Antibody targeting trop2, antibody-drug conjugate, and use thereof

Abstract

Provided are an antibody targeting Trop2, an antibody-drug conjugate, and use thereof. The provided antibody targeting Trop2 is capable of specifically binding to a target cell expressing human Trop2, has high affinity with same, is capable of entering the cell by means of endocytosis and inhibiting tumor growth or progression, and can be used for treating, preventing, and ameliorating conditions in a subject associated with abnormal expression of Trop2 (e.g., breast cancer, urothelial carcinoma, and non-small cell lung cancer).

Description

An antibody targeting Trop2, an antibody-drug conjugate and its applications

[0001] This application claims priority to Chinese patent application 2024114300880, filed on 2024 / 10 / 14. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biomedical technology, and in particular to an antibody targeting Trop2, an antibody-drug conjugate, and their applications. Background Technology

[0003] Trophoblast cell-surface antigen 2 (Trop2), also known as tumor-associated calcium signal transducer 2 (TACSTD2), epidermal glycoprotein 1 (EGP-1), gastrointestinal tumor-associated antigen (GA733-1), or surface marker 1 (M1S1), belongs to the TASCTD family. Trop2 contains 323 amino acids and consists of a hydrophobic leader peptide, an extracellular domain, a transmembrane domain, and a cytoplasmic tail. The extracellular domain of Trop2 includes a unique cysteine-rich domain (CRD), a thyroglobulin type I repeat domain (TY), and a cysteine-deficient domain (CPD) (Liao, S., et al. (2021). "Recent advances in trophoblast cell-surface antigen 2 targeted therapy for solid tumors." Drug Dev Res 82(8):1096-1110.).

[0004] Trop2 regulates multiple signaling molecules and participates in tumorigenesis-related signaling pathways, such as calcium signaling, β-catenin signaling, cyclin expression, and fibronectin adhesion. Upregulation of Trop2 promotes the expression and activation of multiple downstream effector factors, enhancing cell survival and growth. Trop2 can also reduce cell adhesion and promote cell invasion and metastasis via an integrin-dependent pathway. Trop2 expression is significantly elevated in various tumor types, including breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, and ovarian cancer. Its high expression plays a crucial role in tumor growth, invasion, and metastasis and is associated with poor tumor prognosis. Its high expression and differential expression characteristics make it a promising target for ADC drug development.

[0005] Antibody-drug conjugates (ADCs) are a novel form of antibody drug that combines antibodies and small-molecule cytotoxic drugs. They possess both the high target specificity of antibody drugs and the cytotoxic effects of small-molecule compounds, enabling more precise tumor killing and reducing the toxic side effects of cytotoxic drugs. The potential of ADCs in cancer treatment has made them a key focus of antibody drug development in recent years.

[0006] Currently, there are over 20 antibody-drug inhibitors (ADCs) targeting Trop2 in development globally, among which Trodelvy, developed by Immunomedics, has been approved for the treatment of triple-negative breast cancer and urothelial carcinoma. Although the efficacy of Trop2-targeting ADCs has been validated in triple-negative breast cancer and urothelial carcinoma, there is still room for improvement in their efficacy and safety. Therefore, developing high-quality antibodies suitable for Trop2-targeting ADC drug development remains essential. Summary of the Invention

[0007] In order to develop Trop2ADC drugs with better efficacy and safety, this invention provides an antibody targeting Trop2, an antibody-drug conjugate, and their applications.

[0008] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0009] A first aspect of the present invention provides an antibody or antigen-binding fragment thereof targeting Trop2, comprising a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3.

[0010] The LCDR1 contains the amino acid sequence RASESVDSYGNSFX1H (SEQ ID NO:108, X1 is V or M), RASKSVSTSGYSYMH (SEQ ID NO:16), KSSQSLLNSGNQKNYLT (SEQ ID NO:23), RSSQSLVHSNGNTYLH (SEQ ID NO:31), or SASSVSYMY (SEQ ID NO:67); the LCDR2 contains the amino acid sequence X2ASNLES (SEQ ID NO:109, X2 is L, R, or A), WASTRES (SEQ ID NO:24), KVSNRFS (SEQ ID NO:32), or DTSNLAS (SEQ ID NO:68); and the LCDR3 contains the amino acid sequence QQNX3EDPWT (SEQ ID NO:110, X3 is N or I), QNDYSYPX4T (SEQ ID NO:111, X4 is W or F), QHSRELPLT (SEQ ID NO:17), SQSTHVPT (SEQ ID NO:108), or SQSTHVPT (SEQ ID NO:108). NO:33), QQSNEDPRT (SEQ ID NO:54), QHSWEIPWT (SEQ ID NO:60) or QQWSSYPYT (SEQ ID NO:69).

[0011] In some embodiments of the present invention, the LCDR1 comprises an amino acid sequence as shown in SEQ ID NO:8, 16, 23, 31, 43 or 64.

[0012] In some embodiments of the present invention, the LCDR2 comprises an amino acid sequence as shown in SEQ ID NO:9, 24, 32, 50, 56 or 65.

[0013] In some embodiments of the present invention, the LCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 10, 17, 33, 38, 44, 51, 57 or 66.

[0014] In some embodiments of the present invention, the amino acid sequence of LCDR1 is RASESVDSYGNSFX1H (SEQ ID NO:108, X1 is V or M), the amino acid sequence of LCDR2 is LASNLES (SEQ ID NO:9), and the amino acid sequence of LCDR3 is QQNX3EDPWT (SEQ ID NO:110, X3 is N or I).

[0015] In some specific embodiments of the present invention, the amino acid sequence of LCDR1 is as shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:9, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:10; or, the amino acid sequence of LCDR1 is as shown in SEQ ID NO:43, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:9, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:44.

[0016] In some specific embodiments of the present invention, the amino acid sequence of LCDR1 is KSSQSLLNSGNQKNYLT (SEQ ID NO:23), the amino acid sequence of LCDR2 is WASTRES (SEQ ID NO:24), and the amino acid sequence of LCDR3 is QNDYSYPX4T (SEQ ID NO:111, where X4 is W or F).

[0017] In some specific embodiments of the present invention, the amino acid sequence of LCDR1 is shown in SEQ ID NO:16, the amino acid sequence of LCDR2 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:17.

[0018] In some specific embodiments of the present invention, the amino acid sequence of LCDR1 is shown in SEQ ID NO:31, the amino acid sequence of LCDR2 is shown in SEQ ID NO:32, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:33.

[0019] In some specific embodiments of the present invention, the amino acid sequence of LCDR1 is shown in SEQ ID NO:43, the amino acid sequence of LCDR2 is shown in SEQ ID NO:50, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:51.

[0020] In some specific embodiments of the present invention, the amino acid sequence of LCDR1 is shown in SEQ ID NO:16, the amino acid sequence of LCDR2 is shown in SEQ ID NO:56, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:57.

[0021] In some specific embodiments of the present invention, the amino acid sequence of LCDR1 is shown in SEQ ID NO:64, the amino acid sequence of LCDR2 is shown in SEQ ID NO:65, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:66.

[0022] In some embodiments of the present invention, the heavy chain variable region includes any one of the following:

[0023] (1) The amino acid sequence of HCDR1 is GYTFTDYSMH (SEQ ID NO:5); the amino acid sequence of HCDR2 is VISTYYGDAX5 (SEQ ID NO:112, X5 is S or R); and the amino acid sequence of HCDR3 is YGPYX6MDY (SEQ ID NO:113, X6 is A or T); preferably, the amino acid sequence of HCDR1 is as shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:6, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:7; or, the amino acid sequence of HCDR1 is as shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:47, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:48;

[0024] (2) The amino acid sequence of HCDR1 is shown in SEQ ID NO:13, the amino acid sequence of HCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:15.

[0025] (3) The amino acid sequence of HCDR1 is shown in SEQ ID NO:20, the amino acid sequence of HCDR2 is shown in SEQ ID NO:21, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:22.

[0026] (4) The amino acid sequence of HCDR1 is shown in SEQ ID NO:28, the amino acid sequence of HCDR2 is shown in SEQ ID NO:29, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:30.

[0027] (5) The amino acid sequence of HCDR1 is shown in SEQ ID NO:20, the amino acid sequence of HCDR2 is shown in SEQ ID NO:36, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:37.

[0028] (6) The amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:41, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:42.

[0029] (7) The amino acid sequence of HCDR1 is shown in SEQ ID NO:50, the amino acid sequence of HCDR2 is shown in SEQ ID NO:51, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:52.

[0030] (8) The amino acid sequence of HCDR1 is shown in SEQ ID NO:57, the amino acid sequence of HCDR2 is shown in SEQ ID NO:51, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:58; and,

[0031] (9) The amino acid sequence of HCDR1 is shown in SEQ ID NO:64, the amino acid sequence of HCDR2 is shown in SEQ ID NO:65, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:66.

[0032] In some specific embodiments of the present invention, the amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:6, the amino acid sequence of HCDR3 is shown in SEQ ID NO:7, the amino acid sequence of LCDR1 is shown in SEQ ID NO:8, the amino acid sequence of LCDR1 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:10.

[0033] In some specific embodiments of the present invention, the amino acid sequence of HCDR1 is shown in SEQ ID NO:13, the amino acid sequence of HCDR2 is shown in SEQ ID NO:14, the amino acid sequence of HCDR3 is shown in SEQ ID NO:15, the amino acid sequence of LCDR1 is shown in SEQ ID NO:16, the amino acid sequence of LCDR1 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:17.

[0034] In some specific embodiments of the present invention, the amino acid sequence of HCDR1 is shown in SEQ ID NO:20, the amino acid sequence of HCDR2 is shown in SEQ ID NO:21, the amino acid sequence of HCDR3 is shown in SEQ ID NO:22, the amino acid sequence of LCDR1 is shown in SEQ ID NO:23, the amino acid sequence of LCDR1 is shown in SEQ ID NO:24, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:25.

[0035] In some specific embodiments of the present invention, the amino acid sequence of HCDR1 is shown in SEQ ID NO:28, the amino acid sequence of HCDR2 is shown in SEQ ID NO:29, the amino acid sequence of HCDR3 is shown in SEQ ID NO:30, the amino acid sequence of LCDR1 is shown in SEQ ID NO:31, the amino acid sequence of LCDR1 is shown in SEQ ID NO:32, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:33.

[0036] In some specific embodiments of the present invention, the amino acid sequence of HCDR1 is shown in SEQ ID NO:20, the amino acid sequence of HCDR2 is shown in SEQ ID NO:36, the amino acid sequence of HCDR3 is shown in SEQ ID NO:37, the amino acid sequence of LCDR1 is shown in SEQ ID NO:23, the amino acid sequence of LCDR1 is shown in SEQ ID NO:24, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:38.

[0037] In some specific embodiments of the present invention, the amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:41, the amino acid sequence of HCDR3 is shown in SEQ ID NO:42, the amino acid sequence of LCDR1 is shown in SEQ ID NO:43, the amino acid sequence of LCDR1 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:44.

[0038] In some specific embodiments of the present invention, the amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:47, the amino acid sequence of HCDR3 is shown in SEQ ID NO:48, the amino acid sequence of LCDR1 is shown in SEQ ID NO:43, the amino acid sequence of LCDR1 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:44.

[0039] In some specific embodiments of the present invention, the amino acid sequence of HCDR1 is shown in SEQ ID NO:50, the amino acid sequence of HCDR2 is shown in SEQ ID NO:51, the amino acid sequence of HCDR3 is shown in SEQ ID NO:52, the amino acid sequence of LCDR1 is shown in SEQ ID NO:43, the amino acid sequence of LCDR1 is shown in SEQ ID NO:53, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:54.

[0040] In some specific embodiments of the present invention, the amino acid sequence of HCDR1 is shown in SEQ ID NO:57, the amino acid sequence of HCDR2 is shown in SEQ ID NO:51, the amino acid sequence of HCDR3 is shown in SEQ ID NO:58, the amino acid sequence of LCDR1 is shown in SEQ ID NO:16, the amino acid sequence of LCDR1 is shown in SEQ ID NO:59, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:60.

[0041] In some specific embodiments of the present invention, the amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:6, the amino acid sequence of HCDR3 is shown in SEQ ID NO:7, the amino acid sequence of LCDR1 is shown in SEQ ID NO:43, the amino acid sequence of LCDR1 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:44.

[0042] In some specific embodiments of the present invention, the amino acid sequence of HCDR1 is shown in SEQ ID NO:64, the amino acid sequence of HCDR2 is shown in SEQ ID NO:65, the amino acid sequence of HCDR3 is shown in SEQ ID NO:66, the amino acid sequence of LCDR1 is shown in SEQ ID NO:67, the amino acid sequence of LCDR1 is shown in SEQ ID NO:68, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:69.

[0043] In some embodiments of the present invention, the amino acid sequence of HCDR1 is GYTFTDYSMH (SEQ ID NO:5), the amino acid sequence of HCDR2 is VISTYYGDAX5 (SEQ ID NO:112, X5 is S or R), the amino acid sequence of HCDR3 is YGPYX6MDY (SEQ ID NO:113, X6 is A or T), the amino acid sequence of LCDR1 is RASESVDSYGNSFX1H (SEQ ID NO:108, X1 is V or M), the amino acid sequence of LCDR2 is LASNLES (SEQ ID NO:9), and the amino acid sequence of LCDR3 is QQNX3EDPWT (SEQ ID NO:110, X3 is N or I). Specific examples are as follows:

[0044] The amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:6, the amino acid sequence of HCDR3 is shown in SEQ ID NO:7, the amino acid sequence of LCDR1 is shown in SEQ ID NO:8, the amino acid sequence of LCDR1 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:10.

[0045] The amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:47, the amino acid sequence of HCDR3 is shown in SEQ ID NO:48, the amino acid sequence of LCDR1 is shown in SEQ ID NO:43, the amino acid sequence of LCDR1 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:44; or,

[0046] The amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:6, the amino acid sequence of HCDR3 is shown in SEQ ID NO:7, the amino acid sequence of LCDR1 is shown in SEQ ID NO:43, the amino acid sequence of LCDR1 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:44.

[0047] In some embodiments of the present invention, the frame region of the heavy chain variable region and / or the light chain variable region is a mouse-derived frame region.

[0048] In some embodiments of the present invention, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 11, 18, 26, 34, 39, 45, 49, 55, 61, 63 or 70, and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 12, 19, 27, 35, 40, 46, 56, 62, 71 or 114.

[0049] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 11, 18, 26, 34, 39, 45, 49, 55, 61, 63 or 70, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 12, 19, 27, 35, 40, 46, 56, 62, 71 or 114.

[0050] In some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:11, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:12;

[0051] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:18, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19;

[0052] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:26, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:27;

[0053] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:34, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:35;

[0054] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:39, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:40;

[0055] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:45, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:46;

[0056] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:49, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:114;

[0057] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:55, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:56;

[0058] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:61, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:62;

[0059] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:63, and / or, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:46; or

[0060] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:70, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:71.

[0061] In some embodiments of the present invention, the framework region of the heavy chain variable region and / or the light chain variable region is a human-derived framework region.

[0062] In some embodiments of the present invention, the framework region of the heavy chain variable region is derived from the hominid heavy chain IGHV3-23*04; and / or, the framework region of the light chain variable region is derived from the hominid light chain IGLV657*01.

[0063] In some embodiments of the present invention, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:72, 74, 75, 77, 78, 79, 80, 81, 82, 84, 85, 86, 87, 88, 89, 90, 92, 97, 98, 99, 101, 102, 103, 104, 105, 106 or 107, and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:73, 76, 83, 91, 93, 94, 95, 96 or 100.

[0064] In some embodiments of the present invention, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:72, 74, 75, 77, 78, 79, 80 or 81, and / or the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:73 or 76.

[0065] In some embodiments of the present invention, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:82, 84, 85, 86, 87, 88, 89 or 90, and / or the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:73, 76, 83 or 91.

[0066] In some embodiments of the present invention, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 92, 97 or 98, 99, 101, 102, 103, 104, 105, 106 or 107, and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 93, 94, 95 or 96 or 100.

[0067] In some embodiments of the present invention, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:99, 101, 102, 103, 104, 105, 106 or 107, and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:100.

[0068] In some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:72, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:73;

[0069] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:74, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:73;

[0070] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:75, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:73;

[0071] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:75, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:76;

[0072] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:77, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:76;

[0073] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:78, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:76;

[0074] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:79, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:76;

[0075] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:80, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:76;

[0076] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:81, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:76;

[0077] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:82, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:83;

[0078] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:84, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:83;

[0079] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:85, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:83;

[0080] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:86, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:83;

[0081] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:87, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:83;

[0082] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:88, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:83;

[0083] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:89, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:83;

[0084] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:90, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:83;

[0085] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:82, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:91;

[0086] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:92, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:93;

[0087] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:92, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:94;

[0088] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:92, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:95;

[0089] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:92, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:96;

[0090] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:97, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:93;

[0091] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:97, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:94;

[0092] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:97, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:95;

[0093] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:97, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:96;

[0094] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:98, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:93;

[0095] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:99, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:100;

[0096] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:101, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:100;

[0097] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:102, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:100;

[0098] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:103, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:100;

[0099] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:104, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:100;

[0100] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:105, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:100;

[0101] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:106, and / or, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:100; or

[0102] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:107, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:100.

[0103] In some embodiments of the present invention, the antibody is a full-length antibody, Fab, Fab', F(ab')2 or Fv; the Fv is preferably scFv.

[0104] In some embodiments of the present invention, the antibody is a full-length antibody, the heavy chain constant region and / or light chain constant region of which are derived from mouse antibodies or human antibodies.

[0105] In some embodiments of the present invention, the heavy chain constant region and / or the light chain constant region are derived from human antibodies.

[0106] In some embodiments of the present invention, the heavy chain constant region is preferably derived from the human heavy chain IgG1 constant region, the amino acid sequence of which is shown, for example, as SEQ ID NO:3; and / or, the light chain constant region is preferably derived from the human light chain κ chain constant region, the amino acid sequence of which is shown, for example, as SEQ ID NO:4.

[0107] A second aspect of the present invention provides an isolated nucleic acid that encodes an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention.

[0108] A third aspect of the present invention provides a recombinant expression vector comprising the nucleic acid as described in the second aspect of the present invention.

[0109] In some embodiments of the present invention, the recombinant expression vector is a plasmid, granule, bacteriophage, or viral vector, and the viral vector is preferably a retroviral vector, lentiviral vector, adenovirus vector, or adeno-associated virus vector.

[0110] A fourth aspect of the present invention provides a transformant comprising a nucleic acid as described in the second aspect of the present invention or a recombinant expression vector as described in the third aspect of the present invention, wherein the host cell of the transformant is a eukaryotic cell or a prokaryotic cell.

[0111] In some embodiments of the present invention, the eukaryotic cells are mammalian cells, such as 293 cells or Expi-CHO cells.

[0112] A fifth aspect of the present invention provides a method for preparing an antibody or antigen-binding fragment thereof targeting Trop2, the method comprising culturing a transformant as described in the fourth aspect of the present invention.

[0113] A sixth aspect of the present invention provides an antibody-drug conjugate comprising a cytotoxic agent or tag, and an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention.

[0114] In some embodiments of the present invention, the cytotoxic agent is MMAF or MMAE, the tag is a fluorescent agent; and / or, the antibody-drug conjugate further includes a linker, such as MC-VC-PAB.

[0115] In some embodiments of the present invention, the antibody-drug conjugate comprises the antibody or its antigen-binding fragment and MC-VC-PAB-MMAE.

[0116] A seventh aspect of the present invention provides a method for preparing an antibody-drug conjugate as described in the sixth aspect of the present invention, the method comprising reacting the antibody or an antigen-binding fragment thereof with a cytotoxic agent connected with a linker to obtain the antibody-drug conjugate.

[0117] An eighth aspect of the present invention provides a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention or an antibody-drug conjugate as described in the sixth aspect of the present invention, and a pharmaceutically acceptable carrier.

[0118] A ninth aspect of the present invention provides a kit comprising an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, an antibody-drug conjugate as described in the sixth aspect of the present invention, or a pharmaceutical composition as described in the eighth aspect of the present invention.

[0119] The tenth aspect of the present invention provides the use of antibodies or antigen-binding fragments thereof as described in the first aspect of the present invention, nucleic acids as described in the second aspect of the present invention, recombinant expression vectors as described in the third aspect of the present invention, transformants as described in the fourth aspect of the present invention, antibody-drug conjugates as described in the sixth aspect of the present invention, or pharmaceutical compositions as described in the eighth aspect of the present invention in the preparation of medicaments for the diagnosis, prevention, and / or treatment of tumors, or in the preparation of Trop2 inhibitors.

[0120] In some embodiments of the present invention, the tumor is a tumor associated with abnormal Trop2 expression, such as one or more of breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, ovarian cancer, and urothelial carcinoma; preferably lung cancer or gastric cancer.

[0121] The eleventh aspect of the present invention provides a pillbox set, the pillbox set comprising pillbox A and pillbox B, wherein:

[0122] The kit A contains an antibody or its antigen-binding fragment as described in the first aspect of the present invention, an antibody-drug conjugate as described in the sixth aspect of the present invention, or a pharmaceutical composition as described in the eighth aspect of the present invention.

[0123] The kit B contains other antibodies for treating tumors or pharmaceutical compositions containing said other antibodies for treating tumors, and / or other pharmaceuticals for treating tumors.

[0124] In some embodiments of the present invention, the tumor is a tumor associated with abnormal Trop2 expression, such as one or more of breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, ovarian cancer, and urothelial carcinoma; preferably lung cancer or gastric cancer.

[0125] The twelfth aspect of the present invention provides a drug delivery device comprising an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, an antibody-drug conjugate as described in the sixth aspect of the present invention, or a pharmaceutical composition as described in the eighth aspect of the present invention.

[0126] In some embodiments of the invention, the drug delivery device further includes a component for administering the antibody or its antigen-binding fragment, antibody-drug conjugate, or drug composition to a subject, such as a syringe or infusion device.

[0127] The thirteenth aspect of the present invention provides a method for diagnosing, treating, and / or preventing tumors or inhibiting Trop2, the method comprising administering to a subject in need an effective amount of an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, an antibody-drug conjugate as described in the sixth aspect of the present invention, a pharmaceutical composition as described in the eighth aspect of the present invention, or treating the subject in need using a kit as described in the eleventh aspect of the present invention.

[0128] In some embodiments of the present invention, the tumor is a tumor associated with abnormal Trop2 expression, such as one or more of breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, ovarian cancer, and urothelial carcinoma; preferably lung cancer or gastric cancer.

[0129] The fourteenth aspect of the present invention provides a method for detecting Trop2, the method comprising using an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention.

[0130] In some embodiments of the present invention, the method is for non-diagnostic purposes.

[0131] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0132] The reagents and raw materials used in this invention are all commercially available.

[0133] The positive and progressive effects of this invention are as follows:

[0134] The antibody targeting Trop2 provided by this invention can specifically bind to target cells expressing human Trop2, has a high affinity for them, and can enter cells through endocytosis to inhibit tumor growth or progression. It can be used to treat, prevent, and improve diseases related to abnormal Trop2 expression in subjects (such as breast cancer, urothelial carcinoma, non-small cell lung cancer, etc.). Attached Figure Description

[0135] Figures 1A-1D show the binding affinity of candidate molecules (E7, E8, E9 (Figure 1A), E11, E12, E16, E35 (Figure 1B), E1, E4 (Figure 1C), E39, E45 (Figure 1D)) to the huTrop2-His protein.

[0136] Figures 2A-2C show the binding ability of the antibodies of the present invention (E1, E4, E7, E8 (Figure 2A), E9, E11, E12, E16 (Figure 2B), E35, E39 (Figure 2C)) to huTrop2-HEK293 cells.

[0137] Figures 3A-3D show the binding ability of the antibodies of the present invention (E1, E4, E7 (Figure 3A), E8, E9, E11 (Figure 3B), E12, E16 (Figure 3C), E35, E39, E45 (Figure 3D)) to Hacat cells.

[0138] Figures 4A-4D show the cross-binding activity of the antibodies of the present invention (E1, E4 (Figure 4A), E7, E8, E9 (Figure 4B), E11, E12, E16, E35 (Figure 4C), E39, E45 (Figure 4D)) with monkey Trop2 protein.

[0139] Figures 5A-5C show the cross-binding activity of the antibodies of the present invention (E1, E4, E7, E8, E9 (Figure 5A), E11, E12, E16 (Figure 5B), E35, E39, E45 (Figure 5C)) with human Trop1 protein.

[0140] Figures 6A-6E illustrate the activity of the antibodies of the present invention (E1, E4 (Figure 6A), E7, E8 (Figure 6B), E9, E11 (Figure 6C), E12, E16 (Figure 6D), E35, E39 (Figure 6E), E45 (Figure 6F)) in mediating the endocytosis of Trop2 on the surface of HEK293 cells overexpressing Trop2.

[0141] Figures 7A-7I show the binding affinity of the humanized antibodies of the present invention (E1 humanized antibody (Figures 7A and 7B), E4 humanized antibody (Figures 7C and 7D), E7 humanized antibody (Figures 7E and 7F), and E35 humanized antibody (Figures 7G, ​​7H, and 7I)) to huTrop2-his.

[0142] Figures 8A-8F show the binding affinity of the humanized antibodies of the present invention (E1 humanized antibody (Figures 8A and 8B), E4 humanized antibody (Figures 8C and 8D), and E35 humanized antibody (Figures 8E and 8F)) to MDA-MB-231 cells as determined by FACS.

[0143] Figures 9A-9C show the killing activity of the humanized antibody ADCs (E1-H1L2-MMAE, E4-H2L1-MMAE (Figure 9A), E35-H1L1-MMAE (Figure 9B), E7-H1L2-MMAE (Figure 9C)) of the present invention against NCI-H292 cells.

[0144] Figures 10A-10B show the killing activity of the humanized antibody ADCs (E4-H2L1-MMAE, E1-H1L2-MMAE (Figure 10A), E35-H1L1-MMAE (Figure 10B)) of the present invention against HCC827 cells.

[0145] Figures 11A and 11B show the antitumor function and safety of the humanized antibody ADC of the present invention in the NCI-N87 Balb / C nude mouse tumor-bearing model; Figure 11A shows the tumor volume change in mice after treatment with the humanized antibody ADC; Figure 11B shows the body weight change in mice after treatment with the humanized antibody ADC, where the arrows indicate the nodes of drug administration. Detailed Implementation

[0146] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below:

[0147] In this invention, the letters in the amino acid sequence represent single-letter abbreviations of amino acids known in the art, such as those described in J. Biol. Chem, 243, p3558 (1968): alanine: Ala-A, arginine: Arg-R, aspartic acid: Asp-D, cysteine: Cys-C, glutamine: Gln-Q, glutamic acid: Glu-E, histidine: His-H, glycine: Gly-G, asparagine: Asn-N, tyrosine: Tyr-Y, proline: Pro-P, serine: Ser-S, methionine: Met-M, lysine: Lys-K, valine: Val-V, isoleucine: Ile-I, phenylalanine: Phe-F, leucine: Leu-L, tryptophan: Trp-W, threonine: Thr-T.

[0148] In this invention, the amino acid sequences of the listed complementarity determining regions (CDRs) are all shown according to the AbM numbering rules. However, it is well known to those in the art that antibody CDRs can be defined in various ways, such as Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)), based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDapartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT, imgt.cines.fr / ), and Northrop Grumman's method based on affinity propagation clustering using a large number of crystal structures. CDR Definition. Those skilled in the art will understand that, unless otherwise specified, the terms “CDR” and “complementarity-determining region” for a given antibody or its region (e.g., variable region) should be understood to encompass the complementarity-determining region defined as in any of the known embodiments described above by way of the present invention.

[0149] Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations). Although the scope of protection claimed in this invention is based on the sequence defined according to the AbM numbering rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention.

[0150] In this invention, the term "full-length antibody" is used interchangeably to refer to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this invention) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this invention) and a light chain constant region (abbreviated as CL in this invention). The light chain constant region consists of one domain: CL. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulins IgA, IgD, IgE, IgG, and IgM. A complete antibody forms a "Y" shape. The stem of the Y is formed by the second and third constant regions of the two heavy chains (and, for IgE and IgM, a fourth constant region) linked together, and disulfide bonds (interchain) are formed in the hinge. Heavy chains γ, α, and δ have constant regions consisting of three tandem (in a row) Ig domains and hinge regions for increased flexibility; heavy chains μ and ε have constant regions consisting of four immunoglobulin domains. The second and third constant regions are referred to as the "CH2 domain" and the "CH3 domain," respectively. Each arm of Y includes a variable region of a single heavy chain and a first constant region that binds to a variable and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0151] In this invention, a "Fab fragment" consists of a light chain and a heavy chain, comprising the CH1 domain and a variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fc" region contains two heavy chain fragments containing the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain. A "Fab' fragment" contains a portion of a light chain and a heavy chain containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of the two Fab' fragments to form the F(ab')2 molecule. An "F(ab')2 fragment" contains two light chains and two heavy chains containing portions of the constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains. The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody, but lacking the constant region.

[0152] In this invention, scFv refers to a single-chain antibody fragment, which includes a heavy chain variable region, a light chain variable region, and a linker peptide of 15-20 amino acids. The VL and VH domains enable the linker peptides to pair and form monovalent molecules as single polypeptide chains [see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules may have a general structure: NH2-VL-linker peptide-VH-COOH or NH2-VH-linker peptide-VL-COOH.

[0153] In this invention, "nucleic acid" refers to a nucleotide chain of any length and includes DNA and RNA. A nucleotide can be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base, and / or its analogues, or any substrate capable of being incorporated into the chain by DNA or RNA polymerase.

[0154] In this invention, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell, permits the expression of the mRNA, protein, polypeptide, or peptide by the host cell. The vectors of this invention are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of this invention can contain any type of nucleotide, including but not limited to DNA and RNA that can be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. Recombinant expression vectors can contain naturally occurring or non-naturally occurring nucleotide linkages, or both. In an exemplary aspect, modified nucleotides or non-naturally occurring nucleotide linkages do not impede transcription or replication of the vector.

[0155] The recombinant expression vector of the present invention can be any suitable recombinant expression vector capable of being used to transform or transfect one or more genes or sequences of interest into any suitable host cell and preferably to express the genes or sequences in the host cell. Suitable vectors include those designed for amplification and expansion or for expression or both of the above, and examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0156] In this invention, the term "host cell" refers to any type of cell that may contain the nucleic acids or vectors described herein. In exemplary aspects, the host cell is a eukaryotic cell, such as a plant, animal, fungus, or algae; or it may be a prokaryotic cell, such as a bacterium or protozoan.

[0157] In this invention, the pharmaceutical composition may comprise a suitable pharmaceutically acceptable carrier, such as pharmaceutical excipients, including buffers, as known in the art. "Pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous dextran, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Pharmaceutical compositions comprising the invention can be prepared by mixing antibodies of the invention having the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Preferably, the composition is in the form of a lyophilized formulation or an aqueous solution.

[0158] The pharmaceutical compositions of the present invention may also comprise more than one active ingredient required for a specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other active ingredients, such as other antibodies, antiviral agents, small molecule drugs, or immunomodulators. The active ingredients are suitably combined in amounts effective for the intended use. Sustained-release formulations can be prepared, suitable examples of which include a semi-permeable matrix of a solid hydrophobic polymer containing the antibody of the present invention, said matrix being a shaped article, such as a film or microcapsule.

[0159] In this invention, the chimeric antigen receptor (CAR) is an engineered transmembrane protein that combines the specificity of an antigen-specific antibody with the function of a T-cell receptor. Generally, a CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In an exemplary aspect, the extracellular domain of the CAR contains an antigen recognition region, which may be a scFV of an antigen-specific antibody.

[0160] In this invention, the term "antibody-drug conjugate" generally refers to an antibody linked to a biologically active cytotoxic drug via a stable linker unit. The "cytotoxic drug" may include toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant, or animal origin, radioactive isotopes, toxic drugs, chemotherapeutic agents, antibiotics, or ribolysins, or derivatives thereof.

[0161] As used in this article, "tumor" refers to a new growth formed when a cell in a local tissue loses normal regulation of its growth at the gene level under the influence of various carcinogenic factors, resulting in its clonal abnormal proliferation.

[0162] As used herein, the term “Trop2 aberration-associated tumor” refers to tumor cells that aberrately express Trop2, where “aberration-expression” means differential expression of Trop2 relative to other normal cells in the body, such as high / overexpression.

[0163] In this invention, the "non-diagnostic purpose" application scenarios include, but are not limited to: for example, detecting the presence of antigens (proteins containing the extracellular region of Trop2) in vitro in the laboratory; or using it as a positive antibody to screen other antibodies targeting Trop2; or competing with other antibodies targeting Trop2 to detect whether there is competition between the antibodies, i.e., whether the antigen epitopes are the same or similar, etc.

[0164] In this invention, the term "effective amount" refers to the amount of a drug or agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as sought by, for example, an investigator or clinician. Furthermore, the term "effective amount" refers to the amount that causes improved treatment, cure, prevention, or reduction of disease, symptom, or side effects, or reduces the rate of progression of a disease or condition, compared to a corresponding subject who did not receive that amount. Within its scope, the term also includes amounts that effectively enhance normal physiological function.

[0165] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0166] Example 1: Raw Material Preparation and Identification

[0167] 1.1 Preparation and Identification of Trop2 Control Antibody

[0168] Preparation of Trop2 control antibody: In this invention, the anti-Trop2 antibody Sacituzumab (light chain sequence is SEQ ID NO: 1, heavy chain sequence is SEQ ID NO: 2) was used as a positive control antibody. The target fragment was synthesized by General Biotechnology Co., Ltd. according to the sequence disclosed in US9849176. Then, the eukaryotic expression vector pcDNA3.4 (Invitrogen) was constructed using homologous recombination. The constructed recombinant protein expression vector was transformed into *E. coli* DH5α and cultured overnight at 37°C. Plasmid extraction was then performed using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain the desired expression vector for Sacituzumab. The expressed Sacituzumab will be referred to as Sacituzumab below. The expression was measured using ExpiFectamine. TM The CHO transfection kit (Thermo Fisher, A29129) was used to transfect CHO cells with the expression vector to express goxatuzumab. Seven days after transfection, the cell culture supernatant was collected, centrifuged at 15000g for 10 minutes, and the resulting supernatant was filtered through a 0.22μm filter. The antibody in the supernatant was then purified by affinity chromatography using a Protein A / G column. The target antibody was eluted with 100mM glycine (pH 3.0), and the eluted antibody was transferred to PBS buffer via an ultrafiltration concentrator (Millipore, UFC901096).

[0169] Trop2 control antibody identification: The activity of the prepared positive control antibody Sacituzumab (IgG1) was detected using the purified huTrop2-His antigen protein (prepared in Example 1.2). The specific method is as follows: huTrop2-His (2 μg / mL, 30 μL / well) was coated on a 96-well ELISA plate and incubated overnight at 4°C; after washing the plate 3 times, it was blocked with 5% skim milk prepared with PBS at room temperature for 1 hour; after washing the plate 3 times, the control antibody Sacituzumab diluted serially with PBS was added and incubated at room temperature for 1 hour; after washing the plate, the secondary antibody Anti-human-IgG-Kappa+Lambda-HRP (Millipore, AP502P+AP506P) diluted with PBS (1:6000) was added and incubated at room temperature for 1 hour, after washing the plate 6 times, TMB was added for color development for 5-20 minutes, and after stopping the color development, the data were read by OD450 of the microplate reader and processed into graphs using GraphPad Prism. The results showed that the expressed control antibody, Sacituzumab, could bind to the Trop2 protein and had normal anti-Trop2 activity.

[0170] 1.2 Preparation and Identification of Antigen Proteins

[0171] (1) Antigen protein preparation: Through genetic manipulation at the coding gene level, His tags or human Fc (SEQ ID NO: 3) tags were added to the C-terminus of the sequences of human Trop2 protein huTrop2 ECD AA31-274 (Uniprot ID: P09758-1), rhesus monkey Trop2 protein CynoTrop2 ECD AA31-274 (GenBank ID: XP_001114599.1), and human Trop1 protein huTrop1 ECD AA24-265 (GenBank ID: AAH14785.1). The obtained nucleic acid sequences were constructed into pcDNA3.4 vectors, transformed into E. coli DH5α, and cultured overnight at 37°C. Plasmids were then extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01). The obtained plasmids were then subjected to ExpiFectamine. TM 293 Transfection Kit (Gibco) TM A14524) was transiently transfected into HEK293 cells. CRL-1573 TM After 7 days of expression, the cell culture supernatant was collected. The Fc-tagged antigen was purified by affinity purification using COLUMN XK16 / 20 (Cytiva). The purified protein was then eluted with 100 mM glycine (pH 3.0), concentrated, and the buffer was replaced to obtain the antigen proteins (huTrop2-huFc, CynoTrop2-huFc, huTrop1-huFc). The His-tagged antigen was purified by affinity purification using Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), followed by elution with gradient concentrations of imidazole. Each eluted protein was then transferred to PBS buffer via ultrafiltration concentrator (Millipore, UFC901096) to obtain the antigen proteins (huTrop2-His, CynoTrop2-His, huTrop1-His).

[0172] (2) Antigen identification: The prepared antigens (huTrop2-His, huTrop2-huFc, CynoTrop2-His, CynoTrop2-huFc) were detected using the quality-tested control antibody Sacituzumab (IgG1) obtained in step 1.1. The specific method is as follows: ELISA plates were coated with 2 μg / mL huTrop2-His, huTrop2-huFc, CynoTrop2-His, and CynoTrop2-huFc respectively and incubated overnight at 4°C. After washing the plates three times, they were blocked with 5% skim milk prepared with PBS at room temperature for 1 hour. After washing the plates three times, control antibody Sacituzumab, serially diluted with PBS, was added and incubated at room temperature for 1 hour. After washing the plates, secondary antibody Anti-human-IgG-Kappa+Lambda-HRP (Millipore, AP502P+AP506P) diluted 1:6000 with PBS was added and incubated at room temperature for 1 hour. After washing the plates six times, TMB was added for color development for 5-20 minutes to terminate the color development reaction. Data were read using an OD450 microplate reader and processed using GraphPad Prism. The results showed that the antibody Sacitzumab could bind to the constructed and expressed antigens huTrop2-His, huTrop2-huFc, CynoTrop2-His, and CynoTrop2-huFc.

[0173] Example 2: Construction and Identification of Cell Lines Overexpressing Human Trop2

[0174] Construction of HEK293 cell line overexpressing human Trop2 (hereinafter referred to as huTrop2-HEK293): The coding nucleic acid sequence of full-length human Trop2 (UniprotID: P09758-1) was constructed into pLVX-puro plasmid (Clontech, Cat#632164). Then, the obtained plasmid was electroporated into HEK293 cells using an Invitrogen, Neon™ Transfection System, MP922947. CRL-1573 TM After electroporation, the resulting cells were transferred to DMEM medium (Gibco, 11995065) containing 10% FBS (Gibco, 15140-141) and no antibiotics. The cells were then cultured in 10×10 cm cell culture dishes for 48 hours, followed by incubation at an average rate of 10... 4 Cells were aliquoted into 96-well cell culture plates at a density of 1 cell / well, and puromycin was added to a final concentration of 2 μg / mL as a selection pressure. Cell lines that formed clones were picked for identification after about 2 weeks.

[0175] Flow cytometry identification of huTrop2-HEK293 cells: The cells in logarithmic growth phase were digested and plated into 96-well plates. After washing with FACS buffer (1×PBS buffer containing 2% FBS), the cells were incubated with serially diluted primary antibody (Sacituzumab) at 4°C for 30 minutes. After washing, the cells were incubated with the prepared fluorescent secondary antibody anti-human IgG Fc (Abcam, 98596) at 4°C for 30 minutes. Finally, the cells were analyzed by flow cytometry (Beckman, CytoFLEXAOO-1-1102). The results showed that a huTrop2-HEK293 cell line with high expression of human Trop2 was obtained.

[0176] Example 3 Animal Immunization and Immune Bank Construction

[0177] 3.1 Immunization regimen

[0178] Three Balb / C mice (Shanghai Lingchang Biotechnology Co., Ltd.) were cross-immunized with huTrop2-huFc and huTrop2-His antigens via subcutaneous and intraperitoneal injections, with immunizations every two weeks for a total of four immunizations. The initial immunization dose was 100 μg / mouse, and the subsequent three immunizations were administered at a dose of 50 μg / mouse. One week after the fourth immunization, peripheral blood was collected from the mice for immunotiter assay. A final booster immunization with huTrop2-huFc was administered.

[0179] 3.2 Detection of serum antibody titers in mice after immunization

[0180] ELISA plates were coated with 2 μg / mL huTrop2-His and huTrop2-huFc respectively and incubated overnight at 4°C (30 μL / well). After washing three times, the plates were blocked with 5% skim milk prepared in PBS at room temperature for 1 hour. After washing three times, mouse serum diluted serially with PBS was added, along with the antibody Sacituzumab as a positive control, and the plates were incubated at room temperature for 1 hour. After washing, secondary antibodies Goat-anti-mouse-IgG(1+2a+2b+3)-HRP (Jackson, 115-035-164) or Goat-anti-human-Kappa+Lambda-HRP (Millipore, AP502P+AP506P) diluted in PBS were added and incubated at room temperature for 1 hour. After washing six times, TMB was added for color development for 5-20 minutes. After stopping the color development reaction, data were read using an OD450 microplate reader, and the data were processed and plotted using GraphPad Prism. The results showed that the serum titers of all three mice reached the target levels.

[0181] 3.3 Construction of Phage Display Antibody Gene Library

[0182] After immunization, the spleen of the mouse was harvested, and the spleen cells were collected after grinding and filtration. 1 mL of TRIzol was added. TM Reagent (Thermo Fisher, 15596026) lysed spleen cells, and total RNA was extracted using the phenol-chloroform method. The extracted RNA was then reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, 6210A). Subsequently, using the cDNA as a PCR template, specific primers for the mouse antibody sequence (synthesized by Shanghai Platinum Biotech Co., Ltd.) were used to amplify the variable regions of the antibody's light and heavy chains, respectively. The PCR product was digested with NcoI and NotI to obtain the antibody gene fragment, which was then inserted into a phage display vector and ligated using T4 ligase. The ligation product was recovered using a DNA recovery kit (Omega, D6492-02) and finally transformed into competent Escherichia coli SS320 cells (Lucigen, MC1061F) using an electroporator (Bio-Rad, MicroPulser). The electroporated bacteria were plated on 2-YT (C+ / K+2-YT) solid plates containing ampicillin and tetracycline to amplify the correctly transformed SS320 cells with the antibody plasmid. The cells were then packaged using VSCM13 helper phage (purchased from Stratagene) to obtain a phage display library containing the Fab sequence.

[0183] Example 4: Screening of phage display antibody gene libraries

[0184] 4.1 Screening of phage display antibody gene libraries using immunotube and magnetic bead methods

[0185] Both the immunotube method and the magnetic bead method aim to enrich specific antibodies against antigens, and are two complementary and mutually reinforcing experimental methods.

[0186] The principle of immunotube screening is to coat the antigen protein huTrop2-His or huTrop2-huFc onto the surface of an immunotube with high adsorption capacity. A phage display antibody library is added to the immunotube, and the antigen protein adsorbed on the immunotube surface is incubated, washed, and eluted through a panning process. After 2-4 rounds of panning, specific monoclonal antibody Fab against the antigen is finally enriched. In this example, after 3 rounds of panning, monoclonal antibody Fab against human Trop2 was enriched. The specific method refers to step 2.4.2 in Example 2 of patent CN112250763B.

[0187] Magnetic bead screening is based on biotin-labeling of the antigen proteins huTrop2-His and huTrop2-hFc, followed by binding to streptavidin-conjugated magnetic beads. The process involves incubating, washing, and eluting the antigen-bound magnetic beads and the antibody gene phage display library. Typically, 3-4 rounds of screening are performed, resulting in a large enrichment of antigen-specific monoclonal antibodies. In this embodiment, biotin-labeled huTrop2-His and huTrop2-huFc are used for phage display library screening. After 3 rounds of screening, initial screening for Trop2 protein monoclonal antibodies (Fab) is conducted, following step 2.4.1 in Example 2 of patent CN112250763B.

[0188] 4.2 Selection of Monoclonal Cells

[0189] The enrichment effect was evaluated by ELISA detection of the phage pools eluted in each round. Ten clones were randomly selected from each round of phage pools for sequence analysis. The enrichment effect and the reproducibility ratio of the measured sequences were combined to select an appropriate round for single clone selection.

[0190] Using the antigen protein huTrop2-His for initial ELISA monoclonal screening, 11 clones with unique sequences were obtained.

[0191] Example 5 Antibody Construction, Expression and Purification

[0192] 5.1 Plasmid Construction

[0193] The VH coding sequence from the 11 selected monoclonal Fab sequences was ligated with the coding sequence of the heavy chain constant region (SEQ ID NO:3) of human IgG1 to obtain the heavy chain coding sequence of the chimeric antibody. The VL coding sequence from the Fab sequences was ligated with the coding sequence of the Kappa type (SEQ ID NO:4) of the human light chain constant region (CL) to obtain the light chain coding sequence of the chimeric antibody. The coding sequences of the antibody heavy and light chains were inserted into the eukaryotic expression vector plasmid pcDNA3.4 (Invitrogen), and transformed into *E. coli* DH5α, cultured overnight at 37°C. Endotoxin-free plasmid extraction was performed using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free antibody plasmids for eukaryotic expression.

[0194] 5.2 Antibody Expression and Purification

[0195] The full-length antibody sequence obtained above was expressed using the Expi CHO transient transfection system (Thermo Fisher, A29133). The specific method is as follows: On the day of transfection, the CHO cell density was confirmed to be 7 × 10⁶ cells / year. 6 Up to 1×107 With approximately 100 viable cells / mL and a cell viability >98%, the cells were adjusted to a final concentration of 6 × 10⁶ cells / mL using fresh ExpiCHO expression medium pre-warmed to 37°C. 6 Cells / mL. OptiPRO pre-cooled to 4°C TM SFM dilution of the target plasmid (add 1 μg plasmid to 1 mL of the culture medium described above), and simultaneous use of OptiPRO TM SFM diluted ExpiFectamine TM CHO reagent, then mix the two in equal volumes and gently blow to mix thoroughly to prepare ExpiFectamine. TM Incubate the CHO / plasmid DNA mixture at room temperature for 1-5 minutes, then slowly add it to the prepared cell suspension while gently shaking. Finally, place the mixture in a cell culture shaker and culture at 37°C and 8% CO2.

[0196] 18-22 hours after transfection, add ExpiCHO to the culture medium TM Enhancer reagent and ExpiCHO TM Feed the reagent, and incubate the shake flask at 32°C on a shaker with 5% CO2. On day 5 post-transfection, add the same volume of ExpiCHO. TM Feed the reagent slowly while gently mixing the cell suspension. Seven days after transfection, collect the cell culture supernatant expressing the target protein, centrifuge at 15000g for 10 min, and purify the supernatant using MabSelect SuRe LX (GE, 17547403) through affinity chromatography. Then, elute the target protein with 100mM sodium acetate (pH=3.0), neutralize with 1M Tris-HCl, and finally transfer the obtained protein to PBS buffer via ultrafiltration concentrator (Millipore, UFC901096).

[0197] The prepared chimeric antibodies were named using clone numbers, namely E1, E4, E7, E8, E9, E11, E12, E16, E35, E39 and E45. The CDR amino acid sequences of the chimeric antibodies are shown in Table 1. The CDR sequences were determined using the AbM definition method.

[0198] Table 1. Amino acid sequences of the CDR regions of 11 candidate antibodies

[0199] Example 6: Detection of Physicochemical Properties of Candidate Antibodies

[0200] In this embodiment, the relative molecular weight and purity of the antibodies obtained above were detected by SDS-PAGE and SEC-HPLC.

[0201] 6.1 Antibody SDS-PAGE Identification

[0202] Preparation of non-reducing solution: Add 1 μg of each obtained antibody and the quality control IPI (ipilimumab) to 5×SDS loading buffer and 40 mM iodoacetamide, respectively. Heat in a dry bath at 75°C for 10 min, cool to room temperature, and centrifuge at 12000 rpm for 5 min to collect the supernatant.

[0203] Preparation of reducing solution: Add 2 μg of each obtained antibody and the quality control IPI to 5×SDS loading buffer and 5 mM DTT, respectively. Heat in a dry bath at 100℃ for 10 min, cool to room temperature, and centrifuge at 12000 rpm for 5 min to collect the supernatant. Add the supernatant to a Bis-tris 4-15% gradient gel (GenScript) for gel electrophoresis and stain with Coomassie Brilliant Blue to visualize the protein bands.

[0204] Protein gels with chromogenic protein bands were scanned using an EPSON V550 color scanner (decolorized with decolorizing solution until the gel background was transparent). The purity of reduced and non-reduced bands was calculated using ImageJ according to the peak area normalization method.

[0205] The experimental results showed that the bands of each antibody on the non-reducing gel were around 150 kDa, while the bands on the reducing gel were around 55 kDa and 25 kDa, respectively, which were in line with the expected sizes. The SDS-PAGE purity of all 11 antibodies detected by reducing gel was higher than 95% (Table 2).

[0206] 6.2 SEC-HPLC identification of antibody monomer purity

[0207] Materials preparation:

[0208] 1. Mobile phase: 150 mmol / L phosphate buffer, pH 7.4.

[0209] 2. Sample Preparation: Dilute each antibody and the IPI control to 0.5 mg / mL with the mobile phase solution. Use an Agilent HPLC 1100 column (XBridge BEH SEC 3.5 μm, 7.8 mm ID × 30 cm), Waters flow rate set to 0.8 mL / min, injection volume 20 μL, and VWD detector wavelengths of 280 nm and 214 nm. Inject blank solution, IPI control solution, and antibody sample solution sequentially. Calculate the percentages of high molecular weight polymers, antibody monomers, and low molecular weight substances in the samples using the area normalization method.

[0210] The results are shown in Table 2. The purity of the SEC monomers of antibodies E7 and E16 were 85.91% and 84.19%, respectively, while the purity of the SEC monomers of the remaining antibodies was all above 90%.

[0211] Table 2. Antibody expression levels and physicochemical properties obtained in this invention

[0212] Example 7: Detection of antigen-binding activity of candidate antibodies

[0213] In this embodiment, the binding of 11 antibodies (E1, E4, E7, E8, E9, E11, E12, E16, E35, E39, and E45) to the human Trop2 antigen protein huTrop2-His was detected using the ELISA method. The binding affinity of these antibodies (E1, E4, E7, E8, E9, E11, E12, E16, E35, E39, and E45) to huTrop2-HEK293 cells and Hacat cells was also detected using the FACS method. Hacat tumor cells are immortalized human epidermal cells that highly express huTrop2.

[0214] 7.1 ELISA-based detection of antibody binding affinity to antigen protein huTrop2-His

[0215] 96-well ELISA plates were coated with huTrop2-His at a concentration of 2 μg / mL (30 μL / well) and incubated overnight at 4°C. The next day, the plates were washed three times with PBST, blocked with 5% skim milk for 2 h, washed three times with PBST, and then serially diluted antibodies and the positive control antibody Sacituzumab were added and incubated for 1 h. After washing three times with PBST, the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 h. After incubation, the plates were washed six times with PBST and then developed with TMB (SurModics, TMBS-1000-01). Based on the colorimetric results, the reaction was terminated by adding 2M HCl, and the data were read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).

[0216] The results are shown in Figures 1A-1D. All antibodies and antigen proteins huTrop2-His obtained in this invention exhibit good affinity activity. Among them, E4, E8, E9, and E45 bind to huTROP2-His EC32. 50 The concentrations were 0.0040, 0.0051, 0.0041, and 0.0061 μg / mL, respectively, with affinity comparable to the control antibody Sacitzumab.

[0217] 7.2 Detection of antibody binding ability to huTrop2-HEK293 and Hacat cells based on FACS

[0218] In this embodiment, the antibody binding activity was evaluated using two types of human Trop2 overexpressing cells, huTrop2-HEK293 and Hacat cells.

[0219] The specific method is as follows: Prepare a single-cell suspension from huTrop2-HEK293 cells or Hacat cells in the logarithmic growth phase, and adjust the density to 1×10⁻⁶. 6 Cells / mL, 100 μL per well, were added to a 96-well round-bottom plate, centrifuged at 300 g at 4 °C, and the supernatant was removed. Serially diluted 11 antibodies obtained in this invention and the positive control antibody Sacituzumab were added to the corresponding wells, mixed, and incubated at 4 °C for 30 min. After washing the incubated cell mixture three times, 100 μL of 1:300 diluted secondary antibody PE labelled anti-human-Fc (Abcam, 98596) was added, and the mixture was incubated at 4 °C in the dark for 30 min. After washing three times, the cells were analyzed by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

[0220] The results are shown in Figures 2A-2C and 3A-3D. In huTrop2-HEK293 cells, E12, E35, and E39 EC... 50 The effective amounts were 1.081, 0.934, and 1.236 nM, respectively, and the affinity was superior to that of the control antibody Sacituzumab (EC). 50 The affinity of E7 to Trop2 was 1.553 nM, which was comparable to that of the control antibody Sacituzumab. The affinity of the other antibodies to Trop2 was weaker than that of the control antibody Sacituzumab (Figures 2A-2C). On Hacat cells, E1, E35, and E39 showed better affinity to Trop2 than the control antibody Sacituzumab, while E7 and E12 showed comparable affinity to Trop2 to the control antibody Sacituzumab. E8, E9, E11, and E45 showed weaker affinity to Trop2 than the control antibody Sacituzumab (Figures 3A-3D).

[0221] Example 8: Detection of antibody species and homologous cross-activity

[0222] In this embodiment, the cross-reactivity of each candidate antibody in terms of species and family was tested. The monkey Trop2 antigen protein CynoTrop2-His and the human Trop1 antigen protein huTrop1-His prepared in step 1.2 of Example 1 were used to identify the species and family cross-reactivity of the candidate antibodies.

[0223] 8.1 Identification of species cross-reactivity of antibodies

[0224] 96-well ELISA plates were coated with 2 μg / mL CynoTrop2-His (30 μL / well) and incubated overnight at 4°C. The next day, the plates were washed three times with PBST, blocked with 5% skim milk for 2 h, washed three times with PBST, and then serially diluted antibodies and the positive control antibody Sacituzumab were added and incubated for 1 h. After washing three times with PBST, the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 h. After incubation, the plates were washed six times with PBST and then developed with TMB (SurModics, TMBS-1000-01). Based on the colorimetric results, the reaction was terminated by adding 2M HCl, and the data were read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).

[0225] The results are shown in Figures 4A-4D and Table 3. All 11 antibody molecules tested bound to the antigen protein CynoTrop2-His, demonstrating good cross-activity with monkeys. Among them, E8, E9, E11, and E12 showed better binding activity to CynoTrop2-His than the positive control antibody Sacituzumab, while E1, E35, E39, and E49 showed comparable binding activity to CynoTrop2-His as the positive control antibody Sacituzumab.

[0226] 8.2 Antibody homologous cross-reactivity detection of the present invention

[0227] 96-well ELISA plates were coated with 2 μg / mL huTrop1-His (30 μL / well) and incubated overnight at 4°C. The next day, the plates were washed three times with PBST, blocked with 5% skim milk for 2 h, washed three times with PBST, and then serially diluted antibody and positive control antibody Sacituzumab were added and incubated for 1 h. After washing three times with PBST, secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 h. After incubation, the plates were washed six times with PBST and then developed with TMB (SurModics, TMBS-1000-01). Based on the color development results, the reaction was terminated by adding 2M HCl, and the data were read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).

[0228] The results are shown in Figures 5A-5C and Table 3. None of the 11 antibody molecules detected bind to the antigen protein huTrop1-His. Among them, XYDX-S110-BM2 is an antibody that binds to the antigen protein huTrop1-His (Tabs database, antibody name: citatuzumab bogatox).

[0229] Table 3. Antibody species cross-activity and family cross-activity obtained in this invention.

[0230] Example 9 Antibody endocytosis efficiency detection

[0231] In this embodiment, the endocytosis efficiency of the antibody obtained in this invention was detected using the Fab-Zap method. Fab-ZAP is a Fab fragment linked to saporin, a ribosome inhibitor that inhibits protein synthesis and causes cell death. The Fab-ZAP used in this experiment is a Fab fragment that can bind to the human Fc of the chimeric antibody. After incubation with the chimeric antibody, the chimeric antibody is contaminated with a toxin. When the chimeric antibody is endocytosed, the toxin enters the cell along with the chimeric antibody, causing cell death. The endocytosis of the antibody was then detected by measuring cell viability using an MTS (Promega, G3580).

[0232] The specific experimental method is as follows: First, Fab-Zap was diluted to 0.4 nM with DMEM complete medium. Then, each candidate antibody and positive control antibody were serially diluted with 0.4 nM Fab-Zap to prepare antibody dilution solutions. Logarithmic growth phase huTrop2-HEK293 cells were prepared into single-cell suspensions and the density was adjusted to 6 × 10⁻⁶ cells / cells. 6 At a concentration of 50 μL / well, cells were seeded into each well of a 96-well plate. Then, 50 μL of the previously prepared antibody dilution was added to each well, and the mixture was thoroughly pipetted and mixed. The cell culture plates were incubated at 37°C for 48 hours. After incubation, 7.5 μL of Triton X-100 solution was added to each well, and the mixture was gently tugged to mix. The cell culture plates were then incubated at 37°C for 0.5 hours. Next, 20 μL of MTS was added to each well, and the plates were incubated at 37°C for 1–4 hours. Finally, the cell culture plates were centrifuged at 1000 rpm for 5 minutes, and the data were read using a microplate reader at wavelength A492.

[0233] The results are shown in Figures 6A-6F. Antibody molecules E4, E7, E8, E9, E11, and E16 showed better endocytosis than the control antibody Sacituzumab, while the endocytosis of E1, E12, E35, and E39 was comparable to that of the control antibody Sacituzumab.

[0234] Example 10 Antibody Affinity Kinetics (BLI) Analysis

[0235] In this embodiment, the affinity between the candidate antibody and huTrop2-His is detected using a Gator device.

[0236] First, prepare a Q buffer solution using PBS (10mM pH 7.4) (IgG-free, purchased from Jackson Immuno Research Lab) + 0.02% Tween 20 (purchased from Thermo Fisher Scientific) + 0.2% BSA (purchased from Yuanpei). Dilute the storage solution of the antibody to be tested to a final concentration of 30nM using the Q buffer solution. Prepare a multi-fold diluted working solution using the Q buffer solution for the huTrop2-His storage solution. Then, use the Gator instrument and its accompanying software, selecting the Advanced Kinetics experimental mode for detection and analysis.

[0237] The results are shown in Table 4. The KD values ​​of antibodies E1, E4, E7, E16, E35, and E39 are on the same order of magnitude as the control antibody, while the affinity of antibodies E8, E9, E11, E12, and E45 is lower than that of the control antibody.

[0238] Table 4. KD values ​​of the antibodies obtained in this invention.

[0239] Example 11 Humanization of mouse antibodies

[0240] The VH and VL sequences of antibodies E1, E4, E7, and E35 were compared with known human antibody databases to identify the human germline VH and VL sequences with the highest homology to the murine VH and VL sequences, respectively. The frame regions (CDRs and frame regions defined using AbM) of the corresponding human germline VH and VL sequences were selected, and the complementarity-determining regions (CDRs) of these germline genes were replaced with the corresponding CDR sequences from E1, E4, E7, and E35. Then, using computer prediction and simulation, murine amino acids that significantly influence antigen binding in the frame regions of E1, E4, E7, and E35 were retained through reverse mutation. Humanized antibodies were constructed, expressed, and purified using the method described in Example 5. The amino acid sequences of the light chain variable region and heavy chain variable region of the humanized antibodies are shown in Table 5. The humanized antibody proteins were identified by SDS-PAGE and SEC-HPLC.

[0241] Table 5. Amino acid sequences of the variable region of humanized antibodies.

[0242] Example 12: Detection of antigen affinity of humanized antibodies

[0243] In this embodiment, the affinity of the humanized antibody for the human Trop2 antigen protein huTrop2-His was detected using the ELISA method, and the affinity of the humanized antibody for MDA-MB-231 tumor cells was detected using the FACS method. Specific testing methods are described in Example 7.

[0244] The ELISA results are shown in Figures 7A-7I. The affinity of the humanized antibody for the antigen protein is comparable to that of the corresponding parent molecule and the control antibody Sacituzumab.

[0245] The FACS test results are shown in Figures 8A-8F. The humanized antibodies E1 and E35 showed better affinity for the antigenic proteins on MDA-MB-231 tumor cells than the control antibody Sacituzumab. The other humanized molecules showed comparable affinity for the antigenic proteins on MDA-MB-231 tumor cells to the control antibody.

[0246] Example 13 ADC Fabrication

[0247] In this embodiment, antibodies E1-H1L2, E4-H2L1, E7-H1L2, E35-H1L1 and the control antibody Sacituzumab were conjugated to the linker-toxin MC-VC-PAB-MMAE (which has anticancer activity and is composed of MMAE (a tubulin inhibitor) and VC, commercial information: MedChemExpress, HY-15575) at the thiol group of cysteine ​​residues to prepare antibody-drug conjugates (ADCs). IgG1 type antibodies have 16 pairs of cysteine ​​residues, of which 12 are intrachain and 4 are interchain disulfide bonds. The interchain disulfide bonds are solvent-accessible and can be reduced by reducing agents to form eight thiol groups, which then become the conjugation target (McCombs J, Owen S. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. AAPS J. 2015; 17:339-51).

[0248] The specific preparation method is as follows:

[0249] Antibodies E1-H1L2, E4-H2L1, E7-H1L2, E35-H1L1 and control antibody Sacituzumab were removed from the -80℃ freezer, thawed, and transferred to 15mL 30KD ultrafiltration centrifuge tubes. Conjugation buffer (containing 1.78g Na2HPO4·2H2O, 0.31g NaH2PO4·H2O, 7.80g NaCl, 0.35g KCl, and purified water to a final volume of 1000g, pH 7.4) was added to 15mL. The tubes were centrifuged at 3500g for about 20min, concentrated to 2-3mL, and dialyzed again with dialyzed 8-10 times to obtain the antibody stock solution. The antibody concentration after dialyzed was then measured.

[0250] Add the antibody stock solution, coupling buffer, 250mM EDTA stock solution (ethylenediaminetetraacetic acid stock solution, content per 1L: 84.05g EDTA, diluted to 1000g with purified water) and 10mM disulfide reducing agent TCEP stock solution (tris(2-carboxyethyl)phosphonic acid hydrochloride stock solution, content per 1L: C9H) to the reduction reaction system in sequence. 15 2.87 g of O6P·HCl was added, and purified water was brought to a final volume of 1000 g to achieve an antibody concentration of 5 mg / mL, an EDTA concentration of 5 mM, a molar ratio of TCEP to E1-H1L2, E4-H2L1, E7-H1L2, and E35-H1L1 of 2, and a molar ratio of TCEP to Sacituzumab of 2.2. After thorough mixing, the mixture was placed in a 25°C constant-temperature mixer at 200 rpm for 2 hours to allow the reduction reaction to proceed.

[0251] Weigh MC-VC-PAB-MMAE and dissolve it in DMSO to prepare a 10mM MC-VC-PAB-MMAE stock solution. After the reduction reaction is complete, add DMSO (final ratio 10%) and 6 times the antibody equivalent (molar mass multiple) of MC-VC-PAB-MMAE stock solution sequentially to the reaction system in an ice-water bath. After thorough mixing, place the mixture in a 25℃ constant temperature mixer at 200 rpm for 1.5 h of coupling reaction. After coupling is complete, add 12 times the antibody equivalent (molar mass multiple) of 10mM quencher NAC stock solution (N-acetyl-L-cysteine ​​stock solution, content: 1.63g per 1L, diluted with purified water to 1000g) to the reaction system in an ice-water bath for quenching for 10 min.

[0252] After the quenching reaction, the sample was centrifuged and then filtered. The resulting ADC sample was then transferred to a 15 mL 30 KD ultrafiltration centrifuge tube. Dialysis buffer was added to a final volume of 15 mL, and the sample was centrifuged at 3500 g for 20 min to concentrate to 2-3 mL. Dialysis buffer was added again to a final volume of 15 mL, and this process was repeated 8-10 times. After dialysis, the sample was analyzed by SEC-HPLC and HIC-HPLC to determine the concentration and free drug content. The test results are shown in Table 6. The test results show that the purity of the ADCs was consistently above 98%, and the DAR (drug-antibody ratio) values ​​were around 4.

[0253] Table 6 ADC Sample Test Results

[0254] Example 14: Detection of ADC Tumor Cell Killing Effect

[0255] In this embodiment, NCI-H292 and HCC827 cells were used to detect the killing effect of the ADC of the present invention and the control ADC on tumor cells.

[0256] The specific method is as follows: NCI-H292 cells or HCC827 cells in the logarithmic growth phase are prepared into single-cell suspensions, and the cell density is adjusted to 6 × 10⁻⁶. 4 Cells / mL: 50 μL was added to each well of a 96-well cell culture plate and incubated at 37°C with 5% CO2 for 12 h. Then, serially diluted ADC samples were added and incubated at 37°C with 5% CO2 for 72 h. Next, 30 μL of CCK8 (absin / Abisin, abs50003) was added to each well and incubated at 37°C for 1-4 h. The plates were then read at OD450 using a microplate reader. Positive control: Sacitumab-MMAE was used as a positive ADC reference. Cell-only group: The wells contained only cells; no ADC was added, and culture medium was used instead to ensure consistent system volume. Negative control: Isotype antibodies conjugated with the same toxin from the same batch were used to demonstrate that antibodies targeting non-cells could not cause cell death. Cell+triton: Triton was added in the presence of only cells as a positive killing control (100% cell killing) to calculate the killing efficiency of the experimental groups.

[0257] As shown in Figures 9A-9C, in NCI-H292 cells, the cytotoxic activities of E1-H1L2-MMAE and E35-H1L1-MMAE were comparable to those of the control antibody, while the cytotoxic activities of E4-H2L1-MMAE and E7-H1L2-MMAE were weaker than those of the control antibody. As shown in Figures 10A-10B, in HCC827 cells, the cytotoxic activities of E1-H1L2-MMAE and E35-H1L1-MMAE were superior to those of the control antibody, while the cytotoxic activity of E4-H2L1-MMAE was comparable to that of the control antibody.

[0258] Example 15: In vivo efficacy of ADC

[0259] In this embodiment, the NCI-N87 mouse tumor-bearing model was used to detect the inhibitory effects of the ADCs E1-H1L2-MMAE, E4-H2L1-MMAE, E35-H1L1-MMAE and the control ADC on tumor growth.

[0260] The specific method is as follows: NCI-N87 cells in the logarithmic growth phase were prepared into a single-cell suspension, and the cell density was adjusted to 8 × 10⁻⁶. 7 Cells / mL, subcutaneous tumor bearing was performed on 6-8 week old female Balb / C nude mice at 100 μL / mouse, until the tumor grew to 100 mm. 3 The mice were grouped and administered the drugs accordingly. The ADC of this invention and the control ADC were administered to tumor-bearing mice at a single dose of 5 mg / kg, and tumor volume and body weight were monitored weekly.

[0261] The results are shown in Figure 11: In the NCI-N87 tumor-bearing mouse model, ADCs E1-H1L2-MMAE, E4-H2L1-MMAE, E35-H1L1-MMAE and the control antibody ADC can all effectively inhibit tumor growth and their anti-tumor function is comparable to that of the control antibody ADC (Figure 11A). Furthermore, the body weight of mice treated with the ADCs E1-H1L2-MMAE, E4-H2L1-MMAE, and E35-H1L1-MMAE of this invention was not significantly different from that of the control antibody ADC and PBS groups (Figure 11B), indicating that the ADCs of this invention have no obvious toxic side effects.

[0262] The partial sequences used in this invention are as follows:

[0263] 1. Sacituzumab light chain (SEQ ID NO:1)

[0264] 2. Sacituzumab heavy chain (SEQ ID NO:2)

[0265] 3. Human Fc (SEQ ID NO:3)

[0266] 4. Kappa-type coding sequence of the constant region (CL) of the human light chain (SEQ ID NO:4)

[0267] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof targeting Trop2, comprising a heavy chain variable region and a light chain variable region, characterized in that, The light chain variable region includes LCDR1, LCDR2 and LCDR3, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3; The LCDR1 contains an amino acid sequence as shown in SEQ ID NO: 108, 16, 23, 31 or 67; the LCDR2 contains an amino acid sequence as shown in SEQ ID NO: 109, 24, 32 or 68; and the LCDR3 contains an amino acid sequence as shown in SEQ ID NO: 110, 111, 17, 33, 54, 60 or 69. Preferably, the LCDR1 comprises an amino acid sequence as shown in SEQ ID NO:8, 16, 23, 31, 43 or 64; The LCDR2 comprises an amino acid sequence as shown in SEQ ID NO: 9, 24, 32, 50, 56, or 65; and / or, The LCDR3 contains an amino acid sequence as shown in SEQ ID NO: 10, 17, 33, 38, 44, 51, 57 or 66; More preferably, the amino acid sequence of LCDR1 is as shown in SEQ ID NO:108, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:9, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:110; preferably, the amino acid sequence of LCDR1 is as shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:9, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:10; or, the amino acid sequence of LCDR1 is as shown in SEQ ID NO:43, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:9, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:44; The amino acid sequence of LCDR1 is shown in SEQ ID NO:23, the amino acid sequence of LCDR2 is shown in SEQ ID NO:24, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

111. The amino acid sequence of LCDR1 is shown in SEQ ID NO:16, the amino acid sequence of LCDR2 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

17. The amino acid sequence of LCDR1 is shown in SEQ ID NO:31, the amino acid sequence of LCDR2 is shown in SEQ ID NO:32, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

33. The amino acid sequence of LCDR1 is shown in SEQ ID NO:43, the amino acid sequence of LCDR2 is shown in SEQ ID NO:50, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

51. The amino acid sequence of LCDR1 is shown in SEQ ID NO:16, the amino acid sequence of LCDR2 is shown in SEQ ID NO:56, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:57; or, The amino acid sequence of LCDR1 is shown in SEQ ID NO:64, the amino acid sequence of LCDR2 is shown in SEQ ID NO:65, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

66.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, The heavy chain variable region includes any of the following: (1) The amino acid sequence of HCDR1 is shown in SEQ ID NO:5; the amino acid sequence of HCDR2 is shown in SEQ ID NO:112; and the amino acid sequence of HCDR3 is shown in SEQ ID NO:113; preferably, the amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:7; or, the amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:47, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:48; (2) The amino acid sequence of HCDR1 is shown in SEQ ID NO:13, the amino acid sequence of HCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

15. (3) The amino acid sequence of HCDR1 is shown in SEQ ID NO:20, the amino acid sequence of HCDR2 is shown in SEQ ID NO:21, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

22. (4) The amino acid sequence of HCDR1 is shown in SEQ ID NO:28, the amino acid sequence of HCDR2 is shown in SEQ ID NO:29, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

30. (5) The amino acid sequence of HCDR1 is shown in SEQ ID NO:20, the amino acid sequence of HCDR2 is shown in SEQ ID NO:36, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

37. (6) The amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:41, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

42. (7) The amino acid sequence of HCDR1 is shown in SEQ ID NO:50, the amino acid sequence of HCDR2 is shown in SEQ ID NO:51, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

52. (8) The amino acid sequence of HCDR1 is shown in SEQ ID NO:57, the amino acid sequence of HCDR2 is shown in SEQ ID NO:51, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:58; and, (9) The amino acid sequence of HCDR1 is shown in SEQ ID NO:64, the amino acid sequence of HCDR2 is shown in SEQ ID NO:65, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

66.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein, The amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:6, the amino acid sequence of HCDR3 is shown in SEQ ID NO:7, the amino acid sequence of LCDR1 is shown in SEQ ID NO:8, the amino acid sequence of LCDR1 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:

10. The amino acid sequence of HCDR1 is shown in SEQ ID NO:13, the amino acid sequence of HCDR2 is shown in SEQ ID NO:14, the amino acid sequence of HCDR3 is shown in SEQ ID NO:15, the amino acid sequence of LCDR1 is shown in SEQ ID NO:16, the amino acid sequence of LCDR1 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:

17. The amino acid sequence of HCDR1 is shown in SEQ ID NO:20, the amino acid sequence of HCDR2 is shown in SEQ ID NO:21, the amino acid sequence of HCDR3 is shown in SEQ ID NO:22, the amino acid sequence of LCDR1 is shown in SEQ ID NO:23, the amino acid sequence of LCDR1 is shown in SEQ ID NO:24, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:

25. The amino acid sequence of HCDR1 is shown in SEQ ID NO:28, the amino acid sequence of HCDR2 is shown in SEQ ID NO:29, the amino acid sequence of HCDR3 is shown in SEQ ID NO:30, the amino acid sequence of LCDR1 is shown in SEQ ID NO:31, the amino acid sequence of LCDR1 is shown in SEQ ID NO:32, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:

33. The amino acid sequence of HCDR1 is shown in SEQ ID NO:20, the amino acid sequence of HCDR2 is shown in SEQ ID NO:36, the amino acid sequence of HCDR3 is shown in SEQ ID NO:37, the amino acid sequence of LCDR1 is shown in SEQ ID NO:23, the amino acid sequence of LCDR1 is shown in SEQ ID NO:24, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:

38. The amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:41, the amino acid sequence of HCDR3 is shown in SEQ ID NO:42, the amino acid sequence of LCDR1 is shown in SEQ ID NO:43, the amino acid sequence of LCDR1 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:

44. The amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:47, the amino acid sequence of HCDR3 is shown in SEQ ID NO:48, the amino acid sequence of LCDR1 is shown in SEQ ID NO:43, the amino acid sequence of LCDR1 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:

44. The amino acid sequence of HCDR1 is shown in SEQ ID NO:50, the amino acid sequence of HCDR2 is shown in SEQ ID NO:51, the amino acid sequence of HCDR3 is shown in SEQ ID NO:52, the amino acid sequence of LCDR1 is shown in SEQ ID NO:43, the amino acid sequence of LCDR1 is shown in SEQ ID NO:53, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:

54. The amino acid sequence of HCDR1 is shown in SEQ ID NO:57, the amino acid sequence of HCDR2 is shown in SEQ ID NO:51, the amino acid sequence of HCDR3 is shown in SEQ ID NO:58, the amino acid sequence of LCDR1 is shown in SEQ ID NO:16, the amino acid sequence of LCDR1 is shown in SEQ ID NO:59, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:

60. The amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:6, the amino acid sequence of HCDR3 is shown in SEQ ID NO:7, the amino acid sequence of LCDR1 is shown in SEQ ID NO:43, the amino acid sequence of LCDR1 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:44; or, The amino acid sequence of HCDR1 is shown in SEQ ID NO:64, the amino acid sequence of HCDR2 is shown in SEQ ID NO:65, the amino acid sequence of HCDR3 is shown in SEQ ID NO:66, the amino acid sequence of LCDR1 is shown in SEQ ID NO:67, the amino acid sequence of LCDR1 is shown in SEQ ID NO:68, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:

69.

4. The antibody or antigen-binding fragment thereof of claim 2, wherein, The amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:112, the amino acid sequence of HCDR3 is shown in SEQ ID NO:113, the amino acid sequence of LCDR1 is shown in SEQ ID NO:108, the amino acid sequence of LCDR2 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

110. Preferably, the amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:6, the amino acid sequence of HCDR3 is shown in SEQ ID NO:7, the amino acid sequence of LCDR1 is shown in SEQ ID NO:8, the amino acid sequence of LCDR1 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:

10. The amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:47, the amino acid sequence of HCDR3 is shown in SEQ ID NO:48, the amino acid sequence of LCDR1 is shown in SEQ ID NO:43, the amino acid sequence of LCDR1 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:44; or, The amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:6, the amino acid sequence of HCDR3 is shown in SEQ ID NO:7, the amino acid sequence of LCDR1 is shown in SEQ ID NO:43, the amino acid sequence of LCDR1 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:

44.

5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein, The frame region of the heavy chain variable region and / or the light chain variable region is a mouse-derived frame region; Preferably, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 11, 18, 26, 34, 39, 45, 49, 55, 61, 63 or 70, and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 12, 19, 27, 35, 40, 46, 56, 62, 71 or 114; More preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 11, 18, 26, 34, 39, 45, 49, 55, 61, 63 or 70, and / or, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 12, 19, 27, 35, 40, 46, 56, 62, 71 or 114; More preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:11, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:12; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:18, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:26, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:27; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:34, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:35; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:39, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:40; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:45, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:46; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:49, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:114; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:55, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:56; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:61, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:62; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:63, and / or, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:46; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:70, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

71.

6. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein, The framework region of the heavy chain variable region and / or the light chain variable region is a human-derived framework region; Preferably, the framework region of the heavy chain variable region is derived from the human heavy chain IGHV3-23*04; and / or, the framework region of the light chain variable region is derived from the human light chain IGLV657*01; Further preferably, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:72, 74, 75, 77, 78, 79, 80, 81, 82, 84, 85, 86, 87, 88, 89, 90, 92, 97, 98, 99, 101, 102, 103, 104, 105, 106 or 107, and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:73, 76, 83, 91, 93, 94, 95, 96 or 100; More preferably, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:72, 74, 75, 77, 78, 79, 80 or 81, and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:73 or 76. The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:82, 84, 85, 86, 87, 88, 89 or 90, and / or the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:73, 76, 83 or 91. The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:92, 97 or 98, 99, 101, 102, 103, 104, 105, 106 or 107, and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:93, 94, 95 or 96 or 100; or, The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:99, 101, 102, 103, 104, 105, 106 or 107, and / or the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:

100. More preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:72, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:73; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:74, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:73; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:75, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:73; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:75, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:76; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:77, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:76; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:78, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:76; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:79, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:76; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:80, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:76; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:81, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:76; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:82, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:83; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:84, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:83; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:85, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:83; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:86, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:83; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:87, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:83; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:88, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:83; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:89, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:83; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:90, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:83; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:82, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:91; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:92, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:93; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:92, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:94; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:92, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:95; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:92, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:96; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:97, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:93; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:97, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:94; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:97, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:95; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:97, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:96; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:98, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:93; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:99, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:100; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:101, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:100; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:102, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:100; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:103, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:100; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:104, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:100; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:105, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:100; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:106, and / or, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:100; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:107, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

100.

7. The antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein, The antibody is a full-length antibody, Fab, Fab', F(ab')2, or Fv; the Fv is preferably scFv. Preferably, the antibody is a full-length antibody, the heavy chain constant region and / or light chain constant region of which are derived from mouse antibodies or human antibodies; More preferably, the heavy chain constant region and / or the light chain constant region are derived from human antibodies; the heavy chain constant region is preferably derived from the human heavy chain IgG1 constant region, the amino acid sequence of which is shown, for example, as shown in SEQ ID NO:3; and / or, the light chain constant region is preferably derived from the human light chain κ chain constant region, the amino acid sequence of which is shown, for example, as shown in SEQ ID NO:

4.

8. An isolated nucleic acid, comprising, The isolated nucleic acid encodes the antibody or its antigen-binding fragment as described in any one of claims 1 to 7.

9. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 8; preferably, the recombinant expression vector is a plasmid, granule, bacteriophage, or viral vector, and the viral vector is preferably a retroviral vector, lentiviral vector, adenovirus vector, or adeno-associated virus vector.

10. A transformant characterized in that, The transformant comprises the nucleic acid as described in claim 8 or the recombinant expression vector as described in claim 9, and the host cell of the transformant is a eukaryotic cell or a prokaryotic cell; Preferably, the eukaryotic cells are mammalian cells, such as 293 cells or Expi-CHO cells.

11. A method of producing an antibody or antigen-binding fragment thereof targeting Trop2, characterized in that, The method comprises culturing the transformant as described in claim 10.

12. An antibody drug conjugate, characterized in that, The antibody-drug conjugate comprises a cytotoxic agent or tag, and an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 7; Preferably, the cytotoxic agent is MMAF or MMAE, and the tag is a fluorescent agent; and / or, the antibody-drug conjugate further includes a linker, such as MC-VC-PAB; More preferably, the antibody-drug conjugate comprises the antibody or its antigen-binding fragment and MC-VC-PAB-MMAE.

13. A method of preparing the antibody drug conjugate of claim 12, wherein, The method includes reacting the antibody or its antigen-binding fragment with the cytotoxic agent linked with a linker to obtain the antibody-drug conjugate.

14. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 7 and / or an antibody-drug conjugate as described in claim 12, and a pharmaceutically acceptable carrier.

15. A kit comprising, The kit comprises an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 7, an antibody-drug conjugate as described in claim 12, or a pharmaceutical composition as described in claim 14.

16. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 7, the isolated nucleic acid as described in claim 8, the recombinant expression vector as described in claim 9, the transformant as described in claim 10, the antibody-drug conjugate as described in claim 12, or the pharmaceutical composition as described in claim 14 in the preparation of medicaments for the diagnosis, prevention, and / or treatment of tumors, or in the preparation of Trop2 inhibitors; Preferably, the tumor is a tumor associated with abnormal Trop2 expression, such as one or more of breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, ovarian cancer, and urothelial carcinoma; preferably lung cancer or gastric cancer.

17. A kit comprising, The pillbox set includes pillbox A and pillbox B, wherein: The kit A contains an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 7, an antibody-drug conjugate as described in claim 12, or a pharmaceutical composition as described in claim 14; The kit B contains other antibodies for treating tumors or a pharmaceutical composition containing said other antibodies for treating tumors, and / or other drugs for treating tumors; Preferably, the tumor is a tumor associated with abnormal Trop2 expression, such as one or more of breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, ovarian cancer, and urothelial carcinoma; preferably lung cancer or gastric cancer.

18. A drug delivery device characterized in that, The drug delivery device comprises an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 7, an antibody-drug conjugate as described in claim 12, or a pharmaceutical composition as described in claim 14; Preferably, the drug delivery device further includes a component for administering the antibody or its antigen-binding fragment, antibody-drug conjugate, or drug composition to the subject, such as a syringe or infusion device.

19. A method of detecting Trop2, characterized in that, The method includes using an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 7; Preferably, the method is not for diagnostic purposes.

Images