were isolated.

[0260] According to the mutation design shown in Table 9, using the StarMut site-directed mutagenesis kit (Cat.:T111-01, GenStar), site-directed mutagenesis was performed on the light chain (pKN019-h11-4L1) and heavy chain (pKN041-h11-4H1) expression plasmids respectively, and then transferred into Escherichia coli for amplification to obtain the mutant expression plasmids of the light and heavy chains of the h11-4 antibody (h11-4H2~h11-4H7, h11-4L2~h11-4L5), which corresponded to the 11-4 humanized sequences in Table 9 respectively. According to the operation instructions of the transfection reagent 293fectin (Cat.:12347019, Gibco), the light and heavy chain plasmids of the 11-4 humanized antibody were combined as shown in Table 10 and transferred into HEK293 cells for recombinant expression.

[0261] Table 10. Combinations of Humanized 11-4 Light and Heavy Chain Sequences

[0262]

[0263] Note: This table shows the sequences obtained from various combinations of 11-4 light and heavy chains. For example, h11-4-1 indicates that this antibody is composed of the light chain h11-4L1 and the heavy chain h11-4H1 of the 11-4 humanized antibody, and so on.

[0264] At 5 - 6 days after cell transfection, the culture supernatant was collected. The expressed supernatant was purified using a ProA affinity chromatography column to obtain different humanized antibodies of 11 - 4. The Octet QKe system instrument from Fortebio was used to determine the antibody affinity by the method of capturing the antibody Fc segment with a capture antibody (AHC) bio - probe against the human antibody Fc segment. During the determination, the 11 - 4 antibody and the control antibody Sacituzumab were diluted to 4 μg / mL with PBS buffer and flowed through the surface of the AHC probe (Cat.: 18 - 0015, PALL) for 120 s. The human Trop - 2 - His recombinant protein (sequence number: NP_002344.2, 1aa - 274aa) was used as the mobile phase, and the concentration of the Trop - 2 - His recombinant protein was 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 software was used for 1:1 Langmuir binding mode fitting to calculate the kinetic constants of antigen - antibody binding.

[0265] The affinities of the h11 - 4 mutant combination antibody, the chimeric antibody ch11 - 4 and the control antibody Sacituzumab to the human Trop - 2 - His recombinant protein were determined by ForteBio (Table 11).

[0266] Table 11. Results of the affinity determination of different humanized antibodies of 11 - 4 to the recombinant protein of the extracellular region of human Trop - 2

[0267] 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

[0268] Example 10 ELISA was used to detect the species - specificity of the anti - Trop - 2 humanized antibody binding to Trop - 2

[0269] Coat human Trop-2-his recombinant protein (sequence number: NP_002344.2, 1aa - 274aa), cynomolgus monkey Trop-2-His recombinant protein (sequence number: UniProtKB-A0A2K5UE71, 1aa - 272aa), and mouse Trop-2-His recombinant protein (Cat.: 50922-M08H, Sino Biological Inc.) overnight at 4°C at a coating concentration of 1 μg / mL; after washing the plate 3 times with PBS, add 5% BSA in PBS and block at 37°C for 60 min, then wash the plate 3 times with PBST; add h23-12 at different dilution multiples (starting concentration 10 μg / mL, diluted 14 concentrations in 3-fold gradients), h4-3 (starting concentration 3 μg / mL, diluted 12 concentrations in 3-fold gradients), and Sacituzumab (starting concentration 3 μg / mL, diluted 12 concentrations in 3-fold gradients), with one parallel well for each concentration, incubate at 37°C for 60 min, and wash the plate 4 times with PBST; add HRP-anti-human Fc diluted 1:5000 (Cat.: 109-035-098, Jackson Immuno Research), incubate at 37°C for 30 min, and wash the plate 4 times with PBST; add TMB substrate for color development, after incubating at 37°C for 10 min, add 2M HCl to terminate the reaction; using 630 nm as the reference wavelength, read and record the absorbance A450nm - 630nm of the well plate at a wavelength of 450 nm.

[0270] The 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 to recombinant mouse Trop-2 ( Figure 6 , Table 12), providing a basis for the pharmacological and toxicological experiments of the humanized antibody.

[0271] Table 12. EC50 of anti-Trop-2 humanized antibody binding to Trop-2 of different species

[0272]

[0273] Example 11 Affinity analysis of anti-Trop-2 humanized antibody

[0274] Use the Octet QKe system instrument from Fortebio to measure the antibody affinity by the method of capturing the antibody Fc segment with a capture antibody (AHC) specific to the Fc segment of the anti-human antibody.

[0275] When measuring, the antibodies (h23-12, h4-3 and the control antibody Sacituzumab) were diluted to 4 μg / mL with PBS buffer and flowed through the surface of the AHC probe (Cat.: 18-0015, PALL) for 120 s. Human Trop-2-his recombinant protein (sequence number: NP_002344.2, 1aa-274aa) was used as the mobile phase, and the Trop-2-his concentrations corresponding to 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 value was deducted, and the software was used for 1:1 Langmuir binding mode fitting to calculate the kinetic constants of antigen-antibody binding.

[0276] The reaction curves of h23-12, h4-3 and the control antibody Sacituzumab with human Trop-2 recombinant protein are as Figure 7 shown. The fitting curves were made and the affinities were calculated. The affinity (KD) of h23-12 was 6.40E-10 M, the affinity (KD) of h4-3 was 5.45E-10 M, and the affinity (KD) of Sacituzumab was 9.41E-10 M. The detailed kinetic parameters are shown in Table 13. The results show that h23-12 and h4-3 have high affinities with human Trop-2, comparable to the control antibody Sacituzumab, and the dissociation value of h23-12 is better than that of Sacituzumab.

[0277] Table 13. Results of affinity determination of anti-Trop-2 humanized antibodies with human Trop-2 extracellular region recombinant protein

[0278] 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

[0279] Example 12 Internalization activity of anti-human Trop-2 humanized antibody binding to cell surface Trop-2

[0280] Human pancreatic cancer cells BXPC-3 that naturally express human Trop-2 were seeded in a 96-well cell culture plate at a density of 2×10 3 cells / well and cultured for 24 hours. The cells were washed once with PBS and the supernatant was discarded. Using Mix-n-Stain TM CF TMh23-12 labeled with 488A (Cat.: MX488AS100, Sigma) and the 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 an electrothermal incubator at 37°C, and one group was placed in a refrigerator at 4°C as a negative control. After incubating the negative control for 30 minutes, it was washed 3 times with PBS, observed and photographed under a fluorescence microscope. After incubating the experimental group at 37°C for 5 h, it was observed and photographed under a fluorescence microscope.

[0281] The experimental results ( Figure 8 ) showed that both the humanized h23-12 and the control antibody Sacituzumab could be endocytosed mediated by Trop-2 at 37°C and showed a punctate distribution in the cytoplasm. It was suggested that the antibody still maintained its internalization activity after humanization.

[0282] The internalization rate on BxPC-3 cells at 3 h was detected by FACS using the method shown in Example 7. The results are shown in Table 14, suggesting that the internalization rate was comparable to that of Sac after humanization.

[0283] Table 14. Internalization percentage of anti-human Trop-2 antibodies mediated by Trop-2 on the surface of BXPC-3 cells

[0284]

[0285] Example 13 Pharmacokinetic study of a single administration to Balb / C nude mice

[0286] Healthy female 5-week-old Balb / C nude mice were divided into groups of 2. After a single intraperitoneal injection of h23-12 at a single dose (15 mg / kg), sera were collected at the 5th h, 25th h, 48th h, 96th h, 168th h, and 240th h, stored at -20°C, and a control group was set up to compare with the control Sacituzumab at the same dose as the intraperitoneal injection of h23-12 to observe its pharmacokinetic characteristics.

[0287] Healthy female 5-week-old Balb / C nude mice were divided into groups of 4. After a single intraperitoneal injection of h4-3 at a single dose (20 mg / kg), sera were collected at the 4th h, 8th h, 24th h, 48th h, 96th h, 144th h, 192nd h, and 240th h, stored at -20°C, and its pharmacokinetic characteristics were observed.

[0288] The method of using the ELISA coated with human Trop-2-his (sequence number: NP_002344.2, 1aa-274aa) was used to detect the drug concentration in serum, and a standard curve was made at the same time. Taking the concentration of the standard antibody as the Y-axis and the OD value as the X-axis, a linear curve was fitted. Substituting the OD value of the detected serum into the formula, the antibody content in the serum could be obtained, and according to the formula T 1 / 2 =|0.693 / k|, calculate the drug half-life T 1 / 2 .

[0289] The results of the drug-time curve showed that h23-12, h4-3 and the control antibody Sacituzumab all had a relatively long half-life in mice. The drug metabolic half-life T 1 / 2 showed equivalent performance( Figure 9 A, Figure 9 B, Table 15), indicating that there was no obvious inactivation phenomenon of the antibody in vivo and it had good structural stability. Its metabolism conforms to the basic characteristics of monoclonal antibody drugs, and T 1 / 2 was about 170 h.

[0290] Table 15. Pharmacokinetic parameters of anti-Trop-2 antibodies after single-dose administration in nude mice

[0291] <![CDATA[T 1 / 2 (Trop-2 Detection)]]> Sacituzumab (n = 2) 168±14 h23-12 (n = 2) 174±12 h4-3 (n = 4) 180±57

[0292] Example 14 Affinity analysis of anti-Trop-2 naked antibody and its ADC antibody

[0293] Using the Octet QKe system instrument of Fortebio Company, the antibody affinity was measured by the method of using the capture antibody (AHC) bioprobe against the Fc segment of the anti-human antibody to capture the Fc segment of the antibody.

[0294] First, prepare the antibody labeled with the ADC drug SN38. The antibody was reduced with 20 equivalents of dithiothreitol (DTT) in a sodium phosphate buffer at a pH range of 7.0±0.5 for 2 h. Subsequently, the reduced antibody was purified using an ultrafiltration centrifugal tube to remove the excess DTT, and the reduced antibody was replaced into a sodium phosphate buffer at pH 7.0±0.5. Using 7-15% v / v DMSO as a co-solvent, the reduced antibody was incubated with CL2A-SN-38 at ambient temperature for 30 min. Finally, the excess small molecules were removed through an ultrafiltration centrifugal tube. The molecular weight of the antibody-conjugated drug was analyzed by mass spectrometry, and the antibody-drug conjugate ratio (DAR) value of the antibody was calculated. Finally, each antibody carried an average of 7.5 SN38 molecules.

[0295] When measuring, 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 the AHC probe (Cat.: 18-0015, PALL) for 120 s. Human Trop-2-his recombinant protein (sequence 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 software was used for 1:1 Langmuir binding mode fitting to calculate the kinetic constants of antigen-antibody binding.

[0296] As shown in Table 16, compared with the naked antibodies, the affinities of the ADC antibodies labeled with SN38 for the human Trop-2 recombinant protein did not change significantly.

[0297] Table 16. Results of affinity determination of anti-Trop-2 naked antibodies and their ADC antibodies for human Trop-2 recombinant protein

[0298]

[0299] Example 15 FACS detection of internalization of anti-Trop-2 naked antibodies and their ADC antibodies mediated by BXPC-3

[0300] According to the method described in Example 7, the internalization rates on human pancreatic cancer cells BXPC-3 and human gastric cancer cells NCI-N87 were detected respectively. The antibodies to be tested included the 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 the naked antibodies h232-12, ch4-3, ch11-4, the positive control antibody Sacituzumab, and the negative isotype control antibody NC-IgG1, 10 μg / ml.

[0301] As shown in Table 17 and Table 18, the internalization ratios of the naked antibody of h23-12 and the ADC antibody labeled with SN38 were similar, and the internalization degree was similar to that of the control antibody; while the internalization ratios of the ADC antibodies labeled with SN38 of ch4-3 and ch11-4 were higher than those of the naked antibodies.

[0302] Table 17. Percentage of internalization of anti-human Trop-2 antibodies mediated by Trop-2 on the surface of NCI-N87 cells

[0303]

[0304]

[0305] Table 18. Percentage of internalization of anti-human Trop-2 antibody mediated by Trop-2 on the surface of BXPC-3 cells

[0306]

[0307] Example 16 Detection of the killing activity of anti-Trop-2-ADC antibody against cells

[0308] Human pancreatic cancer cells (BxPC-3) were taken and inoculated into 96-well cell culture plates at 2×10 3 cells / well, and cultured overnight in an incubator at 37°C with 5% CO 2 After overnight culture, different concentrations of SN38-labeled anti-Trop-2 antibody samples were added according to Table 19 (two parallel wells were set for each concentration and blank cell wells (untreated) were set), and the cells were cultured in an incubator at 37°C with 5% CO 2 After 3 hours of culture, the medium was replaced with fresh complete medium. The treatment method of the previous day was repeated the next day, and after continuous treatment for 4 days, the killing activity of the SN38-labeled anti-Trop-2 antibody against the cells was detected using the Cell Counting Kit-8 (CCK-8) kit.

[0309] Table 19. Anti-Trop-2-ADC antibody and its working concentration

[0310]

[0311] The results showed ( Figure 10 , Table 20) that each anti-Trop-2 ADC antibody specifically killed the target cells, and there was no significant difference in the killing activity compared with the control antibody Sacituzumab-SN38.

[0312] Table 20. Inhibitory activity of anti-Trop-2 ADC antibody on cell growth

[0313] 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

[0314] Example 17 Pharmacodynamic evaluation of anti-Trop2-ADC antibody in the N87 subcutaneous xenograft tumor model

[0315] Five-week-old female BALB / c nude mice were taken and subcutaneously inoculated with 3×10 6 human gastric cancer cells (NCI-N87). When the tumors grew to 150 mm 3Random grouping was performed at around [specific time], with 6 mice per group. The grouping, dosing dose, and frequency are shown in Table 21. Each group was intravenously injected twice a week, and the tumor volume and mouse body weight were measured during dosing. When the mouse body weight decreased by more than 15%, or the tumor volume of a single animal exceeded 3000 mm 3 or the average tumor volume of a group of animals exceeded 2000 mm 3 the experiment was terminated and the mice were euthanized.

[0316] Table 21. Grouping, dosing dose, and frequency of nude mice

[0317]

[0318] As Figure 11 , Figure 12 shown, the anti-Trop2-ADC antibody has a dose-dependent inhibitory effect on tumor growth. At high doses (10 mg / kg), no difference in the efficacy of each ADC antibody was observed, and no obvious toxic effect of the ADC small molecule SN38 was observed. The body weights of the animals in each experimental group increased steadily, with no significant difference from the control.

[0319] Example 18 Pharmacodynamic evaluation of the combination of anti-Trop2 antibody and anti-CD47 antibody in the SKOV3 subcutaneous xenograft tumor model

[0320] Five-week-old female BALB / c nude mice were subcutaneously inoculated with 3×10 6 human ovarian cancer cells (SKOV3) on the right hypochondrium of each mouse. When the tumor grew to about 150 mm 3 random grouping was performed, with 6 mice per group. The grouping, dosing dose, and frequency are shown in Table 22. Each group was intraperitoneally injected twice a week for a total of 5 times. The tumor volume and mouse body weight were measured during dosing, and the mouse status was observed. After the last dosing of the experiment, the mice were euthanized. The anti-CD47 antibody refers to the patent application publication US20150183874A1 and is a humanized 5F9 version 2.

[0321] Table 22. Grouping, dosing dose, and frequency of nude mice

[0322] Group Drug Dosing Dose Dosing 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

[0323] As Figure 13 , Figure 14 shown, compared with the negative control hIgG4, the h23-12 + Anti-CD47 combination group showed certain antitumor activity, while the Anti-CD47 and h23-12 single drug groups had no obvious 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, and thus have a synergistic antitumor effect.

[0324] Example 19 Pharmacodynamic Evaluation of Anti-Trop2-ADC Antibody in N87 Subcutaneous Xenograft Model

[0325] Five-week-old female BALB / c nude mice were subcutaneously inoculated with 3×10 6 human gastric cancer cells (NCI-N87). When the tumors grew to about 100 mm 3 in size, they were randomly divided into groups of 6 mice per group. The grouping, dosing doses, and frequencies are shown in Table 23. Each group was intravenously injected with the drug twice a week for a total of 6 weeks. During drug administration, the tumor volume and mouse body weight were measured. When the mouse body weight decreased by more than 15%, or the tumor volume of a single animal exceeded 3000 mm 3 or the average tumor volume of a group of animals exceeded 2000 mm 3 the experiment was stopped and the mice were euthanized.

[0326] Table 23. Grouping and Dosing Doses and Frequencies of Nude Mice

[0327] Group Drug Dosing Dose Dosing 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

[0328] As Figure 15 , Figure 16 shown, the anti-Trop2-ADC antibody had a dose-dependent inhibitory effect on tumor growth. At a dose of 5 mg / kg, the ADC antibodies ch3-11-SN38 and ch11-4-SN38 had slightly better efficacy than Sacituzumab-SN38, and no obvious toxic effects of the ADC small molecule SN38 were observed.

[0329] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or deformations according to the present invention, as long as they do not depart from the spirit of the present invention, they shall fall within the scope of the appended claims of the present invention. Sequence Listing <110> Mabwell (Shanghai) Biotech Co., Ltd. Beijing Kenuo Xincheng Technology Co., Ltd. <120> Anti-Human Trop-2 Antibody and Its Application <130> LC19110043 <160> 60 <170> SIPOSequenceListing 1.0 <210> 1 <211> 123 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <223> Heavy chain variable region <400> 15 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Thr Pro Gly Asp Asn Tyr Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly His Gly Asn Tyr Val Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 16 <211> 119 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <223> Light chain variable region <400> 18 Asp Val Val Val Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Phe Gly 1 5 10 15 Asp Gln Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Thr Asn Ser 20 25 30 Tyr Gly Asn Thr Phe Leu Ser Trp Tyr Leu His Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Leu Tyr Gly Ile Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Asn Thr Ile Lys Pro Glu Asp Leu Gly Met Tyr Tyr Cys Phe Gln Ser 85 90 95 Thr His Gln Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 19 <211> 106 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <223> Light chain variable region <400> 21 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 22 <211> 106 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Light chain variable region <400> 22 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 23 <211> 106 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Light chain variable region <400> 23 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 24 <211> 107 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <223> Light chain variable region <400> 29 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Ile Gly Thr Ser 20 25 30 Ile Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Glu Phe Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 30 <211> 112 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <223> light chain constant region <400> 38 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 39 <211> 451 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <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> <221> PEPTIDE <222> ()..() <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> <221> gene <222> ()..() <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> <221> gene <222> ()..() <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> <221> gene <222> ()..() <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> <221> gene <222> ()..() <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> <221> gene <222> ()..() <223> Heavy chain variable region <400> 45 gaggtgcagc tggtgcagtc tggacccgag gtgaagaagc ctggagcctc cgtgaaggtg 60 tcctgcaagg cctccggctt caccttcacc gactacgtga tcggctgggt gcgacaggct 120 cctggccagg gactggagtg gatcggcgag atctacctgg gctccggcac catctactac 180 accgagaagt tcaagggacg ggtgaccatg acagccgaca cctccacctc caccgcctac 240 atggagctgt cctccctgcg gtccgaggac accgccgtgt actactgcgc tcgaggctcc 300 atcttcccct tcgactactg gggccagggc accctggtga ccgtgtcctc t 351 <210> 46 <211> 318 <212> DNA <213> Artificial <220> <221> gene <222> ()..() <223> Light chain variable region <400> 46 gacatccagc tgacccagtc tccctcctcc ctgtctgcct ccgtgggcga cagggtgacc 60 atcacctgct ctgcctcctc ctccgtgtcc tacatgtact ggtaccagca gaagcctggc 120 aaggctccca agctgctgat ctacgacacc tccaccctgg cctctggcgt gccctccagg 180 ttctctggct ccggatctgg caccgacttc accctgacca tctcctccct gcagcccgag 240 gacttcgcca cctactactg ccagcagtgg tcctcctacc cctacacctt cggacagggc 300 accaagctgg agatcaag 318 <210> 47 <211> 351 <212> DNA <213> Artificial <220> <221> gene <222> ()..() <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> <221> gene <222> ()..() <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> <221> gene <222> ()..() <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> <221> gene <222> ()..() <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> <221> gene <222> ()..() <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> <221> gene <222> ()..() <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> <221> gene <222> ()..() <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> <221> gene <222> ()..() <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> <221> gene <222> ()..() <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> <221> gene <222> ()..() <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> <221> gene <222> ()..() <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> <221> gene <222> ()..() <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> <221> gene <222> ()..() <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> <221> gene <222> ()..() <223> Light chain constant region <400> 60 agaaccgtgg cggcgccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 agaaccgtgg cggcgccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggtaccgcta gcgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 ggtaccgcta gcgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg t 321 agcttcaaca ggggagagtg t 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 the heavy chain variable region and the light chain variable region are selected from the following combinations: (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 shown in SEQ ID NO: 14; and, the amino acid sequence shown in SEQ ID NO: 34; (7) The amino acid sequence shown in SEQ ID NO: 14; and, the amino acid sequence shown in SEQ ID NO: 35; (8) The amino acid sequence shown in SEQ ID NO: 15; and, the amino acid sequence 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 shown in SEQ ID NO: 16; and, the amino acid sequence 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 shown in SEQ ID NO: 3; and, the amino acid sequence shown in SEQ ID NO: 20; (13) The amino acid sequence shown in SEQ ID NO: 5; and, the amino acid sequence shown in SEQ ID NO:

24.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment is a monoclonal antibody, single-chain antibody, bifunctional antibody, fully or partially humanized antibody, chimeric antibody, dsFv, (dsFv) 2 , Fab, Fab', F(ab') 2 or Fv.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein, the bispecific antibody is BsFv.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein, the antibody or antigen-binding fragment thereof further comprises a human or murine constant region.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein, the antibody or antigen-binding fragment thereof comprises a human or murine light chain constant region (CL) or heavy chain constant region (CH).

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein, The antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from IgG, IgA, IgM, IgD or IgE, or a light chain constant region of the κ or λ type.

7. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein, the antibody is a monoclonal antibody.

8. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein, the antibody is a murine, chimeric or humanized monoclonal antibody.

9. The antibody or antigen-binding fragment thereof according to claim 7, wherein, the heavy chain constant region of the monoclonal antibody is of the IgG1 or IgG4 subtype, and the light chain constant region is of the κ type.

10. The antibody or antigen-binding fragment thereof according to claim 7, wherein, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:

37.

11. The antibody or antigen-binding fragment thereof according to claim 7, wherein, the light chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:

38.

12. The antibody or antigen-binding fragment thereof according to claim 10, wherein, the light chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:

38.

13. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

14. A vector comprising the nucleic acid molecule according to claim 13.

15. A host cell, which comprises the nucleic acid molecule according to claim 13 or the vector according to claim 14, or the host cell is transformed or transfected with the nucleic acid molecule according to claim 13 or the vector according to claim 14.

16. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, the nucleic acid molecule according to claim 13, the vector according to claim 14, or the host cell according to claim 15, and optionally a pharmaceutically acceptable excipient.

17. The pharmaceutical composition according to claim 16, wherein, the pharmaceutical composition further comprises other antibody drugs.

18. The pharmaceutical composition according to claim 17, wherein, the antibody drug is a macrophage immune checkpoint antibody.

19. The pharmaceutical composition according to claim 17, wherein, the antibody drug is an anti-CD47 antibody.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, the nucleic acid molecule according to claim 13, the vector according to claim 14, the host cell according to claim 15, or the pharmaceutical composition according to any one of claims 16 to 19 in the preparation of a drug for treating gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial cancer, triple-negative breast cancer or cervical cancer.

21. A kit, the kit comprising the antibody molecule or its antigen-binding fragment according to any one of claims 1 to 12, the nucleic acid molecule according to claim 13, the vector according to claim 14, the host cell according to claim 15, or the pharmaceutical composition according to any one of claims 16 to 19.

22. A conjugate, the conjugate comprising the antibody or its antigen-binding fragment according to any one of claims 1 to 12 and a drug conjugated thereto, wherein the drug is a cytotoxic agent.

23. The conjugate according to claim 22, the conjugate being an antibody-drug conjugate (ADC) represented by the following formula: (the antibody or its antigen-binding fragment according to any one of claims 1 to 12)-(linker)-(cytotoxic agent).

24. The conjugate according to claim 23, wherein, the cytotoxic agent is a tubulin inhibitor or a DNA replication inhibitor.

25. The conjugate according to claim 24, wherein, the tubulin inhibitor is paclitaxel or docetaxel.

26. The conjugate according to claim 24, wherein, the DNA replication inhibitor is irinotecan or its metabolically active metabolite SN-38.

27. Use of the antibody or its antigen-binding fragment according to any one of claims 1 to 12, the nucleic acid molecule according to claim 13, the vector according to claim 14, or the host cell according to claim 15 in the preparation of an antibody-drug conjugate (ADC) for the treatment of gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial cancer, triple-negative breast cancer or cervical cancer.

28. Use of the conjugate according to any one of claims 22 to 26 in the preparation of a drug for the treatment of gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial cancer, triple-negative breast cancer or cervical cancer.

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