Frα-targeting antibody-drug conjugate and use thereof

Abstract

Provided are an anti-FRα monoclonal antibody, a biparatopic antibody and an antibody-drug conjugate formed by means of conjugating the antibody with a drug, and the use of the antibody and antibody-drug conjugate in the preparation of a drug for treating diseases associated with FRα expression, such as cancer.

Description

An antibody-drug conjugate targeting FRα and its uses Technical Field

[0001] This disclosure pertains to the field of biomedicine, specifically relating to an anti-FRα antibody or its antigen-binding fragment, a bispecific antibody and its antibody-drug conjugates coupled to a drug, and their tautomers, stereoisomers or pharmaceutically acceptable salts, as well as the use of said antibody or its antigen-binding fragment, bispecific antibody, antibody-drug conjugates and their tautomers, stereoisomers or pharmaceutically acceptable salts in medicaments for treating FRα-expression-related diseases such as cancer. Background Technology

[0002] In 2020, there were 19.29 million new cancer cases worldwide, of which 4.57 million were in China, accounting for 23.7% of the global total. As the world's most populous country, China's number of new cancer cases far exceeds that of other countries. Globally, due to the increasing aging population, the cancer burden is projected to increase by 50% by 2040 compared to 2020, with the number of new cancer cases reaching nearly 30 million at that time (Zheng, RS et al. Zhonghua Zhongliuzazhi. 46, 3(2024): 221-231.).

[0003] Folic acid receptor alpha (FRα), also known as folate-binding protein, is a 38kD glycosylphosphatidylinositol (GPI) anchoring protein encoded by the folate receptor 1 (FOLR1) gene. It has a high affinity for folate and reduced folate derivatives and mediates the delivery of 5-methyltetrahydrofolate and folate analogues into cells. FRα plays a crucial role in embryonic development and is also an accomplice to tumor cells. After binding to folate, FRα mediates downstream signaling pathways, regulating signals related to tumor cell growth, development, and division, and promoting tumor cell proliferation and metastasis.

[0004] FRα is almost not expressed in normal cells, but it is widely and highly expressed in solid tumors, such as mesothelioma (72-100%), triple-negative breast cancer (35-68%), ovarian cancer (76-89%), and non-small cell lung cancer (14-74%), and is involved in tumor invasion, metastasis, and development. While FRα expression levels are low in normal cells, they are significantly elevated in certain cancer cells, thus making it an important target for cancer diagnosis and treatment in recent years.

[0005] Currently, investigational FRα antibody-drug conjugates include IMGN853 (US20220160889A1), STRO-002 (CN111655726A), MORAb202 (patent US10322192B2), and the biepisode antibody-drug conjugate IMGN151 (US20200362029A1). These specific drugs have been evaluated in clinical trials in cancer patients with intermediate to high FRα levels, showing positive efficacy in patients with high FRα expression, but have limitations in achieving progression-free survival in a broader cancer patient population. Therefore, there is still a need for folate receptor-targeted ADC drugs with higher ADC delivery capabilities to achieve more effective therapeutic goals. Summary of the Invention

[0006] To provide patients with more diverse treatment options, this disclosure discloses antibodies or antigen-binding fragments thereof that recognize FRα with higher affinity, and compared to investigational drugs, the antibody-drug conjugates of this disclosure have a greater ability to kill tumor cells in vitro. Furthermore, in vivo animal experimental data confirm that the antibody-drug conjugates obtained in this disclosure can more effectively inhibit the proliferation of tumor cells.

[0007] In a first aspect, this disclosure provides an anti-FRα antibody or its antigen-binding fragment, comprising a heavy chain variable region, said heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein:

[0008] (1) The sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.1; the sequence of HCDR2 is the sequence shown in SEQ ID NO.2 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.2; and the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.3.

[0009] (2) The sequence of HCDR1 is the sequence shown in SEQ ID NO.9 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.9; the sequence of HCDR2 is the sequence shown in SEQ ID NO.10 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.10; and the sequence of HCDR3 is the sequence shown in SEQ ID NO.11 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.11.

[0010] (3) The sequence of HCDR1 is the sequence shown in SEQ ID NO.17 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.17; the sequence of HCDR2 is the sequence shown in SEQ ID NO.18 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.18; and the sequence of HCDR3 is the sequence shown in SEQ ID NO.19 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.19.

[0011] (4) The sequence of HCDR1 is the sequence shown in SEQ ID NO.25 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.25; the sequence of HCDR2 is the sequence shown in SEQ ID NO.26 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.26; and the sequence of HCDR3 is the sequence shown in SEQ ID NO.27 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.27.

[0012] (5) The sequence of HCDR1 is the sequence shown in SEQ ID NO.32 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.32; the sequence of HCDR2 is the sequence shown in SEQ ID NO.33 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.33; and the sequence of HCDR3 is the sequence shown in SEQ ID NO.34 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.34.

[0013] (6) The sequence of HCDR1 is the sequence shown in SEQ ID NO.40 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.40; the sequence of HCDR2 is the sequence shown in SEQ ID NO.41 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.41; and the sequence of HCDR3 is the sequence shown in SEQ ID NO.42 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.42.

[0014] (7) The sequence of HCDR1 is the sequence shown in SEQ ID NO.48 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.48; the sequence of HCDR2 is the sequence shown in SEQ ID NO.49 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.49; and the sequence of HCDR3 is the sequence shown in SEQ ID NO.50 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.50.

[0015] (8) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 56 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 56; the sequence of HCDR2 is the sequence shown in SEQ ID NO. 57 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 57; and the sequence of HCDR3 is the sequence shown in SEQ ID NO. 58 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 58; or

[0016] (9) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 64 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 64; the sequence of HCDR2 is the sequence shown in SEQ ID NO. 65 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 65; and the sequence of HCDR3 is the sequence shown in SEQ ID NO. 34 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 34.

[0017] Secondly, this disclosure provides an anti-FRα antibody or its antigen-binding fragment, including a light chain variable region, wherein the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein:

[0018] (1) The sequence of LCDR1 is the sequence shown in SEQ ID NO.4 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.4; the sequence of LCDR2 is the sequence shown in SEQ ID NO.5 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.5; and the sequence of LCDR3 is the sequence shown in SEQ ID NO.6 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.6.

[0019] (2) The sequence of LCDR1 is the sequence shown in SEQ ID NO.12 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.12; the sequence of LCDR2 is the sequence shown in SEQ ID NO.13 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.13; and the sequence of LCDR3 is the sequence shown in SEQ ID NO.14 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.14.

[0020] (3) The sequence of LCDR1 is the sequence shown in SEQ ID NO.20 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.20; the sequence of LCDR2 is the sequence shown in SEQ ID NO.21 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.21; and the sequence of LCDR3 is the sequence shown in SEQ ID NO.22 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.22.

[0021] (4) The sequence of LCDR1 is the sequence shown in SEQ ID NO.28 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.28; the sequence of LCDR2 is the sequence shown in SEQ ID NO.21 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.21; and the sequence of LCDR3 is the sequence shown in SEQ ID NO.29 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.29.

[0022] (5) The sequence of LCDR1 is the sequence shown in SEQ ID NO.35 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.35; the sequence of LCDR2 is the sequence shown in SEQ ID NO.36 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.36; and the sequence of LCDR3 is the sequence shown in SEQ ID NO.37 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.37.

[0023] (6) The sequence of LCDR1 is the sequence shown in SEQ ID NO.43 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.43; the sequence of LCDR2 is the sequence shown in SEQ ID NO.44 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.44; and the sequence of LCDR3 is the sequence shown in SEQ ID NO.45 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.45.

[0024] (7) The sequence of LCDR1 is the sequence shown in SEQ ID NO.51 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.51; the sequence of LCDR2 is the sequence shown in SEQ ID NO.52 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.52; and the sequence of LCDR3 is the sequence shown in SEQ ID NO.53 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.53.

[0025] (8) The sequence of LCDR1 is the sequence shown in SEQ ID NO. 59 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 59; the sequence of LCDR2 is the sequence shown in SEQ ID NO. 60 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 60; and the sequence of LCDR3 is the sequence shown in SEQ ID NO. 61 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 61; or

[0026] (9) The sequence of LCDR1 is the sequence shown in SEQ ID NO.35 or has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.35; the sequence of LCDR2 is the sequence shown in SEQ ID NO.36 or has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.36; and the sequence of LCDR3 is the sequence shown in SEQ ID NO.66 or has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.66.

[0027] Thirdly, this disclosure provides an anti-FRα antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein:

[0028] (1) The sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.1; the sequence of HCDR2 is the sequence shown in SEQ ID NO.2 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.2; the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.3; the sequence of LCDR1 is the sequence shown in SEQ ID NO.4 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.4; and the sequence of LCDR2 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.4. The sequence shown in NO.5 has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.6, and the sequence of LCDR3 is the sequence shown in SEQ ID NO.6 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.6;

[0029] (2) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 9 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 9; the sequence of HCDR2 is the sequence shown in SEQ ID NO. 10 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 10; the sequence of HCDR3 is the sequence shown in SEQ ID NO. 11 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 11; the sequence of LCDR1 is the sequence shown in SEQ ID NO. 12 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 12; and the sequence of LCDR2 is the sequence shown in SEQ ID NO. 13 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 10. The sequence shown in NO.13 has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.14, and the sequence of LCDR3 is the sequence shown in SEQ ID NO.14 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.14;

[0030] (3) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 17 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 17; the sequence of HCDR2 is the sequence shown in SEQ ID NO. 18 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 18; the sequence of HCDR3 is the sequence shown in SEQ ID NO. 19 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 19; the sequence of LCDR1 is the sequence shown in SEQ ID NO. 20 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 20; and the sequence of LCDR2 is the sequence shown in SEQ ID NO. 21 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 17. The sequence shown in NO.21 has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.22, and the sequence of LCDR3 is the sequence shown in SEQ ID NO.22 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.22;

[0031] (4) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 25 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 25; the sequence of HCDR2 is the sequence shown in SEQ ID NO. 26 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 26; the sequence of HCDR3 is the sequence shown in SEQ ID NO. 27 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 27; the sequence of LCDR1 is the sequence shown in SEQ ID NO. 28 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 28; and the sequence of LCDR2 is the sequence shown in SEQ ID NO. 21 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 25. The sequence shown in NO.21 has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.29, and the sequence of LCDR3 is the sequence shown in SEQ ID NO.29 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.29;

[0032] (5) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 32 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 32; the sequence of HCDR2 is the sequence shown in SEQ ID NO. 33 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 33; the sequence of HCDR3 is the sequence shown in SEQ ID NO. 34 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 34; the sequence of LCDR1 is the sequence shown in SEQ ID NO. 35 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 35; and the sequence of LCDR2 is the sequence shown in SEQ ID NO. 36 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 32. The sequence shown in NO.36 has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.37, and the sequence of LCDR3 is the sequence shown in SEQ ID NO.37 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.37;

[0033] (6) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 40 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 40; the sequence of HCDR2 is the sequence shown in SEQ ID NO. 41 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 41; the sequence of HCDR3 is the sequence shown in SEQ ID NO. 42 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 42; the sequence of LCDR1 is the sequence shown in SEQ ID NO. 43 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 43; and the sequence of LCDR2 is the sequence shown in SEQ ID NO. 44 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 43. The sequence shown in NO.44 has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.45, and the sequence of LCDR3 is the sequence shown in SEQ ID NO.45 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.45;

[0034] (7) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 48 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 48; the sequence of HCDR2 is the sequence shown in SEQ ID NO. 49 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 49; the sequence of HCDR3 is the sequence shown in SEQ ID NO. 50 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 50; the sequence of LCDR1 is the sequence shown in SEQ ID NO. 51 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 51; and the sequence of LCDR2 is the sequence shown in SEQ ID NO. 52 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 48. The sequence shown in NO.52 has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.53, and the sequence of LCDR3 is the sequence shown in SEQ ID NO.53 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.53;

[0035] (8) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 56 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 56; the sequence of HCDR2 is the sequence shown in SEQ ID NO. 57 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 57; the sequence of HCDR3 is the sequence shown in SEQ ID NO. 58 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 58; the sequence of LCDR1 is the sequence shown in SEQ ID NO. 59 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 59; and the sequence of LCDR2 is the sequence shown in SEQ ID NO. 60 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 59. The sequence shown in SEQ ID NO. 60 has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 61, and the sequence of LCDR3 is the sequence shown in SEQ ID NO. 61 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 61; or

[0036] (9) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 64 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 64; the sequence of HCDR2 is the sequence shown in SEQ ID NO. 65 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 65; the sequence of HCDR3 is the sequence shown in SEQ ID NO. 34 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 34; the sequence of LCDR1 is the sequence shown in SEQ ID NO. 35 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 35; and the sequence of LCDR2 is the sequence shown in SEQ ID NO. 36 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 64. The sequence shown in NO.36 has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.66, and the sequence of LCDR3 is the sequence shown in SEQ ID NO.66 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.66.

[0037] In this disclosure, the CDR sequence is defined according to Kabat.

[0038] The anti-FRα antibody or its antigen-binding fragment described in this disclosure is a murine, chimeric, or humanized antibody or its antigen-binding fragment. Preferably, the anti-FRα antibody or its antigen-binding fragment described in this disclosure is a humanized antibody or its antigen-binding fragment.

[0039] Fourthly, this disclosure provides an anti-FRα antibody or an antigen-binding fragment thereof, wherein the anti-FRα antibody or the antigen-binding fragment thereof includes a heavy chain variable region, wherein:

[0040] (1) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 69 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 69;

[0041] (2) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.71 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.71;

[0042] (3) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.73 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.73;

[0043] (4) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.75 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.75;

[0044] (5) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.77 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.77;

[0045] (6) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.79 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.79;

[0046] (7) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.81 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.81;

[0047] (8) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.7 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.7;

[0048] (9) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.15 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.15;

[0049] (10) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.23 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.23;

[0050] (11) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.30 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.30;

[0051] (12) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.38 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.38;

[0052] (13) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.46 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.46;

[0053] (14) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.54 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.54;

[0054] (15) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 62 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 62; or

[0055] (16) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.67 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.67.

[0056] Fifthly, this disclosure provides an anti-FRα antibody or an antigen-binding fragment thereof, wherein the anti-FRα antibody or the antigen-binding fragment thereof includes a light chain variable region, wherein:

[0057] (1) The light chain variable region sequence is the sequence shown in SEQ ID NO.70 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.70;

[0058] (2) The light chain variable region sequence is the sequence shown in SEQ ID NO.72 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.72;

[0059] (3) The light chain variable region sequence is the sequence shown in SEQ ID NO.74 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.74;

[0060] (4) The light chain variable region sequence is the sequence shown in SEQ ID NO.76 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.76;

[0061] (5) The light chain variable region sequence is the sequence shown in SEQ ID NO.78 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.78;

[0062] (6) The light chain variable region sequence is the sequence shown in SEQ ID NO.80 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.80;

[0063] (7) The light chain variable region sequence is the sequence shown in SEQ ID NO.82 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.82;

[0064] (8) The light chain variable region sequence is the sequence shown in SEQ ID NO.8 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.8;

[0065] (9) The light chain variable region sequence is the sequence shown in SEQ ID NO.16 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.16;

[0066] (10) The light chain variable region sequence is the sequence shown in SEQ ID NO.24 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.24;

[0067] (11) The light chain variable region sequence is the sequence shown in SEQ ID NO.31 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.31;

[0068] (12) The light chain variable region sequence is the sequence shown in SEQ ID NO.39 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.39;

[0069] (13) The light chain variable region sequence is the sequence shown in SEQ ID NO.47 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.47;

[0070] (14) The light chain variable region sequence is the sequence shown in SEQ ID NO.55 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.55;

[0071] (15) The light chain variable region sequence is the sequence shown in SEQ ID NO. 63 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 63; or

[0072] (16) The light chain variable region sequence is the sequence shown in SEQ ID NO.68 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.68.

[0073] Sixthly, this disclosure provides an anti-FRα antibody or an antigen-binding fragment thereof, wherein the anti-FRα antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:

[0074] (1) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 69 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 69, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 70 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 70;

[0075] (2) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.71 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.71, and the light chain variable region sequence is the sequence shown in SEQ ID NO.72 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.72;

[0076] (3) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.73 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.73, and the light chain variable region sequence is the sequence shown in SEQ ID NO.74 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.74;

[0077] (4) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.75 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.75, and the light chain variable region sequence is the sequence shown in SEQ ID NO.76 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.76;

[0078] (5) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.77 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.77, and the light chain variable region sequence is the sequence shown in SEQ ID NO.78 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.78;

[0079] (6) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.79 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.79, and the light chain variable region sequence is the sequence shown in SEQ ID NO.80 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.80;

[0080] (7) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 81 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 81; the light chain variable region sequence is the sequence shown in SEQ ID NO. 82 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 82; or

[0081] (8) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.7 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.7, and the light chain variable region sequence is the sequence shown in SEQ ID NO.8 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.8;

[0082] (9) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.15 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.15, and the light chain variable region sequence is the sequence shown in SEQ ID NO.16 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.16;

[0083] (10) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.23 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.23, and the light chain variable region sequence is the sequence shown in SEQ ID NO.24 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.24;

[0084] (11) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.30 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.30, and the light chain variable region sequence is the sequence shown in SEQ ID NO.31 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.31;

[0085] (12) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.38 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.38, and the light chain variable region sequence is the sequence shown in SEQ ID NO.39 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.39;

[0086] (13) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.46 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.46, and the light chain variable region sequence is the sequence shown in SEQ ID NO.47 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.47;

[0087] (14) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.54 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.54, and the light chain variable region sequence is the sequence shown in SEQ ID NO.55 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.55;

[0088] (15) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 62 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 62; the light chain variable region sequence is the sequence shown in SEQ ID NO. 63 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 63; or

[0089] (16) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 67 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 67, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 68 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 68.

[0090] In some embodiments of aspects one through six, the anti-FRα antibody or its antigen-binding fragment specifically binds to human FRα. In some specific embodiments, the anti-FRα antibody or its antigen-binding fragment binds to a conserved FRα protein epitope from different species (e.g., between human and cynomolgus monkey FRα).

[0091] In this disclosure, the antigen-binding fragment is Fv, VHH, scFv, Fab, Fab', or (Fab')2.

[0092] In some embodiments of aspects one through six, the anti-FRα antibody or its antigen-binding fragment has ADCC activity.

[0093] In some embodiments of aspects one through six, the anti-FRα antibody or its antigen-binding fragment has CDC activity.

[0094] In some embodiments of aspects one through six, the anti-FRα antibody or its antigen-binding fragment has ADCP activity.

[0095] In some implementations of aspects one through six, the anti-FRα antibody is a full-length antibody.

[0096] In some embodiments of aspects one through six, the anti-FRα antibody is a monoclonal antibody.

[0097] In some embodiments of aspects one through six, the anti-FRα antibody is an IgM, IgD, IgG, IgA, or IgE antibody. In some specific embodiments, the anti-FRα antibody is an IgG antibody. In some more specific embodiments, the anti-FRα antibody is an IgG1 antibody.

[0098] In some embodiments of aspects one through six, the anti-FRα antibody is an isotype of IgG1, IgG2, or IgG4.

[0099] In some embodiments of aspects one through six, the anti-FRα antibody comprises a light chain constant region of the κ or λ subtype.

[0100] In some embodiments of aspects one through six, the anti-FRα antibody comprises a human IgG1 heavy chain constant region and a human κ light chain constant region.

[0101] In some embodiments of aspects one through six, the anti-FRα antibody comprises a wild-type Fc region.

[0102] In some embodiments of the first to sixth aspects, the anti-FRα antibody or its antigen-binding fragment is engineered to have enhanced ADCC, CDC, and / or ADCP activities. In some specific embodiments, the anti-FRα antibody includes an engineered Fc region, said engineered Fc region causing the antibody to have enhanced ADCC, CDC, and / or ADCP effects.

[0103] In some embodiments of aspects one through six, the anti-FRα antibody or its antigen-binding fragment is engineered to have attenuated ADCC, CDC, and / or ADCP activities. In some specific embodiments, the anti-FRα antibody comprises an engineered Fc region, said engineered Fc region causing the antibody to have attenuated ADCC, CDC, and / or ADCP effects. For the target FRα of this disclosure and for the in vivo application of the antibody, antibodies with attenuated or even eliminated ADCC, CDC, and / or ADCP activities may be advantageous, as an excessively strong Fc effect may affect or even weaken the efficacy of the antibody. Methods for attenuating or eliminating the Fc effect are known in the art, such as 1) amino acid residue mutation modification (primarily attenuating binding to the relevant receptor), 2) glycosylation modification, 3) IgG4 type antibody modification, etc.

[0104] In this disclosure, as a non-limiting example, the Fc region of the anti-FRα antibody may contain an amino acid mutation that reduces ADCC, CDC, and / or ADCP effects; preferably, the mutation is selected from one or more of IgG1 L234A, L235A, P329A, P329G, or P331S, more preferably, the mutation is IgG1 L234A-L235A-P329A-P331S or IgG1 L234A-L235A-P329G.

[0105] In a seventh aspect, this disclosure provides isolated nucleic acid molecules that encode the anti-FRα antibodies or antigen-binding fragments thereof described in the first to sixth aspects.

[0106] In some embodiments, this disclosure provides combinations of isolated polynucleotides comprising a polynucleotide encoding a light chain of an antibody of the present disclosure or an antigen-binding fragment thereof, and a polynucleotide encoding a heavy chain of an antibody of the present disclosure or an antigen-binding fragment thereof. In some embodiments, the polynucleotide is operatively linked to a regulatory sequence that can be recognized by host cells transformed with the vector.

[0107] Eighthly, this disclosure provides a carrier containing the nucleic acid molecules described in the seventh aspect.

[0108] In some embodiments, the expression vectors of this disclosure comprise the nucleic acid molecules or combinations of polynucleotides described herein, wherein the polynucleotides are efficiently linked to a regulatory sequence that allows the polypeptides they encode to be expressed in host cells or cell-free expression systems. The choice of expression vector depends on the choice of host cell and can be selected to achieve the desired expression and regulatory characteristics in the chosen host cell.

[0109] The nucleic acid in the vector can be operatively linked to one or more expression control sequences. As used herein, "operatively linked" means incorporated into the genetic construct such that the expression control sequence effectively controls the expression of the target coding sequence. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. A promoter is an expression control sequence consisting of a region of the DNA molecule typically located within 100 nucleotides upstream of the transcription start site (usually near the start site of RNA polymerase II). For the coding sequence to be under the control of the promoter, the translation start site of the polypeptide translation reading frame must be located between 1 and 50 nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function at different distances from the transcription start site. Enhancers can also be located downstream of the transcription start site. When RNA polymerase is able to transcribe the coding sequence into mRNA, and then the mRNA can be translated into the protein encoded by the coding sequence, the coding sequence is "operatively linked" to and "under the control" of the expression control sequence in the cell.

[0110] Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophages, baculoviruses, tobacco mosaic virus, herpesviruses, cytomegaloviruses, retroviruses, vaccinia virus, adenoviruses, and adeno-associated viruses. Many vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (LaJolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0111] Expression vectors may include tag sequences. Tag sequences are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted at any position within the polypeptide, including the carboxyl or amino terminus. Examples of useful tags include, but are not limited to, Fc fragments, polyhistidine, green fluorescent protein (GFP), glutathione S-transferase (GST), c-myc, hemagglutinin, Flag™ tags (Kodak, New Haven, CT), maltose E-binding protein, and protein A. In some embodiments, the nucleic acid molecule encoding the FRα fusion polypeptide is contained in a vector of nucleic acid containing one or more domains encoding the constant region of the Ig heavy chain, such as amino acid sequences corresponding to the hinge region, CH2 region, and CH3 region of the human immunoglobulin Cγ1 chain (Fc fragment).

[0112] Ninthly, this disclosure provides a host cell containing the nucleic acid molecule described in the seventh aspect or the vector described in the eighth aspect.

[0113] In some embodiments, the host cell can be a prokaryotic host cell, a eukaryotic host cell, or a bacteriophage. Prokaryotic host cells can be *Escherichia coli*, *Bacillus subtilis*, *Streptomyces*, or *Proteus mirabilis*, etc. Eukaryotic host cells can be fungi such as *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharomyces cerevisiae*, and *Trichoderma*, insect cells such as armyworms, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the host cell is preferably a mammalian cell, more preferably a BHK cell, CHO cell, NSO cell, or COS cell.

[0114] In a tenth aspect, this disclosure provides a bispecific antibody having a first antigen-binding site and a second antigen-binding site, wherein one antigen-binding site comprises an anti-FRα antibody or an antigen-binding fragment thereof as described in the first to sixth aspects; preferably, the first antigen-binding site of the bispecific antibody comprises an anti-FRα antibody or an antigen-binding fragment thereof as described in the first to sixth aspects, and the second antigen-binding site comprises another anti-FRα antibody or an antigen-binding fragment thereof as described in the first to sixth aspects; more preferably, the first antigen-binding site and the second antigen-binding site of the bispecific antibody bind to different epitopes of the same antigen FRα.

[0115] In its eleventh aspect, this disclosure provides a bispecific antibody, wherein,

[0116] (1) The first antigen binding site includes a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO.1 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.1; the HCDR2 sequence is the sequence shown in SEQ ID NO.2 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.2; and the HCDR3 sequence is the sequence shown in SEQ ID NO.3 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.3. In the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO.4 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.4; and the LCDR2 sequence is the sequence shown in SEQ ID NO.5 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.4. The sequence shown in SEQ ID NO. 5 has at least 85%, 90%, 95%, 98%, or 99% identity, and the LCDR3 sequence is the sequence shown in SEQ ID NO. 6 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 6; the second antigen binding site includes a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO. 9 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 9, the HCDR2 sequence is the sequence shown in SEQ ID NO. 10 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 10, and the HCDR3 sequence is the sequence shown in SEQ ID NO. 11 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 10. The sequence shown in NO. 11 has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence. In the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO. 12 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 12; the LCDR2 sequence is the sequence shown in SEQ ID NO. 13 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 13; and the LCDR3 sequence is the sequence shown in SEQ ID NO. 14 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 14.

[0117] (2) The first antigen binding site includes a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO.40 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.40; the HCDR2 sequence is the sequence shown in SEQ ID NO.41 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.41; and the HCDR3 sequence is the sequence shown in SEQ ID NO.42 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.42. In the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO.43 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.43; and the LCDR2 sequence is the sequence shown in SEQ ID NO.44 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.43. The sequence shown in SEQ ID NO. 44 has at least 85%, 90%, 95%, 98%, or 99% identity, and the LCDR3 sequence is the sequence shown in SEQ ID NO. 45 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 45; the second antigen binding site includes a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO. 32 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 32, the HCDR2 sequence is the sequence shown in SEQ ID NO. 33 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 33, and the HCDR3 sequence is the sequence shown in SEQ ID NO. 34 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 33, and the LCDR3 sequence is the sequence shown in SEQ ID NO. 34 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 45; The sequence shown in NO. 34 has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 35. In the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO. 35 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 35; the LCDR2 sequence is the sequence shown in SEQ ID NO. 36 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 36; and the LCDR3 sequence is the sequence shown in SEQ ID NO. 37 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 37.

[0118] (3) The first antigen binding site includes a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO. 64 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 64; the HCDR2 sequence is the sequence shown in SEQ ID NO. 65 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 65; and the HCDR3 sequence is the sequence shown in SEQ ID NO. 34 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 34. In the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO. 35 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 35; and the LCDR2 sequence is the sequence shown in SEQ ID NO. 36 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 64. The sequence shown in SEQ ID NO. 36 has at least 85%, 90%, 95%, 98%, or 99% identity, and the LCDR3 sequence is the sequence shown in SEQ ID NO. 66 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 66; the second antigen binding site includes a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO. 40 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 40, the HCDR2 sequence is the sequence shown in SEQ ID NO. 41 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 41, and the HCDR3 sequence is the sequence shown in SEQ ID NO. 42 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 41. The sequences shown in SEQ ID NO. 42 have at least 85%, 90%, 95%, 98%, or 99% identity. Within the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO. 43 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 43; the LCDR2 sequence is the sequence shown in SEQ ID NO. 44 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 44; and the LCDR3 sequence is the sequence shown in SEQ ID NO. 45 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 45.

[0119] Preferably, the antibody is a humanized antibody.

[0120] Furthermore, this disclosure provides a bispecific antibody, wherein,

[0121] (1) The first antigen-binding site includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 69 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 69, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 70 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 70; the second antigen-binding site includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 71 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 71, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 72 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 72; or

[0122] (2) The first antigen-binding site includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 77 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 77, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 78 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 78; the second antigen-binding site includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 75 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 75, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 76 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 76.

[0123] (3) The first antigen binding site includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 81 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 81, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 82 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 82; the second antigen binding site includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 77 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 77, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 78 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 78.

[0124] Preferably, the bispecific antibody disclosed herein is an IgG-like bispecific antibody, wherein the left arm is the first antigen binding site and the right arm is the second antigen binding site.

[0125] In one embodiment, the first antigen-binding site of the bispecific antibody disclosed herein comprises a first heavy chain and a first light chain; the second antigen-binding site comprises a second heavy chain and a second light chain.

[0126] In one embodiment, the first antigen-binding site of the bispecific antibody disclosed herein comprises a first heavy chain and a first light chain; the second antigen-binding site comprises an scFv-Fc chain.

[0127] In one embodiment, the bispecific antibody of this disclosure is a fusion protein of an IgG-like antibody with an N-terminal (amino-terminus) or C-terminal (carboxyl-terminus) scFv fragment linked to it; preferably, the IgG-like antibody targets a first antigen, and the scFv fragment linked to the N-terminus (amino-terminus) or C-terminus (carboxyl-terminus) targets a second antigen; preferably, the scFv fragment is linked to the IgG-like antibody via a linker; more preferably, the linker is a G... n S m , where G is glycine, S is serine, n is an integer selected from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and m is an integer selected from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0128] Preferably, the heavy chain variable region and / or light chain variable region of the scFv fragment in this disclosure contain a mutation to cysteine ​​(Cys); preferably, the mutation to Cys is located at position 44 of the heavy chain region and / or position 100 or 101 of the light chain variable region; more preferably, the mutation to Cys in the heavy chain variable region is G44C, and / or the mutation to Cys in the light chain variable region is Q100C or Q101C.

[0129] In one embodiment, the constant region of the bispecific antibody is the constant region of the IgG1 antibody.

[0130] Preferably, the Fc region of the bispecific antibody described in this disclosure contains an amino acid mutation that reduces ADCC, CDC and / or ADCP effects, wherein the mutation is selected from one or more mutations of IgG1 L234A, L235A, P329G, P329A, and P331S; more preferably, the mutation is L234A-L235A-P329A-P331S or L234A-L235A-P329G.

[0131] More preferably, the Fc region of the bispecific antibody described in this disclosure includes amino acid mutations in the CH3 region that facilitate the purification of the bispecific antibody, forming a mortar and / or a sac; more preferably, the mutations forming a sac are Y349C, T366S, L368A and Y407V, and the mutations forming a mortar are S354C and T366W.

[0132] Preferably, the bispecific antibody described in this disclosure carries CH1-CL chain exchange between the first heavy chain and the first light chain or between the second heavy chain and the second light chain.

[0133] More preferably, this disclosure provides a bispecific antibody, wherein,

[0134] (1) The bispecific antibody comprises a first heavy chain, a second heavy chain, a first light chain and a second light chain, wherein the amino acid sequence of the first heavy chain is shown in SEQ ID NO.98, the amino acid sequence of the second heavy chain is shown in SEQ ID NO.100, the amino acid sequence of the first light chain is shown in SEQ ID NO.96, and the amino acid sequence of the second light chain is shown in SEQ ID NO.102.

[0135] (2) The bispecific antibody is a fusion protein of an IgG-like antibody with an N-terminal (amino-terminus) or C-terminal (carboxyl-terminus) scFv fragment linked to it, comprising two identical heavy chains linked to the scFv fragment and two identical light chains, wherein the amino acid sequence of the heavy chain linked to the scFv fragment is as shown in SEQ ID NO. 92 or 97, and the amino acid sequence of the light chain is as shown in SEQ ID NO. 96; or

[0136] (3) The first antigen-binding site of the bispecific antibody includes one heavy chain and one light chain, and the second antigen-binding site of the bispecific antibody is an scFv-Fc chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO.105, the amino acid sequence of the light chain is shown in SEQ ID NO.96, and the amino acid sequence of the scFv-Fc chain is shown in SEQ ID NO.104; or the amino acid sequence of the heavy chain is shown in SEQ ID NO.88, the amino acid sequence of the light chain is shown in SEQ ID NO.90, and the amino acid sequence of the scFv-Fc chain is shown in SEQ ID NO.83; or the amino acid sequence of the heavy chain is shown in SEQ ID NO.110, the amino acid sequence of the light chain is shown in SEQ ID NO.112, and the amino acid sequence of the scFv-Fc chain is shown in SEQ ID NO.106.

[0137] In a twelfth aspect, this disclosure provides a nucleic acid molecule encoding the aforementioned bispecific antibody.

[0138] In some embodiments, this disclosure provides combinations of isolated polynucleotides comprising a polynucleotide encoding a light chain of an antibody of the present disclosure or an antigen-binding fragment thereof, and a polynucleotide encoding a heavy chain of an antibody of the present disclosure or an antigen-binding fragment thereof. In some embodiments, the polynucleotide is operatively linked to a regulatory sequence that can be recognized by host cells transformed with the vector.

[0139] In a thirteenth aspect, this disclosure provides an expression vector comprising the nucleic acid molecule described in the twelfth aspect.

[0140] In some embodiments, the expression vectors of this disclosure comprise the nucleic acid molecules or combinations of polynucleotides described herein, wherein the polynucleotides are efficiently linked to a regulatory sequence that allows the polypeptides they encode to be expressed in host cells or cell-free expression systems. The choice of expression vector depends on the choice of host cell and can be selected to achieve the desired expression and regulatory characteristics in the chosen host cell.

[0141] The nucleic acid in the vector can be operatively linked to one or more expression control sequences. As used herein, "operatively linked" means incorporated into the genetic construct such that the expression control sequence effectively controls the expression of the target coding sequence. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. A promoter is an expression control sequence consisting of a region of the DNA molecule typically located within 100 nucleotides upstream of the transcription start site (usually near the start site of RNA polymerase II). For the coding sequence to be under the control of the promoter, the translation start site of the polypeptide translation reading frame must be located between 1 and 50 nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function at different distances from the transcription start site. Enhancers can also be located downstream of the transcription start site. When RNA polymerase is able to transcribe the coding sequence into mRNA, and then the mRNA can be translated into the protein encoded by the coding sequence, the coding sequence is "operatively linked" to and "under the control" of the expression control sequence in the cell.

[0142] Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophages, baculoviruses, tobacco mosaic virus, herpesviruses, cytomegaloviruses, retroviruses, vaccinia virus, adenoviruses, and adeno-associated viruses. Many vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (LaJolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0143] Expression vectors may include tag sequences. Tag sequences are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted at any position within the polypeptide, including the carboxyl or amino terminus. Examples of useful tags include, but are not limited to, Fc fragments, polyhistidine, green fluorescent protein (GFP), glutathione S-transferase (GST), c-myc, hemagglutinin, Flag™ tags (Kodak, New Haven, CT), maltose E-binding protein, and protein A. In some embodiments, the nucleic acid molecule encoding the FRα fusion polypeptide is contained in a vector of nucleic acid containing one or more domains encoding the constant region of the Ig heavy chain, such as amino acid sequences corresponding to the hinge region, CH2 region, and CH3 region of the human immunoglobulin Cγ1 chain (Fc fragment).

[0144] In a fourteenth aspect, this disclosure provides a host cell comprising the expression vector described in the thirteenth aspect.

[0145] In some embodiments, the host cell can be a prokaryotic host cell, a eukaryotic host cell, or a bacteriophage. Prokaryotic host cells can be *Escherichia coli*, *Bacillus subtilis*, *Streptomyces*, or *Proteus mirabilis*, etc. Eukaryotic host cells can be fungi such as *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharomyces cerevisiae*, and *Trichoderma*, insect cells such as armyworms, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the host cell is preferably a mammalian cell, more preferably a BHK cell, CHO cell, NSO cell, or COS cell.

[0146] In a fifteenth aspect, this disclosure provides antibody-drug conjugates comprising the aforementioned anti-FRα antibody or its antigen-binding fragment or the aforementioned bispecific antibody, as well as tautomers, stereoisomers or pharmaceutically acceptable salts thereof.

[0147] Preferably, the antibody-drug conjugate is obtained by conjugating a therapeutic agent to the aforementioned anti-FRα antibody or its antigen-binding fragment or the aforementioned bispecific antibody.

[0148] In this disclosure, the therapeutic agent may be an antitumor drug, such as a cytotoxic drug, an immune enhancer, or a radioactive isotope.

[0149] In some implementations, the types of cytotoxic drugs include microtubule inhibitors, DNA topoisomerase inhibitors, DNA damaging agents, antimetabolites, or antitumor antibiotics.

[0150] In some implementations, microtubule inhibitors include, but are not limited to, orrisstatin derivatives (e.g., MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F)) or maytansine alkaloid derivatives (e.g., DM1, DM4, ansamitocin, maytansine, or dolastatin and its derivatives).

[0151] In some embodiments, the DNA topoisomerase inhibitor is a camptothecin analog or a DNA topoisomerase I inhibitor and its derivatives, such as DXD, SN38, irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, 22-hydroxyeclipticine, topotecan, letopotecan, belotetane, eczetane, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-pyranoxylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3, 4-Dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-acrylamide, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxyphenylpropyl)-(E)-2-acrylamide, 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acrylamide dihydrochloride, N-[2-(dimethylamino)ethyl]-4-acrylamide.

[0152] In some implementations, DNA damaging agents include, but are not limited to, calicheamicin, duocarmycin, and pyrrolobenzodiazepine (PBD) derivatives of astromycin.

[0153] In some implementations, antimetabolites include, but are not limited to, methotrexate, 6-mercaptopurine, or 5-fluorouracil.

[0154] In some implementations, antitumor antibiotics include, but are not limited to, peptide antibiotics (such as actinomycin D or bleomycin) or anthraquinone drugs (such as doxorubicin or mitoxantrone hydrochloride).

[0155] In some implementations, the immune enhancer includes, but is not limited to, levamisole, pidotimod, imiquimod, isoproinosine, polyinosinic-cytosine, or polyinosinuric acid.

[0156] In some embodiments, the radionuclide is a diagnostic radionuclide or a therapeutic radionuclide. According to embodiments of this disclosure, the diagnostic radionuclide is at least one selected from 68Ga, 64Cu, 18F, 86Y, 89Zr, 111In, 99mTc, 11C, 123I, 125I, and 124I. According to embodiments of this disclosure, the therapeutic radionuclide is at least one selected from 177Lu, 90Y, 125I, 131I, 211At, 111In, 153Sm, 186Re, 188Re, 67Cu, 225Ac, 213Bi, 212Bi, and 212Pb.

[0157] In some embodiments, the antibody-drug conjugates of this disclosure have the structure shown in formula (I):

[0158] A-(LD) d (I)

[0159] Wherein A is the anti-FRα antibody or its antigen-binding fragment as described in the first to sixth aspects of this disclosure, or the bispecific antibody as described in the tenth to eleventh aspects of this disclosure;

[0160] L is the connecting sub-part, with one end connected to A and the other end connected to the therapeutic agent D;

[0161] d represents the molar ratio of the therapeutic agent to A (also known as DAR, or drug-antibody conjugate ratio), selected from integers or decimals from 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0162] When d is a decimal, it refers to the average number of connector-therapeutic agents (LDs) per A-joint.

[0163] In some embodiments, A is selected from antibodies Cab-48C6C6H5, Cab-51F8C10C10, Cab-52E3B5G2, Cab-60E8C3F5, Cab-66D5GSE6, Cab-66H5D4E6, Cab-68G7A4B2, Cab-71F11D9D9, Cab-82C11E8F4, Hab48C6C6H5, and Hab51F8. C10C10, Hab52E3B5G2, Hab66D5GSE6, Hab66H5D4E6, Hab71F11D9D9, Hab82C11E8F4, 66H5+66D 5(S1), 51F8+48C6(S4), 51F8+48C6(S5), 51F8+48C6(S6), 51F8+48C6(S7) and 82C11+66H5(S8).

[0164] In some embodiments, the anti-FRα antibody or its antigen-binding fragment of the present disclosure is covalently linked to a therapeutic agent via a linker.

[0165] In some implementations, L is a cuttable connector.

[0166] In some implementations, L can be cleaved under intracellular conditions or under microenvironmental conditions.

[0167] In one implementation, L is hydrolyzable at a pH less than 5.5.

[0168] In some implementations, L can be cleaved by intracellular proteases.

[0169] In some implementations, L is a cathepsin-cleavable linker.

[0170] In some implementations, L includes a dipeptide or a tetrapeptide.

[0171] In some embodiments, the dipeptide is selected from valine-citrulline (Cit) and valine-alanine (Ala), and the tetrapeptide is selected from glycine-glycine-phenylalanine (Phe)-glycine (Gly).

[0172] In some implementations, the antibody is linked to the linker via the antibody's cysteine ​​thiol group.

[0173] In one embodiment, the antibody is linked to a linker via the amino group of the antibody (specifically, the amino group of a glutamine residue).

[0174] In some embodiments, L is a cleavable linker, including, but not limited to, acid-labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers containing amino acids, such as valine and / or citrulline, such as citrulline-valine or phenylalanine-lysine), light-labile linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to avoid resistance mediated by multidrug transporters.

[0175] In some embodiments, -L- is selected from -L1-L2-L3-L4-, where L1 is a covalent linking unit covalently linked to A, L2 is an extension unit, L3 is selected from peptide residues consisting of 2-8 amino acids, and L4 is a bond or self-cleaving fragment.

[0176] In some implementations, L1 is selected from (or its open-loop form) ), The asterisk (*) indicates a connection to A.

[0177] In some implementations, L2 is selected from -L2a -、-L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2b Each is independently selected from -C1-C8 alkylene-, -C1-C8 alkylene-C3-C8 cycloalkylene-, -C1-C8 ynylene-, -C6-C18 arylene-, -5 to 12 heteroarylene-, -C6-C18 arylene-C1-8 alkylene-, -5 to 12 heteroarylene-C1-8 alkylene-, 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 1) 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, preferably 9-30, more preferably 9-26) carbon atoms The L2 is a straight-chain or branched heteroalkylene group, wherein each of the alkylene, cycloalkylene, arylene, heteroarylene, and heteroalkylene groups is optionally substituted by one or more substituents independently selected from C1-C6 alkyl, heteroalkyl with 1-6 carbon atoms, C1-C6 alkoxy, hydroxyl, amino, carboxyl, or C3-C8 cycloalkyl groups, wherein the heteroalkyl or heteroalkylene group contains 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, preferably 1-8, more preferably 3-8) heteroatoms, and the heteroatoms of the heteroalkyl, heteroalkylene, and heteroarylene groups are selected from one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) of N, O, or S (preferably O), and L2 is optionally substituted by R1, wherein R1 is a polar hydrophilic group, and the hydrophilic polar group is a group containing polyethylene glycol, preferably m is selected from integers from 3 to 50, preferably integers from 8 to 24, such as 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24.

[0178] In some implementations, L2 is selected from -L 2a -、-L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2b Each is independently selected from -C1-C6 alkylene-, -C1-C3 alkylene-C3-C6 cycloalkylene-, -ethynyl-C1-C6 alkylene-, and -(CH2CH2O). n -、-(CH2CH2O)n C 1-3 alkylene-, -C 1-3 Alkylene (CH2CH2O) n C 1-3 alkylene-, -C 1-3 Alkylene-OC 1-3 alkylene-, phenylene-, phenylene-C 1-3 Alkylene-, where n is an integer selected from 1 to 12 (preferably 3 to 8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0179] In some implementations, L2 is selected from -L 2a -、-L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2b Each is independently selected from -(CH2)5-, phenylene, -phenyl-CH2-, and -CH2-O-CH2-.

[0180] In some implementations, L2 is selected from: (CH2CH2O) n CH2C(O)-、-(CH2CH2O) n CH2CH2-、 The * position is connected to L1, and n is defined as above.

[0181] In some embodiments, L3 is selected from peptide residues consisting of 2-8 amino acids, wherein the amino acids are selected from phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, and glycine. Preferably, L3 is selected from:

[0182] The * position is connected to L2.

[0183] In some implementations, L4 is selected from: bond, -*NH-CH2-, The * position is connected to L3, and R1 is selected from hydrogen,

[0184] R2 is selected from:

[0185] Among them, r, s, t, and u are each independently selected from integers from 1 to 50 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50), and r is preferably an integer from 8 to 24.

[0186] In some implementations, R2 is selected from the following structures:

[0187] In some implementations, L4 is selected from -*NHCH2-,

[0188] In some implementations, L4 is selected from:

[0189] -*NHCH2-、

[0190] In some implementation schemes, L is selected from:

[0191] The * position is connected to A.

[0192] In some implementation schemes, L is selected from:

[0193] The * position is connected to A.

[0194] In some implementation schemes, D is selected from:

[0195] Where X is selected from key, R3 and R4 are each independently selected from H, deuterium, and C. 1-3 Alkyl (preferably methyl, ethyl, n-propyl, isopropyl), 3-6 membered cycloalkyl (preferably cyclopropane, cyclobutane, cyclopentane, cyclohexane), or R3 and R4 together with one or more carbon atoms attached thereto form a 3-6 membered cycloalkyl (preferably cyclopropane, cyclobutane, cyclopentane, cyclohexane), where n' is selected from an integer of 1-6 (e.g., 1, 2, 3, 4, 5, or 6), wherein The location is connected to L4.

[0196] In some implementations, X is selected from key,

[0197] In some implementation schemes, D is selected from:

[0198] In some embodiments, antibody-drug conjugates having the structure shown in Formula (I) and their tautomers, stereoisomers, or pharmaceutically acceptable salts are provided: Formula I has the following structure:

[0199] The definitions of R2 and d are as described above.

[0200] In some embodiments, the antibody-drug conjugate of Formula I has the following structure:

[0201] Where r is selected from integers 1-50, preferably integers 8-24, and preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24. The definition of d is as described above.

[0202] In some embodiments, in the antibody-drug conjugate of Formula I and its tautomers, stereoisomers or pharmaceutically acceptable salts, A is selected from Hab48C6C6H5, Hab51F8C10C10, Hab52E3B5G2, Hab66D5GSE6, Hab66H5D4E6, Hab71F11D9D9, Hab82C11E8F4, 66H5+66D5 (S1), 51F8+48C6 (S4), 51F8+48C6 (S5), 51F8+48C6 (S6), 51F8+48C6 (S7) and 82C11+66H5 (S8).

[0203] In some embodiments, the antibody-drug conjugate of Formula I, its tautomers, stereoisomers, or pharmaceutically acceptable salts, has the following structure:

[0204] Wherein, A is selected from Hab48C6C6H5, Hab51F8C10C10, Hab52E3B5G2, Hab66D5GSE6, Hab66H5D4E6, Hab71F11D9D9, Hab82C11E8F4, 66H5+66D5(S1), 51F8+48C6(S4), 51F8+48C6(S5), 51F8+48C6(S6), 51F8+48C6(S7), and 82C11+66H5(S8), that is, each formula represents A as a specific antibody-drug conjugate of the above antibodies; d is selected from 2, 4, 6, or 8.

[0205] In a sixteenth aspect, this disclosure provides a fusion protein comprising the anti-FRα antibody or its antigen-binding fragment as described in the first to sixth aspects.

[0206] Examples of fusion proteins described in this disclosure include Fab fusion proteins, Fc fusion proteins, and single-chain antibody (scFv) fusion proteins, which are named according to the different sites at which effector proteins (e.g., cytokines) are fused.

[0207] In a seventeenth aspect, this disclosure provides a chimeric antigen receptor polypeptide (CAR) comprising the aforementioned anti-FRα antibody or its antigen-binding fragment, or the bispecific antibody according to any one of claims 16-26.

[0208] In an eighteenth aspect, this disclosure provides cells expressing the aforementioned chimeric antigen receptor (CAR) gene modification, said cells being selected from autologous or allogeneic T cells, B cells, NK cells, macrophages, monocytes, dendritic cells, neutrophils, basophils, eosinophils, mast cells, NK-T cells, MAIT cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, and erythrocytes.

[0209] In a nineteenth aspect, this disclosure provides a pharmaceutical composition comprising the aforementioned anti-FRα antibody or its antigen-binding fragment, the aforementioned bispecific antibody, the aforementioned antibody-drug conjugate and its tautomers, stereoisomers or pharmaceutically acceptable salts, the aforementioned fusion protein, the aforementioned antigen receptor polypeptide or the aforementioned gene-modified cells.

[0210] In this disclosure, the pharmaceutical composition of the seventeenth aspect further includes a pharmaceutically acceptable carrier.

[0211] In a twentieth aspect, this disclosure provides the use of the aforementioned anti-FRα antibody or its antigen-binding fragment, the aforementioned bispecific antibody or the aforementioned antibody-drug conjugate and its tautomers, stereoisomers or pharmaceutically acceptable salts, the aforementioned fusion protein, the aforementioned antigen receptor polypeptide, the aforementioned genetically modified cell or the aforementioned pharmaceutical composition in the preparation of a medicament for treating tumors.

[0212] In some implementations, the tumor is a tumor whose tumor cells express FRα (FRα+).

[0213] In some embodiments, the tumor is a tumor in which tumor cells highly express FRα (FRα+). In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 60% of the tumor cells in a tumor cell population expressing FRα. In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 70% of the tumor cells in a tumor cell population expressing FRα. In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 80% of the tumor cells in a tumor cell population expressing FRα. In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 90% of the tumor cells in a tumor cell population expressing FRα. In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 95% of the tumor cells in a tumor cell population expressing FRα. In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 98% of the tumor cells in a tumor cell population expressing FRα. In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 99% of the tumor cells in a tumor cell population expressing FRα.

[0214] In some implementations, the tumor is a solid tumor.

[0215] In some implementations, the tumor is a hematologic malignancy.

[0216] In some implementations, the tumor is a malignant tumor.

[0217] In some implementations, the tumor is cancer.

[0218] In some implementations, the tumor is ovarian cancer, oral cancer, and more preferably ovarian cancer.

[0219] In a nineteenth aspect, this disclosure provides a method for treating tumors in an individual, particularly tumors on which FRα is expressed on the surface of tumor cells, the method comprising administering to the individual a therapeutically effective amount of the aforementioned anti-FRα antibody or its antigen-binding fragment, the aforementioned bispecific antibody, the aforementioned antibody-drug conjugate and its tautomers, stereoisomers or pharmaceutically acceptable salts, the aforementioned fusion protein, the aforementioned antigen receptor polypeptide, the aforementioned genetically modified cells or the aforementioned pharmaceutical composition.

[0220] In some implementations, the tumor is a tumor whose tumor cells express FRα (FRα+).

[0221] In some embodiments, the tumor is a tumor in which tumor cells highly express FRα (FRα+). In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 60% of the tumor cells in a tumor cell population expressing FRα. In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 70% of the tumor cells in a tumor cell population expressing FRα. In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 80% of the tumor cells in a tumor cell population expressing FRα. In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 90% of the tumor cells in a tumor cell population expressing FRα. In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 95% of the tumor cells in a tumor cell population expressing FRα. In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 98% of the tumor cells in a tumor cell population expressing FRα. In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 99% of the tumor cells in a tumor cell population expressing FRα.

[0222] In some implementations, the tumor is a solid tumor.

[0223] In some implementations, the tumor is a hematologic malignancy.

[0224] In some implementations, the tumor is a malignant tumor.

[0225] In some implementations, the tumor is cancer.

[0226] In some implementations, the tumor is ovarian cancer, oral cancer, and more preferably ovarian cancer.

[0227] In a twentieth aspect, this disclosure provides the use of therapeutically effective amounts of the aforementioned anti-FRα antibody or its antigen-binding fragment, the aforementioned bispecific antibody, the aforementioned antibody-drug conjugate and its tautomers, stereoisomers or pharmaceutically acceptable salts, the aforementioned fusion protein, the aforementioned antigen receptor polypeptide, the aforementioned genetically modified cells or the aforementioned pharmaceutical composition for treating tumors in an individual, particularly tumors on which FRα (FRα+) is expressed on the surface of tumor cells.

[0228] In some implementations, the tumor is a tumor whose tumor cells express FRα (FRα+).

[0229] In some embodiments, the tumor is a tumor in which tumor cells highly express FRα (FRα+). In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 60% of the tumor cells in a tumor cell population expressing FRα. In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 70% of the tumor cells in a tumor cell population expressing FRα. In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 80% of the tumor cells in a tumor cell population expressing FRα. In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 90% of the tumor cells in a tumor cell population expressing FRα. In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 95% of the tumor cells in a tumor cell population expressing FRα. In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 98% of the tumor cells in a tumor cell population expressing FRα. In some embodiments, a tumor highly expressing FRα (FRα+) is defined as at least 99% of the tumor cells in a tumor cell population expressing FRα.

[0230] In some implementations, the tumor is a solid tumor.

[0231] In some implementations, the tumor is a hematologic malignancy.

[0232] In some implementations, the tumor is a malignant tumor.

[0233] In some implementations, the tumor is cancer.

[0234] In some implementations, the tumor is ovarian cancer, oral cancer, and more preferably ovarian cancer.

[0235] In a twentieth aspect, this disclosure provides the aforementioned anti-FRα antibody or its antigen-binding fragment, the aforementioned bispecific antibody, the aforementioned antibody-drug conjugate and its tautomers, stereoisomers or pharmaceutically acceptable salts, the aforementioned fusion protein, the aforementioned antigen receptor polypeptide, the aforementioned genetically modified cell or the aforementioned pharmaceutical composition, which are used as drugs or for treatment.

[0236] Terminology Definition

[0237] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art. For definitions and terminology in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in this field to refer to one of the 20 commonly used L-amino acids.

[0238] Although the numerical ranges and parameter approximations shown in the broad scope of this disclosure are intended to be as accurate as possible in the specific embodiments, any numerical value inherently contains a certain degree of error due to the standard deviation present in their respective measurements. Furthermore, all ranges disclosed herein should be understood to encompass any and all subranges contained therein. For example, the stated range “1 to 10” should be considered to include any and all subranges between the minimum value 1 and the maximum value 10 (inclusive); that is, all subranges beginning with a minimum value of 1 or greater, such as 1 to 6.1, and subranges ending with a maximum value of 10 or less, such as 5.5 to 10. Additionally, any references referred to as “incorporated herein” should be understood to be incorporated herein in their entirety.

[0239] The term “object” or “individual” as used in this article refers to mammals, such as humans, but can also refer to other animals, such as wild animals, livestock, or laboratory animals (e.g., chimpanzees, monkeys, rats, mice, rabbits, guinea pigs, marmots, ground squirrels, etc.).

[0240] As used herein, the term "antigen" refers to a predetermined target to which an antibody can selectively bind. Examples of antigens include, but are not limited to, peptides, sugars, nucleic acids, lipids, haptens, or other naturally occurring or synthetic compounds.

[0241] In a broad sense, an "antibody" can refer to an immunoglobulin molecule that can specifically bind to a target via at least one antigen recognition site located in the variable region of an immunoglobulin molecule. Therefore, it encompasses complete antibodies / full-length antibodies, single-chain antibodies, or any antigen-binding fragment of an antibody (also known as an "antigen-binding moiety"). When "antibody" and "antigen-binding fragment / antigen-binding moiety" appear in the same context, "antibody" can be understood as the complete entity relative to the "antigen-binding fragment / antigen-binding moiety," and both correspond to the broad concept of antibody.

[0242] The term "anti-FRα antibody" or "FRα-binding antibody" includes antibodies capable of binding to FRα with sufficient affinity, such that the antibody can be used as a diagnostic and / or therapeutic agent when targeting FRα. In some embodiments, the anti-FRα antibody binds to less than about 10% of unrelated non-FRα proteins, as determined, for example, by fluorescence activated cell sorting (FACS) analysis or immunoassays (e.g., radioimmunoassay (RIA)). For antibodies that "specifically bind" to or are "specific" to FRα, in some embodiments, the dissociation constant (KD) of the FRα-binding antibody is less than or equal to 500 nM, 100 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. In some implementations, the anti-FRα antibody binds to conserved FRα protein epitopes from different species (e.g., between human and cynomolgus monkey FRα).

[0243] A "full-length antibody" refers to a protein containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL) and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL regions can be further subdivided into multiple highly variable regions called complementarity-determining regions (CDRs), interspersed with multiple more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (Clq) of the classical complement system. Full-length antibodies can be any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or subclasses mentioned above), but the antibody does not need to belong to any specific class. Immunoglobulins can be designated into different classes based on the antibody amino acid sequence of the constant region of the heavy chain. Typically, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant regions of the heavy chain corresponding to different immunoglobulin classes are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known. Chimeric or humanized antibodies are also included in the antibodies according to this disclosure. It is well known to those skilled in the art that complementarity-determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of an antibody. There are several common ways to define the CDR amino acid sequence of VH or VL, such as the Kabat definition, the IMGT definition, and the Chothia definition. For a given antibody's variable region amino acid sequence, the CDR amino acid sequence in the VH and VL amino acid sequences can usually be determined according to different definitions. In the embodiments of this disclosure, the Kabat definition of the CDR amino acid sequence is used. For a given antibody's variable region amino acid sequence, the CDR amino acid sequence in the variable region amino acid sequence can be analyzed in various ways.

[0244] The term "mouse antibody" refers to an antibody derived from the fusion of B cells and myeloma cells of immunized mice. Mouse hybrid fusion cells that can proliferate indefinitely and secrete antibodies are selected, and then screened, prepared, and purified to obtain antibodies. Mouse antibodies generally have immunogenicity, so they need to be humanized in the subsequent process.

[0245] The term "humanized antibody" refers to an antibody obtained by grafting a CDR sequence derived from another mammalian species, such as a mouse line, onto a human framework sequence. To preserve binding affinity, certain residues in the backbone (called FR) segment can be modified. Humanized antibodies or fragments thereof according to this disclosure can be prepared using techniques known to those skilled in the art.

[0246] The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, for example, an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody. Chimeric antibodies or fragments thereof according to this disclosure can be prepared using gene recombination technology. For example, the chimeric antibody can be produced by cloning recombinant DNA containing a promoter and a sequence encoding the variable region of a non-human, particularly mouse, monoclonal antibody according to this disclosure, and a sequence encoding the constant region of a human antibody. The chimeric antibody of this disclosure encoded by such a recombinant gene will be, for example, a mouse-human chimera, whose specificity is determined by the variable region derived from mouse DNA and whose isotype is determined by the constant region derived from human DNA.

[0247] The term "monoclonal antibody" refers to an antibody obtained from a population of essentially homogeneous antibodies, meaning that the individual antibodies that make up the population are identical, except that naturally occurring mutations may exist in a small number of individuals.

[0248] As used herein, the terms “antigen-binding fragment” or “antigen-binding moiety” or “antigen-binding region” are used interchangeably and refer to a portion of an antibody containing amino acid residues that interact with the antigen and confer specificity and affinity to the antigen, particularly antibody fragments such as Fv, Fab, F(ab')2, or Fab', optionally containing chemically modified fragments that add half-life, such as the addition of poly(alkylene) glycols like polyethylene glycol (“PEGylated”) (a PEGylated fragment referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) (“PEG” stands for polyethylene glycol), which has FRα binding activity. Preferably, the antigen-binding fragment will consist of or contain a portion of the heavy or light chain variable chain of its source antibody, the portion of which is sufficient to retain the same binding specificity and sufficient affinity as its source antibody, and such antigen-binding fragment will contain at least 5 amino acids, preferably 10, 15, 25, 50, and 100 consecutive amino acids of its source antibody sequence. Examples of antigen-binding fragments include, but are not limited to: (1) Fab fragments, which may be monovalent fragments having VL-CL chains and VH-CH1 chains; (2) F(ab')2 fragments, which may be divalent fragments having two Fab' fragments connected by disulfide bridges (i.e., Fab' dimers) in the hinge region; (3) Fv fragments with VL and VH domains on a single arm of antibody; and (4) VHH fragments composed of VH domains.

[0249] The term "single-chain antibody (scFv)" refers to a single polypeptide chain consisting of VH and VL domains linked by peptide linkers. (scFv)2 contains two VH domains and two VL domains linked by peptide linkers, with the two VL domains combined with the two VH domains via disulfide bridges.

[0250] The terms "Fc fragment," "Fc region," "Fc domain," "Fc part," or similar terms refer to a portion of the constant region of the antibody heavy chain, including the hinge region, the CH2 segment, and the CH3 segment of the constant region. The Fc region of an anti-FRα antibody can be engineered or modified, including effector-related modifications, to reduce or eliminate antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), which can be achieved by introducing one or more amino acid substitutions / mutations into the Fc region of the antibody.

[0251] As used in this article, "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope.

[0252] The term "bispecific antibody" refers to an antibody that simultaneously binds to two antigenic epitopes. These two epitopes can be on different antigens or on the same antigen. Bispecific antibodies can have various structural configurations. For example, a bispecific antibody can consist of two Fc fragments and two antigen-binding fragments fused to them, with a structure similar to natural antibodies, the difference being that the two arms bind different antigenic targets or epitopes; this is also called an IgG-like bispecific antibody. Non-IgG-like bispecific antibodies generally use scFv fragments or Fab fragments as basic modules. By utilizing appropriately modified peptide linkers, scFv fragments or Fab fragments can form dimers, trimers, tetramers, pentamers, or even higher-order oligomers.

[0253] Generally, to prepare monoclonal antibodies or their functional fragments, especially murine monoclonal antibodies or their functional fragments, one can refer to the techniques described in particular in the manual "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988) or refer to the techniques described by Kohler and Milstein for preparation from hybridoma cells (Nature, 256: 495-497, 1975).

[0254] In describing some embodiments involving amino acid or nucleic acid sequence mutations, this disclosure uses the designation "XaaaY" (e.g., L234A), where "aaa" represents the sequence position of an amino acid or base (when a specific reference sequence exists, "aaa" represents the residue order position in that reference sequence; or it can be numbered according to the position numbering system commonly used in the art, such as the EU numbering system), "X" represents the original amino acid or base at "aaa", and "Y" represents the changed amino acid or base at "aaa". As an example, when describing the L234A mutation in the human heavy chain constant region, it means that, according to the EU numbering system for the human heavy chain constant region, leucine (L) at position 234 is mutated to alanine (A).

[0255] The term "isolated" refers to biological components (e.g., nucleic acids, proteins (including antibodies), or organelles) that have been substantially separated from or purified from other biological components (i.e., other and extra chromosomal DNA and RNA, proteins, and organelles) in their natural environment (e.g., cells). "Isolated" nucleic acids and proteins include those purified using standard purification methods. The term also includes nucleic acids and proteins prepared through recombinant expression in host cells, as well as chemically synthesized nucleic acids.

[0256] An "expression vector" is a vector that includes one or more expression control sequences. An "expression control sequence" is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence.

[0257] The term "antibody-drug conjugate" or "antibody-conjugated drug" (ADC) refers to the linking of a target ligand, such as an antibody (e.g., a monoclonal antibody), or an antibody fragment to a biologically active molecule via a linker or adapter.

[0258] Unless otherwise specified, the terms "pharmaceutically acceptable salt" or "medicinal salt" refer to a salt that, within the bounds of reasonable medical judgment, is suitable for contact with the tissues of mammals, particularly humans, without excessive toxicity, irritation, allergic reactions, etc., and is commensurate with a reasonable benefit / risk ratio. Medically acceptable salts of amines, carboxylic acids, and other types of compounds are well known in the art. The salts may be prepared in situ during the final isolation and purification of the compounds disclosed herein, or solely by reacting a free base or free acid with a suitable reagent. Exemplary salts include, but are not limited to: sulfates, trifluoroacetates, citrates, acetates, oxalates, hydrochlorides, hydrobromides, hydroiodates, nitrates, hydrogen sulfates, phosphates, acidic phosphates (-H₂PO₄) salts, phosphites, isonicotinates, lactates, salicylates, acidic citrates, tartrates, oleates, tannates, pantothenates, hydrogen tartrates, ascorbic acid salts, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, potassium salts, sodium salts, ammonium salts, calcium salts, etc. Additionally, pharmaceutically acceptable salts have more than one charged atom in their structure. Examples where multiple charged atoms are part of a pharmaceutically acceptable salt can have multiple counter atoms. For example, a pharmaceutically acceptable salt has one or more charged atoms and / or one or more counter atoms.

[0259] Unless otherwise specified, the compounds described in this disclosure (including antibody-drug conjugates) also contain "isotope labels," which refer to compounds of this disclosure that can exist in an isotopically traced or enriched form, containing one or more atoms whose atomic weight or mass number differs from the atomic weight or mass number of the most abundant atoms found in nature. The isotope can be radioactive or non-radioactive. Commonly used isotopes for isotope labeling include hydrogen isotopes. 2 H and 3 H; Carbon isotopes: 13 C and 14 C; Chlorine isotopes: 35 Cl and 37 Cl; Fluorine isotopes: 18 F; Iodine isotopes: 123 I and 125 I; Nitrogen isotopes: 13 N and15 N; oxygen isotopes: 15 O, 17 O and 18 O and sulfur isotopes 35 S. These isotope-labeled compounds can be used to study the distribution of pharmaceutical molecules in tissues. Especially 2 H and 13 C, due to their ease of labeling and detection, are more widely used. Substitution of certain heavy isotopes, such as deuterium (2H), can enhance metabolic stability and prolong half-life, thereby achieving a therapeutic advantage through dose reduction. Isotope-labeled compounds are generally synthesized starting from a labeled starting material, using known synthetic techniques similar to those used to synthesize non-isotope-labeled compounds.

[0260] Unless otherwise specified, the compounds described in this disclosure (including antibody-drug conjugates) also comprise “solvates,” where “solvate” means the physical association of the disclosed compound with one or more solvent molecules (organic or inorganic). This physical association includes hydrogen bonding. In some cases, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate can be separated. The solvent molecules in the solvate may be present in a regular and / or disordered arrangement. Solvates may contain stoichiometric or non-stoichiometric solvent molecules. “Solvate” encompasses both solution phases and separable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0261] Unless otherwise specified, the term "stereoisomer" refers to compounds having the same chemical structure but with different spatial arrangements of atoms or groups. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometric isomers (cis / trans) isomers, and inhibited isomers. Any mixture of stereoisomers obtained can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on differences in the physicochemical properties of the components, for example, by chromatography and / or fractional crystallization.

[0262] The term "linker-drug" refers to the partial structure of an "antibody-drug conjugate" consisting of a linker and a bioactive compound.

[0263] The term "linker" refers to a chemical structural fragment, denoted by L, that is linked at one end to an antibody and at the other end to a cytotoxic drug. In some embodiments of this disclosure, the "linker" is formed by linking an antibody to a "linker-drug compound." In other embodiments, a portion of the "linker" is first linked to an antibody, and another portion is linked to a bioactive compound, and then the linker is formed by covalently linking the different portions of the linker. The linker in this disclosure includes a covalently linked unit linked to the antibody, an extension unit, peptide residues, and optionally a self-cleaving fragment.

[0264] The linker-drug compound and the antibody described in this disclosure are linked by conventional coupling methods in the art, including: lysine coupling, inter-light and heavy chain reducing disulfide bond coupling, and directional coupling. This disclosure preferably uses inter-light and heavy chain reducing disulfide bond coupling, i.e., the coupling is achieved by reacting one or more of the disulfide bond sites between the light and heavy chains (two sites between the heavy chain and two sites between the heavy and light chains) with a thiol group (sulfur atom of a cysteine ​​residue) formed after reduction. In this disclosure, the linker L and the antibody linking portion are represented by L1, which is formed by the reaction of the L1' group in the linker compound with the antibody, for example... or The asterisk (*) indicates a link to the thiol group of the antibody, and the doublet (**) indicates a link to the spacer.

[0265] The term "extension unit" refers to the linking group between the covalent linker unit in linker L and the polypeptide sequence. This linker can be any divalent organic group, such as a chemical bond, C... 1-10 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-10 cycloalkyl, C 6-12 aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic, or a combination of two or more of these groups; the C 1-10 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-10 cycloalkyl, C 6-12 Aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic, or a combination of two or more of these groups, optionally separated by carbonyl, O, S, or N atoms; the C 1-10 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-10 cycloalkyl, C 6-12 Aryl, 5-12-membered heteroaryl, and 5-12-membered heterocyclic groups can be optionally C-shaped. 1-6 Alkyl, C 3-6 Cycloalkyl, halogen atom, halogenated C 1-6Alkyl substitution; preferably, the extension unit comprises hydrophilic segment substitution or insertion; preferably, the extension unit is as defined in L2.

[0266] The term "peptide residue" is well known in the art and is selected from a divalent peptide group comprising 2 to 8 optionally substituted protein or non-protein amino acids, L-type or D-type amino acid residues, each of which may be the same or different and is independently selected from residues of the following amino acids: alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), pentaneous (Nva), or leucine (Nle), or analogues of the above amino acids. The peptide residues in this disclosure may include substituted or derived amino acid residues, such as amino acid residues with side-chain amino groups specifically substituted as defined in CN115279417A, such as:

[0267] The term "self-cleaving fragment" is well known in the art, such as the p-aminobenzyl carbonate fragment or -NH-CH2- fragment commonly used in the art. In specific embodiments of this disclosure, the "self-cleaving fragment" includes the modification of "hydrophilic fragment".

[0268] The term "hydrophilic fragment" is well known in the art. The hydrophilic fragment may contain a polyethylene glycol group, such as a methoxy-terminated polyethylene glycol group, or further connect other hydrophilic fragments via a polyethylene glycol group. The hydrophilic fragment may also contain a polyamino acid fragment, such as polyglycine or polysarcosine, which may be combined with a polyethylene glycol group. The hydrophilic fragment may also be a monosaccharide, disaccharide, or oligosaccharide, which may be a chain-like or cyclic sugar and may contain glycosamines, sugar acids, or phosphorylated sugars. Preferably, the sugar group is combined with a polyethylene glycol fragment or a polyamino acid fragment; more preferably, at least two sugar groups are introduced through a polyvalent linker, such as a linker based on aspartic acid, glutamic acid, or lysine. It may also contain polycarboxylic acid groups, polysulfonic acid groups, or chelating groups; the structure of the polycarboxylic acid group is as follows:

[0269] The structure of the polysulfonic acid group is as follows:

[0270] The chelating group can be, for example, a DOTA group or a NOTA group.

[0271] Unless otherwise specified, the term "heteroatom" refers to nitrogen, oxygen, sulfur, and halogen atoms.

[0272] Unless otherwise specified, the term "alkyl" refers to a monovalent saturated aliphatic hydrocarbon group, a straight-chain or branched group comprising 1-20 carbon atoms, preferably comprising 1-10 carbon atoms (i.e., C10). 1-10 Alkyl groups, more preferably containing 1-8 carbon atoms (C64- ... 1-8 Alkyl groups, more preferably containing 1-6 carbon atoms (i.e., C64-C ... 1-6 Alkyl group). For example, "C 1-6 "Alkyl" refers to a group that is alkyl and has 1 to 6 carbon atoms in its carbon chain (specifically 1, 2, 3, 4, 5, or 6). Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, etc.

[0273] Unless otherwise specified, the terms "-alkyl-" or "alkylene" refer to a saturated straight-chain or branched divalent hydrocarbon group. For example, C1-C8 alkylene refers to a straight-chain or branched alkylene having 1-8 carbon atoms.

[0274] Unless otherwise specified, the term "heteroalkyl" means that one or more carbon atoms in an alkyl group are replaced by heteroatoms.

[0275] Unless otherwise specified, the term "heteroalkyl" refers to a divalent "heteroalkyl" with other groups attached to both ends.

[0276] Unless otherwise specified, the term "phenylene" refers to a divalent "phenyl" with other groups attached to both ends.

[0277] Unless otherwise specified, the term "cycloalkyl" refers to a fully saturated carbon ring that can exist as a monocyclic, bridged, or spirocyclic ring. Preferably, it contains 3-12 carbon atoms (i.e., C3-12 cycloalkyl), more preferably 3-10 carbon atoms (C3-10 cycloalkyl), even more preferably 3-7 carbon atoms (C3-7 cycloalkyl), 4-6 carbon atoms (C4-6 cycloalkyl), or 5-6 carbon atoms (C5-6 cycloalkyl). Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclopropyl, 2-ethyl-cyclopentyl, dimethylcyclobutyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, adamantyl, etc.

[0278] Unless otherwise specified, the term "cycloalkylene" refers to a divalent "cycloalkyl" with other groups attached to both ends.

[0279] As used herein, the term "pharmaceutical composition" refers to a combination of at least one drug substance and optionally a pharmaceutically acceptable carrier or excipient, which are combined together to achieve a particular purpose. In some embodiments, the pharmaceutical composition comprises combinations that are separate in time and / or space, provided that they can work together to achieve the purpose of this disclosure. For example, the components contained in the pharmaceutical composition (e.g., antibodies, nucleic acid molecules, combinations of nucleic acid molecules, and / or conjugates according to this disclosure) may be administered to an individual as a whole or separately. When the components contained in the pharmaceutical composition are administered to an individual separately, the components may be administered to the individual simultaneously or sequentially. Preferably, the pharmaceutically acceptable carrier is water, a buffered aqueous solution, an isotonic saline solution such as PBS (phosphate-buffered saline), glucose, mannitol, dextran glucose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or a polyalkylene glycol such as polypropylene glycol, triglycerides, etc. The type of pharmaceutically acceptable carrier used depends particularly on whether the composition according to this disclosure is formulated for oral, nasal, intradermal, subcutaneous, intramuscular, or intravenous administration. The compositions according to this disclosure may contain wetting agents, emulsifiers, or buffering substances as additives. The pharmaceutical compositions or formulations according to this disclosure may be administered via any suitable route, such as oral, nasal, intradermal, subcutaneous, intramuscular, or intravenous administration.

[0280] The term "therapeutic effective dose" or "effective dose" as used herein refers to a dose sufficient to demonstrate its benefit to the individual to which it is administered. The actual amount administered, as well as the rate and duration of administration, will depend on the individual's condition and severity. Prescribing treatment (e.g., determining the dosage) is ultimately the responsibility of and depends on the general practitioner and other physicians, who typically consider the disease being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the physician.

[0281] EC 50 EC50 primarily refers to the concentration of a drug, antibody, or toxin that, after a specific exposure time, achieves 50% of its maximum biological effect. In pharmaceutical science, besides characterizing the activation capacity of agonists in in vitro experiments, it can also be used to indicate the blood drug concentration required to achieve half of the maximum biological effect in vivo. In some literature, EC50... 50 It is also used to characterize the potency of a compound at the cellular level (including agonist and antagonist), and can be measured by methods such as ELISA to determine EC. 50 value.

[0282] As used herein, the term "fusion protein" generally refers to a protein composed of at least two domains that are not naturally associated, are encoded by separate genes, and are transcribed and translated as a whole to produce a single protein. In the context of this disclosure, a "fusion protein" containing an antibody or antigen-binding fragment refers to a product obtained by fusing an antibody or antigen-binding fragment with another bioactive protein using genetic engineering techniques. Such antibody fusion proteins possess both the antigen-binding capacity of the antibody and the unique biological characteristics of the bioactive protein fused with the antibody; for example, fusion with an albumin-binding fragment can prolong the in vivo half-life of the antibody.

[0283] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed fusion polypeptide that generally includes an antigen-binding region (e.g., an antibody or its antigen-binding moiety), a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. CARs utilize the antigen-binding properties of monoclonal antibodies to specifically and reactively redirect T cells and other immune cells to selected targets in a non-MHC-restricted manner. In this disclosure, the antigen-binding region may be in the form of the antibody or a bispecific antibody ScFv as described herein.

[0284] The term "identity / homology / consistency" in relation to amino acid or nucleic acid sequences is defined as the percentage of identical residues in an amino acid or nucleotide sequence variant after sequence alignment and vacancy introduction, reaching the maximum percentage of identity if desired. The methods and computer programs used for alignment are well known in the art.

[0285] As used in this article, "tumor" refers to a growth or solid lesion formed by abnormal cell growth. Tumors can be benign, pre-malignant, or malignant.

[0286] The term “malignant tumor” as used in this article refers to or describes a physiological condition in mammals characterized by uncontrolled cell growth. Attached Figure Description

[0287] Figure 1: Results of the binding experiment between the chimeric antibody and the soluble human FRα protein.

[0288] Figure 2: Results of the binding experiment between the chimeric antibody and the soluble human FOLR2 protein.

[0289] Figure 3: Results of the binding experiment between the chimeric antibody and the soluble human FOLR3 protein.

[0290] Figure 4: Results of the chimeric antibody binding experiment with KB-1 cells.

[0291] Figure 5: Results of the chimeric antibody binding experiment with SKOV-3 cells.

[0292] Figure 6: Results of the binding experiment between humanized antibody and soluble human FRα protein.

[0293] Figure 7: Results of the binding experiment between humanized antibody and soluble cynomolgus monkey FRα protein.

[0294] Figure 8: Results of the binding experiment between humanized antibody and soluble mouse FRα protein.

[0295] Figure 9: Results of the humanized antibody binding experiment with KB-1 cells.

[0296] Figure 10: Results of the experiment on the binding of humanized antibody to SKOV-3 cells.

[0297] Figure 11: Results of endocytosis experiment of humanized antibody on KB-1 cells.

[0298] Figure 12: Results of the humanized antibody conjugate killing KB-1 cells.

[0299] Figure 13: Results of tumor suppression experiments of humanized antibody conjugates in mice.

[0300] Figures 14-1, 14-2, 14-3, and 14-4: Structural diagrams of FRα dual epitope antibodies.

[0301] Figure 15: Results of the binding experiment between the dual epitope antibody and soluble human FRα protein.

[0302] Figure 16: Results of the experiment on the binding of the dual epitope antibody to KB-1 cells.

[0303] Figure 17: Results of the experiment on the binding of the dual epitope antibody to SKOV-3 cells.

[0304] Figures 18A and 18B: Results of endocytosis experiments of dual epitope antibodies on FRα-positive cells.

[0305] Figure 19: Results of the killing effect of the biepisode antibody (JSSW001) conjugate on KB-1 cells.

[0306] Figure 20: Results of the killing experiment on KB-1 cells by the biepisode antibody (045B) conjugate.

[0307] Figure 21: In vivo tumor suppression experiment results of the dual epitope antibody conjugate (045B). Detailed Implementation

[0308] The present disclosure is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this disclosure. The preferred embodiments and materials shown herein are for illustrative purposes only.

[0309] Any non-essential improvements and adjustments made to the implementation scheme by those skilled in the art based on the above-described invention shall still fall within the scope of protection of this disclosure.

[0310] Example 1: Obtaining anti-human FRα monoclonal antibody

[0311] Healthy female BALB / c mice, aged 6-8 weeks, were selected, and recombinant human FRα-His protein (ACRO, FO1-H52H1) was used as the immunogen. For the primary immunization, a mixture of Freund's complete adjuvant and immunoprotein was injected subcutaneously at multiple points on the back and groin of the mice, with an immunogen-to-adjuvant ratio of 1:1, at a dose of 100 μg / mouse. Before the primary immunization, a small amount of blood was collected from the mouse's eyeball as a negative control for serum antibody titer testing. 7-10 days after the primary immunization, a mixture of Freund's incomplete adjuvant and immunoprotein was administered via the same route as the primary immunization. Subsequently, booster immunizations were performed 2-3 times every 7-10 days, with an immunogen-to-adjuvant ratio of 1:1, at a dose of 50 μg / mouse. Peripheral serum from the mice was collected and analyzed using enzyme-linked immunosorbent assay (ELISA). Mice with high serum antibody titers were selected for cell fusion. Three days prior to fusion, mice were given a pulse immunization with 50 μg of unadjuvanted immunogen via tail vein or intraperitoneal injection. On the day of fusion, mouse spleens were harvested and a single-cell suspension was prepared using RPMI 1640 basal medium (GIBCO). SP2 / 0 cells and spleen cells were mixed at a ratio of 1:3 to 1:5 and fused using electrofusion. Cells were cultured in HAT selective medium for 5-7 days, then switched to HT medium and cultured in a 37°C CO2 incubator.

[0312] When hybridoma cells reached a certain quantity, the hybridoma supernatant was screened using ELISA to select positive clones. Subclonal selection was then performed using limiting dilution, ultimately yielding single-clone positive cell lines: 48C6C6H5, 51F8C10C10, 52E3B5G2, 60E8C3F5, 66H5D4E6, 66D5GSE6, 68G7A4B2, 71F11D9D9, and 82C11E8F4. Next-generation sequencing of the hybridoma cells yielded nine antibodies, as follows:

[0313] 48C6C6H5

[0314] Heavy chain CDR1: SSWMN (SEQ ID NO.1)

[0315] Heavy chain CDR2: RIYPGHGDTNYNGKFKG (SEQ ID NO.2)

[0316] Heavy chain CDR3: NLILRYYGLDY (SEQ ID NO.3)

[0317] Heavy chain variable region sequence

[0318] Light chain CDR1: RASESVDNYGISFMN (SEQ ID NO.4)

[0319] Light chain CDR2: AASNQGS (SEQ ID NO.5)

[0320] Light chain CDR3: QQSKEVPWT (SEQ ID NO.6)

[0321] Light chain variable region sequence

[0322] 51F8C10C10

[0323] Heavy chain CDR1: NYWIQ (SEQ ID NO.9)

[0324] Heavy chain CDR2: TVYPGNGDTGYTQKFND (SEQ ID NO.10)

[0325] Heavy chain CDR3: RTGGYLDF (SEQ ID NO.11)

[0326] Heavy chain variable region sequence

[0327] Light chain CDR1: RASENIYSVLA (SEQ ID NO.12)

[0328] Light chain CDR2: DATNLAD (SEQ ID NO.13)

[0329] Light chain CDR3: QHFWSTPFT (SEQ ID NO.14)

[0330] Light chain variable region sequence

[0331] 52E3B5G2

[0332] Heavy chain CDR1: DYVIS (SEQ ID NO.17)

[0333] Heavy chain CDR2: EIYPGSGSQNYNAKFKG (SEQ ID NO.18)

[0334] Heavy chain CDR3: RDFYGSLDY (SEQ ID NO.19)

[0335] Heavy chain variable region sequence

[0336] Light chain CDR1: SVSSSISSSNLH (SEQ ID NO.20)

[0337] Light chain CDR2: GTSNLAS (SEQ ID NO.21)

[0338] Light chain CDR3: QQWSSYPWT (SEQ ID NO.22)

[0339] Light chain variable region sequence

[0340] 60E8C3F5

[0341] Heavy chain CDR1: DYGMH (SEQ ID NO.25)

[0342] Heavy chain CDR2: VISTYYGNTNYNQKFKG (SEQ ID NO.26)

[0343] Heavy chain CDR3: SDGYYYAMDY (SEQ ID NO.27)

[0344] Heavy chain variable region sequence

[0345] Light chain CDR1: SVSSSISSSKLH (SEQ ID NO.28)

[0346] Light chain CDR2: GTSNLAS (SEQ ID NO.21)

[0347] Light chain CDR3: QQWSSYPLT (SEQ ID NO.29)

[0348] Light chain variable region sequence

[0349] 66D5GSE6

[0350] Heavy chain CDR1: DTYMH (SEQ ID NO.32)

[0351] Heavy chain CDR2: NIDPANGNTKYDSKFQG (SEQ ID NO.33)

[0352] Heavy chain CDR3: VLTGTSAFAY (SEQ ID NO.34)

[0353] Heavy chain variable region sequence

[0354] Light chain CDR1: RASQNINNNLH (SEQ ID NO.35)

[0355] Light chain CDR2: YASPSMS (SEQ ID NO.36)

[0356] Light chain CDR3: QQSHIWPQLT (SEQ ID NO.37)

[0357] Light chain variable region sequence

[0358] 66H5D4E6

[0359] Heavy chain CDR1: SYWIN (SEQ ID NO.40)

[0360] Heavy chain CDR2: NIYPSDSYTFYNQEFKD (SEQ ID NO.41)

[0361] Heavy chain CDR3: HGNYPDWYFDV (SEQ ID NO.42)

[0362] Heavy chain variable region sequence

[0363] Light chain CDR1: KASQDVITAVA (SEQ ID NO.43)

[0364] Light chain CDR2: SASYRYT (SEQ ID NO.44)

[0365] Light chain CDR3: QQHYSTPRT (SEQ ID NO.45)

[0366] Light chain variable region sequence

[0367] 68G7A4B2

[0368] Heavy chain CDR1: NYDMS (SEQ ID NO.48)

[0369] Heavy chain CDR2: TISGGGDYTYYPDIVKG (SEQ ID NO.49)

[0370] Heavy chain CDR3: HWDSGSIYWYFDV (SEQ ID NO.50)

[0371] Heavy chain variable region sequence

[0372] Light chain CDR1: RASKSIYKYLV (SEQ ID NO.51)

[0373] Light chain CDR2: SGSTLQS (SEQ ID NO.52)

[0374] Light chain CDR3: QQHSEYPLT (SEQ ID NO.53)

[0375] Light chain variable region sequence

[0376] 71F11D9D9

[0377] Heavy chain CDR1: NYWMH (SEQ ID NO.56)

[0378] Heavy chain CDR2: YINPSTDYTDYNEKFRD (SEQ ID NO.57)

[0379] Heavy chain CDR3: SYDYAYYYAMDY (SEQ ID NO.58)

[0380] Heavy chain variable region sequence

[0381] Light chain CDR1: SASSTVSYMH (SEQ ID NO.59)

[0382] Light chain CDR2: DTSKLAS (SEQ ID NO.60)

[0383] Light chain CDR3: QQWNSNPWT (SEQ ID NO.61)

[0384] Light chain variable region sequence

[0385] 82C11E8F4

[0386] Heavy chain CDR1: DTYIH (SEQ ID NO.64)

[0387] Heavy chain CDR2: NIDPANGNTKYDPKFQG (SEQ ID NO.65)

[0388] Heavy chain CDR3: VLTGTSAFAY (SEQ ID NO.34)

[0389] Heavy chain variable region sequence

[0390] Light chain CDR1: RASQNINNNLH (SEQ ID NO.35)

[0391] Light chain CDR2: YASPSMS (SEQ ID NO.36)

[0392] Light chain CDR3: QQGHIWPQLT (SEQ ID NO.66)

[0393] Light chain variable region sequence

[0394] Note: In the antibody sequences above, the underlined sequences are CDR sequences (determined and annotated according to the KABAT numbering system).

[0395] Example 2: FRα chimeric antibody affinity detection

[0396] Based on the variable region sequence information of the monoclonal antibody encoding gene, a human-mouse chimeric antibody expression vector was constructed by ligating it into the constant region of human hIgG1. The obtained eukaryotic expression vector was transiently transfected into CHO-K1 cells and cultured for 5 to 7 days. Cell supernatant was collected by centrifugation and purified using a Protein A column to obtain the target antibodies Cab-48C6C6H5, Cab-51F8C10C10, Cab-52E3B5G2, Cab-60E8C3F5, Cab-66D5GSE6, Cab-66H5D4E6, Cab-68G7A4B2, Cab-71F11D9D9, and Cab-82C11E8F4. Fortebio assays were used to detect the affinity of the chimeric antibodies for human FRα protein.

[0397] Using the ProA biosensor, anti-FRα chimeric antibody was immobilized onto the biosensor to a binding response threshold of 0.3 nm. After a 120-s baseline step, the sensor was immersed in human FRα-His protein (ACRO, FO1-H52H1) diluted with 0.02% PBST buffer. The initial protein concentration was 100 nM, with 3-fold serial dilutions and 7 gradients. The binding and dissociation times of the immobilized material with the analyte were 120 s and 300 s, respectively. The binding curves were analyzed using regression calculations to determine the antigen-antibody affinity. The results are shown in Table 1. The chimeric antibodies Cab-48C6C6H5, Cab-51F8C10C10, Cab-52E3B5G2, Cab-66D5GSE6, Cab-66H5D4E6, Cab-68G7A4B2, Cab-71F11D9D9 and Cab-82C11E8F4 all showed superior binding activity compared to the control molecule MoRab-003 (US2020 / 0297860A1, SEQ ID NO1, 6).

[0398] Table 1. Affinity of chimeric antibody to human FRα protein as determined by Fortebio

[0399] Example 3: Cross-reactivity of chimeric antibodies with human FOLR2 and FOLR3 proteins

[0400] The affinity of chimeric antibodies for recombinant human FRα, FOLR2, and FOLR3 proteins was determined using ELISA. FRα-His, FOLR2 (ACRO, FO2-H5223), and FOLR3 (ACRO, FO3-H52H3) proteins were diluted to 500 ng / ml and coated onto ELISA plates. After blocking, serially diluted antibodies (0-3.5 nM) were added, followed by incubation with secondary antibodies and colorimetric readings. The affinity results obtained by fitting with GraphPad are shown in Figures 1, 2, and 3. The experimental results showed that the samples bound to FRα-His but not to recombinant human FOLR2 or FOLR3 proteins.

[0401] Example 4: Binding of chimeric antibodies to FRα-positive cells

[0402] The binding affinity of the chimeric antibody to the FRα-overexpressing cell line KB-1 and the FRα-neutralizing cell line SKOV3 was evaluated. Cells were cultured at a concentration of 1 × 10⁻⁶ cells / year. 6At a density of 1 / ml, 100 μl / well was seeded into 96-well plates. Serially diluted antibodies (0-200 nM) were added and incubated at 4°C for 1 h. Goat anti-human secondary antibody was added, and the cells were labeled at 4°C in the dark for 45 min. After cell collection and washing, the fluorescence intensity of the cells was read by flow cytometry. The experimental results are shown in Figures 4 and 5. The results indicate that the chimeric antibodies can bind to FRα-positive cells in a dose-dependent manner, and the maximum binding capacity of Cab-51F8C10C10, Cab-48C6C6H5, and Cab-82C11E8F4 molecules on both highly and moderately expressed cells is superior to the control molecules.

[0403] Example 5: Humanization of FRα Antibody

[0404] Using Discovery Studio and Antibody Modeling is used for homology modeling. Through structural simulation and rational design, a human frame region that most closely resembles the mouse antibody frame region is obtained.

[0405] Humanized antibodies are obtained by inserting the CDRs of the light and heavy chains into the framework sequences of matching light and heavy chain genes, respectively. Then, they are processed using Discovery Studio and... Antibody Modeling was used to construct 3D models and analyze whether there were any sites where replacing mouse amino acids with human amino acids at the framework position would affect binding and / or CDR conformation. Reverse mutations were performed, and the sequences are shown in Table 2.

[0406] Table 2. Sequences with reversion mutations (CDR number: kabat) Note: In the antibody sequences above, the underlined sequences are CDR sequences (determined and annotated according to the KABAT numbering system).

[0407] Example 6: Detection of Affinity for Humanized Anti-FRα Antibody

[0408] 6.1 Fortebio assay for the affinity between humanized antibodies and human FRα protein

[0409] Using the ProA biosensor, the humanized antibody obtained in Example 5 was immobilized on the biosensor to a binding response threshold of 0.3 nm. After a 120-second baseline step, the sensor was immersed in FRα protein diluted with 0.02% PBST buffer. The initial protein concentration was 100 nM, with 3-fold serial dilutions and 7 gradients. The binding and dissociation times of the immobilized material with the analyte were 120 s and 300 s, respectively. The affinity data of the antigen and antibody were calculated by regression analysis using analytical software. Among all detected antibodies, Hab-48C6C6H5, Hab-51F8C10C10, Hab-52E3B5G2, Hab-66D5G5E6, Hab-66H5D4E6, Hab-71F11D9D9, and Hab-82C11E8F4 all showed superior affinity to MORab-003, as shown in Table 3.

[0410] Table 3. Affinity of humanized antibodies to human FRα protein as determined by Fortebio

[0411] 6.2 ELISA detection of the affinity between humanized antibody and FRα protein

[0412] The affinity of the humanized antibody obtained in Example 5 for human FRα-His protein (ACRO, FO1-H52H1), cynomolgus monkey FRα-His protein (ACRO, FO1-C52H8), and mouse FRα-His protein (ACRO, FO1-M5225) was determined by ELISA. FRα protein was diluted to 500 ng / ml and coated onto the ELISA plate. After blocking, 3-fold serially diluted antibodies (0-34 nM) were added. After incubation with secondary antibody, colorimetric readings were performed. The affinity results obtained by fitting with GraphPad are shown in Figures 6, 7, and 8 and Table 4. Experimental results showed that the humanized antibodies had human-monkey recognition ability, and that Hab-48C6C6H5, Hab-51F8C10C10, Hab-52E3B5G2, Hab-66H5D4E6, and Hab-82C11E8F4 bound to FRα protein better than the control molecule MORab-003. The humanized antibody Hab-82C11E8F4 bound weakly to mouse FRα protein.

[0413] Table 4. EC50 of humanized antibody affinity detected by ELISA 50 value

[0414] Example 7: Binding of humanized antibodies to human FRα-expressing cells

[0415] The binding affinity of humanized antibodies to FRα on the cell surface was assessed using KB-1 and SKOV-3 cells. Cells were cultured at a density of 2 × 10⁶ cells / year. 6 At a density of 1 / ml, 100 μl / well was seeded into 96-well plates. The antibody (0-100 nM) was added in 3-fold serial dilutions and incubated at 4°C for 1 h. After washing away the antibody, goat anti-human secondary antibody was added, and the cells were labeled at 4°C in the dark for 45 min. Cells were collected, washed, and their fluorescence intensity was read by flow cytometry. The experimental results are shown in Figures 9 and 10. The results indicate that the humanized antibody can bind to the FRα protein expressed on the cell line in a dose-dependent manner. Hab-48C6C6H5, Hab-51F8C10C10, Hab-52E3B5G2, Hab-66H5D4E6, and Hab-82C11E8F4 showed superior recognition ability compared to the control molecule MoRab-003.

[0416] Example 8: Endocytotic activity of humanized antibodies

[0417] The endocytic rate of the humanized FRα antibody was determined using the FACS method, and the endocytic capacity of the humanized antibody after binding to FRα on the cell surface was evaluated using the KB-1 cell line. Cells were cultured at a rate of 2 × 10⁻⁶ cells / year. 6 Cells were seeded at a density of 1 / ml in 96-well plates, and antibody was added to a final concentration of 34.2 nM. Cells were incubated at 4°C for 1 h. After washing with FACS buffer, cells were incubated at 4°C and 37°C for 2 h each. Goat anti-human secondary antibody was added, and cells were labeled at 4°C in the dark for 45 min. After collecting and washing the cells, the fluorescence intensity was read by flow cytometry.

[0418] The calculated antibody endocytosis rates within two hours are shown in Figure 11 and Table 5. The results indicate that the humanized antibodies disclosed herein possess good endocytic activity. On KB-1 cells, the endocytic abilities of Hab-51F8C10C10, Hab-66D5G5E6, Hab-66H5D4E6, Hab-71F11D9D9, and Hab-82C11E8F4 are superior to the control molecule MoRab-003.

[0419] Table 5. Humanized antibody endocytosis rate at 2 hours

[0420] Example 9: Killing activity of FRα humanized antibody conjugate

[0421] To verify that FRα antibody conjugates can mediate the delivery of cytotoxic agents to living cells, FRα ADCs were prepared by conjugating JSSW-001 toxin with FRα antibodies and in vitro cell killing experiments were performed. JSSW-001 (Shanghai Haoyuan Biomedical Technology Co., Ltd.; preparation can be referred to CN116135232B, Example 1) was conjugated with humanized antibodies Hab-66D5G5E6, Hab-71F11D9D9, and a control antibody using a cysteine ​​conjugation method to obtain ADC products Hab-66D5-JSSW001, Hab-71F11-JSSW001, and MORAb-003-JSSW001. The procedure was as follows: tris(2-carboxyethyl)phosphine (TCEP) (Bailingwei Technology Co., Ltd.) was added to the humanized antibody at a molar ratio of 1:10 and incubated at 37°C for 2 h. JSSW-001 was then added to the above solution at a molar ratio of 1:12 and reacted at 25°C for 30 min. Next, N-ethylmaleimide (Bailinwei Technology Co., Ltd.) was added at a molar ratio of 1:14, and the reaction was carried out at room temperature for 20 min to terminate the coupling reaction. The above antibody-drug conjugate was purified by affinity chromatography using an AKTA purifier to obtain the conjugated antibody-drug conjugate (ADC) molecules, which were then exchanged into PBS buffer. SEC and LC-MS assays showed that the JSSW-001 molecules were successfully conjugated. The free small molecule content of the conjugated ADC was less than 0.0015%, the SEC purity was greater than 95%, and the DAR value of the conjugated ADCs was approximately 8.

[0422] In vitro cytotoxicity was assessed using the cancer cell line KB-1. Cells were grown at a rate of 1 × 10⁶ cells / year. 3 / wells were seeded in 96-well plates. 3-fold serially diluted antibodies (0-650 nM) were added, and the plates were co-incubated for 7 days. Cell Titer Glo was then used to count viable cells to determine the ability of the ADC molecules to kill tumor cells.

[0423] The results obtained by fitting with GraphPad are shown in Figure 12 and Table 6. Hab-66D5-JSSW001 and Hab-71F11-JSSW001 have better killing ability than the control molecule MORAb-003-JSSW001.

[0424] Table 6. Killing KB-1 EC cells by FRα antibody conjugates 50 value

[0425] Example 10: In vivo biological activity of FRα humanized antibody conjugate in animals

[0426] Using the method described in Example 9, JSSW-001 (Shanghai Haoyuan Biomedical Technology Co., Ltd.; preparation can be referred to CN116135232B, Example 1) was conjugated with humanized antibodies Hab-48C6C6H5, Hab-51F8C10C10, Hab-66D5G5E6, Hab-66H5D4E6, Hab-71F11D9D9, and Hab-82C11E8F4 to obtain ADC products Hab-48C6-JSSW001, Hab-51F8-JSSW001, and Hab-66C6-001. The monotherapy efficacy of humanized antibody ADCs D5-JSSW001, Hab-66H5-JSSW001, Hab-71F11-JSSW001, and Hab-82C11-JSSW001 in tumor xenograft models was evaluated. (The text also mentions evaluating the single-therapeutic efficacy of 1×10⁻⁶ humanized antibody ADCs in tumor xenograft models.) 6 One KB-1 human oral epidermal carcinoma cell line was inoculated into NU / NU mice. The tumors were allowed to grow to approximately 150 mm². 3 At approximately 10:00 AM, 1.5 mg / kg of the ADC sample was injected intravenously, while a control solvent group was set up. A single injection was administered to investigate the in vivo antitumor effect of the FRα humanized antibody ADC. The experimental results are shown in Figure 13 and Table 7. Compared with the solvent group, Hab-51F8-JSSW001, Hab-66D5-JSSW001, Hab-71F11-JSSW001, and Hab-82C11-JSSW001 all significantly inhibited tumor growth. The Hab-82C11-JSSW001 group resulted in a 51.7% tumor weight inhibition rate and a tumor size reduction of 592.8 ± 248.5 mm. 3 The tumor volume.

[0427] Table 7. In vivo tumor suppression results of FRα antibody conjugates

[0428] Example 11 FRα antibody epitope detection

[0429] To further determine the epitope classification of antibodies, we used Octet epitope pairing to detect the antigenic epitope classification of candidate molecules. Human FRα-His protein was loaded onto the HIS1K sensor at 5 μg / ml, and then exposed to both a primary and a secondary antibody. The binding signal of the secondary antibody was used to determine whether the two antibodies recognized the same epitope. Hab-48C6C6H5, Hab-51F8C10C10, Hab-52E3B5G2, Hab-66D5G5E6, Hab-66H5D4E6, Hab-71F11D9D9, and Hab-82C11E8F4 are abbreviated as 48C6, 51F8, 52E3, 66D5, 66H5, 71F11, and 82C11, respectively.

[0430] Results: The identification epitopes of 66H5 and 66D5, 48C6 and 71F11, 48C6 and 51F8, 82C11 and 66H5, and 66H5 and 71F11 are different.

[0431] Example 12: Obtaining FRα dual epitope antibody

[0432] To explore the effects of different FRα biepisode antibodies on antibody function, different epitope antibodies were combined and biepisode molecules with different structures were expressed, including scFv+Fab 1:1 structure (see Figure 14-1, S1, S7, S8), scFv+Fab 2:2 structure (see Figure 14-2, S4; Figure 14-3, S5), and Fab+Fab (see Figure 14-4, S6), for a total of 6 biepisode antibodies. The sequences of the anti-FRα biepisode antibodies are shown in Table 8.

[0433] Table 8. FRα biepisotope antibody sequences

[0434] Example 13: ELISA detection of the affinity between FRα dual epitope antibody and FRα protein.

[0435] The affinity of the biepisode antibody for recombinant human FRα protein was determined using ELISA. Human FRα-His protein (ACRO, FO1-H52H1) was diluted to 500 ng / ml and coated onto an ELISA plate. After blocking, a four-fold serial dilution of antibody (0-60 nM) was added. After incubation with secondary antibody, the results were analyzed and the affinities were measured. The results, fitted with GraphPad, are shown in Figure 15 and Table 9. The experimental results showed that the affinities of biepisode molecules S4 and S6 were superior to the positive control IMGN-151 (CN 117730098A, SEQ ID NO: 61, 62, 56), while the affinities of S1, S5, and S7 were comparable to those of the positive control IMGN-151.

[0436] Table 9. EC50 for ELISA detection of affinity of biepisode antibody molecules 50 value

[0437] Example 14: Binding of FRα dual epitope antibody to human FRα-expressing cells

[0438] The binding ability of the biepisode antibody to FRα on the cell surface was assessed using KB-1 and SKOV-3 cells. Cells were cultured at 2 × 10⁶ cells per cell line. 6 At a density of 1 / ml, 100 μl / well was seeded into 96-well plates. The antibody (0-150 nM) was added in 4-fold serial dilutions and incubated at 4°C for 1 h. Goat anti-human secondary antibody was added, and the cells were labeled at 4°C in the dark for 45 min. After cell collection and washing, the fluorescence intensity of the cells was read by flow cytometry. The experimental results are shown in Figures 16 and 17 and Table 10. The results indicate that the biepisode antibody can bind to the FRα protein expressed on the cell line in a dose-dependent manner. In KB-1 cells, antibodies S1, S4, S5, and S6 recognized ECs with FRα affinity on the cell surface. 50 The values ​​were superior to the control molecule IMGN-151. In SKOV-3 cells, antibodies S1, S4, S5, and S7 recognized ECs with FRα affinity on the cell surface. 50 The value was better than that of the control molecule IMGN-151.

[0439] Table 10. Binding assay of FRα biepisotope antibody to human FRα-expressing cells

[0440] Example 15: FRα dual epitope antibody endocytosis activity

[0441] The endocytosis rate of FRα biepisotope antibodies was determined using the FACS method. The endocytosis capacity of biepisotope antibodies relative to single-episotope humanized antibodies after binding to FRα on the cell surface was evaluated using KB-1, SKOV3, H441, and OVCAR3 cell lines. Cells were cultured at 2 × 10⁻⁶ cells / year. 6Cells were seeded at a density of 1 / ml in 96-well plates, and antibody was added to a final concentration of 34.2 nM. Cells were incubated at 4°C for 1 h. After washing with FACS buffer, cells were incubated at 4°C and 37°C for 2 h, respectively. After washing, goat anti-human secondary antibody was added, and the cells were labeled at 4°C in the dark for 45 min. Cells were collected, washed, and their fluorescence intensity was read by flow cytometry. The antibody uptake and ratio over two hours are shown in Figures 18A and 18B. The results indicate that the dual-epitope antibody has good endocytic activity.

[0442] Example 16: In vitro cytotoxic activity of FRα dual epitope antibody conjugate

[0443] 16.1 In vitro cytotoxic activity of the FRα biepitaxy antibody-JSSW001 conjugate

[0444] To verify that the FRα biepisode antibody conjugate can mediate the delivery of cytotoxic agents to living cells, an FRα ADC was prepared by conjugating the FRα biepisode antibody with JSSW-001 toxin and in vitro cell killing experiments were performed. JSSW-001 (Shanghai Haoyuan Biomedical Technology Co., Ltd., synthesis method can be found in CN116135232B, Example 1) was conjugated with the biepisode antibody and the control antibody IMGN-151 using a cysteine ​​conjugation method. The procedure was as follows: TCEP (Bailingwei Technology Co., Ltd.) was added to the biepisode antibody at a molar ratio of antibody to TCEP of 1:10, and the mixture was incubated at 37°C for 2 h. JSSW-001 was then added to the above solution at a molar ratio of antibody to toxin of 1:12, and the reaction was carried out at 25°C for 30 min. Next, N-ethylmaleimide (Bailingwei Technology Co., Ltd.) was added at a molar ratio of 1:14, and the reaction was carried out at room temperature for 20 min to terminate the conjugation reaction. The above antibody-drug conjugate was purified by affinity chromatography using an AKTA purifier to obtain the conjugated antibody-drug conjugate (ADC) molecule, which was then exchanged into PBS buffer. SEC and LC-MS assays confirmed that the JSSW-001 molecule was successfully conjugated. The free small molecule content of the conjugated ADC was less than 0.0015%, and the SEC purity was greater than 95%.

[0445] In vitro cytotoxicity was assessed using the cancer cell line KB-1. Cells were grown at a rate of 1 × 10⁶ cells / year. 3 / wells were seeded in 96-well plates. Antibody-drug conjugates (0-500 nM) were added in 3-fold serial dilutions and incubated for seven days. Cell Titer Glo was then used to count viable cells to determine the ability of the ADC molecules to kill tumor cells. The results, fitted with GraphPad, are shown in Figure 19 and Table 11. S6-JSSW001 and S8-JSSW001 showed superior KB-1 killing activity compared to single epitope Hab48-JSSW001 and Hab51-JSSW001 (Hab48-JSS2W001 and Hab51-JSSW001 are Hab-48C6-JSSW001 and Hab-51F8-JSSSW001, respectively).

[0446] Table 11. Killing of KB-1 cells by EC50 cells with FRα biepisotope antibody-JSSW001 conjugate 50 value

[0447] In vitro cytotoxic activity of the FRα biepisotope antibody-045B conjugate.

[0448] The concentration of the FRα dual epitope antibody was adjusted to 10 g / L using phosphate buffer (pH 7.0). The FRα dual epitope antibody solution after buffer replacement was placed in a centrifuge tube, and 10.0 equivalents of 5 mM TCEP (Adamas-beta, Ltd.) were added. The reaction was carried out at 37°C for 1 hour to reduce the disulfide bonds between the antibody chains to thiol groups. Subsequently, a solution of 20 mM of compound 045B dissolved in DMSO (its synthesis can be found in WO2024222870A1, Examples 13-14) was added (12 equivalents of 045B per antibody). The reaction was carried out at 25°C for 1 hour. After coupling, the solution was subjected to ultrafiltration (Cobetter, Ltd.) to remove residual linker toxins and stored in PBS solution at pH 7.4.

[0449] The control molecule IMGN-151-045B was prepared by replacing the antibody with the method described above.

[0450] The obtained FRα biepisotope antibody-045B ADC was used in an in vitro cell killing assay to evaluate its ability to kill FRα-expressing KB-1 cells. Cells were grown at a rate of 2 × 10⁻⁶ cells / year. 4At a density of 0.5 μl / well, the antibody was seeded at a density of 0.5 μl / ml in 96-well plates. A 4-fold serial dilution of the antibody (0-250 nM) was added, and the plates were incubated for seven days. Cell Titer Glo was then used to count viable cells to determine the ability of the ADC molecules to kill tumor cells. The in vitro killing activity of the biepisode antibody-045B conjugate obtained after GraphPad fitting is shown in Figure 20 and Table 12. The S6-045B and S7-045B molecules showed significantly better killing ability against KB-1 tumor cells than the control molecule IMGN-151-045B.

[0451] Table 12. Killing assay of FRα biepisotope antibody conjugates (EC) 50 value

[0452] Example 17: In vivo killing activity of FRα dual epitope antibody conjugate

[0453] The monotherapy efficacy of Hab82-045B, S6-045B, S7-045B, and IMGN151-045B ADCs was evaluated in a tumor xenograft model. (1×10⁻⁶) 6 Personal oral epidermal carcinoma cells (OV90) were inoculated into NOD / SCID mice. The tumors were allowed to grow to approximately 127 mm². 3 At approximately 10:00 AM, the in vivo antitumor effect of FRα-ADC was investigated by intravenous injection of 0.9 mg / kg of the ADC sample and control, using a single injection. The experimental results are shown in Figure 21 and Table 13. Compared with the solvent group, the Hab82-045B, S6-045B, and S7-045B groups all significantly inhibited tumor growth.

[0454] Table 13. In vivo tumor suppression results of the FRα biepisotope antibody-045B conjugate.

Claims

An anti-FRα antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein: (1) The sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.1; the sequence of HCDR2 is the sequence shown in SEQ ID NO.2 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.2; the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.3; the sequence of LCDR1 is the sequence shown in SEQ ID NO.4 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.4; and the sequence of LCDR2 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

4. The sequence shown in NO.5 has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.6, and the sequence of LCDR3 is the sequence shown in SEQ ID NO.6 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.6; (2) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 9 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 9; the sequence of HCDR2 is the sequence shown in SEQ ID NO. 10 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 10; the sequence of HCDR3 is the sequence shown in SEQ ID NO. 11 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 11; the sequence of LCDR1 is the sequence shown in SEQ ID NO. 12 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 12; and the sequence of LCDR2 is the sequence shown in SEQ ID NO. 13 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

10. The sequence shown in NO.13 has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.14, and the sequence of LCDR3 is the sequence shown in SEQ ID NO.14 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.14; (3) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 17 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 17; the sequence of HCDR2 is the sequence shown in SEQ ID NO. 18 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 18; the sequence of HCDR3 is the sequence shown in SEQ ID NO. 19 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 19; the sequence of LCDR1 is the sequence shown in SEQ ID NO. 20 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 20; and the sequence of LCDR2 is the sequence shown in SEQ ID NO. 21 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

17. The sequence shown in NO.21 has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.22, and the sequence of LCDR3 is the sequence shown in SEQ ID NO.22 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.22; (4) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 25 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 25; the sequence of HCDR2 is the sequence shown in SEQ ID NO. 26 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 26; the sequence of HCDR3 is the sequence shown in SEQ ID NO. 27 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 27; the sequence of LCDR1 is the sequence shown in SEQ ID NO. 28 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 28; and the sequence of LCDR2 is the sequence shown in SEQ ID NO. 21 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

25. The sequence shown in NO.21 has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.29, and the sequence of LCDR3 is the sequence shown in SEQ ID NO.29 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.29; (5) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 32 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 32; the sequence of HCDR2 is the sequence shown in SEQ ID NO. 33 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 33; the sequence of HCDR3 is the sequence shown in SEQ ID NO. 34 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 34; the sequence of LCDR1 is the sequence shown in SEQ ID NO. 35 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 35; and the sequence of LCDR2 is the sequence shown in SEQ ID NO. 36 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

32. The sequence shown in NO.36 has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.37, and the sequence of LCDR3 is the sequence shown in SEQ ID NO.37 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.37; (6) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 40 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 40; the sequence of HCDR2 is the sequence shown in SEQ ID NO. 41 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 41; the sequence of HCDR3 is the sequence shown in SEQ ID NO. 42 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 42; the sequence of LCDR1 is the sequence shown in SEQ ID NO. 43 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 43; and the sequence of LCDR2 is the sequence shown in SEQ ID NO. 44 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

43. The sequence shown in NO.44 has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.45, and the sequence of LCDR3 is the sequence shown in SEQ ID NO.45 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.45; (7) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 48 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 48; the sequence of HCDR2 is the sequence shown in SEQ ID NO. 49 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 49; the sequence of HCDR3 is the sequence shown in SEQ ID NO. 50 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 50; the sequence of LCDR1 is the sequence shown in SEQ ID NO. 51 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 51; and the sequence of LCDR2 is the sequence shown in SEQ ID NO. 52 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

48. The sequence shown in NO.52 has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.53, and the sequence of LCDR3 is the sequence shown in SEQ ID NO.53 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.53; (8) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 56 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 56; the sequence of HCDR2 is the sequence shown in SEQ ID NO. 57 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 57; the sequence of HCDR3 is the sequence shown in SEQ ID NO. 58 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 58; the sequence of LCDR1 is the sequence shown in SEQ ID NO. 59 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 59; and the sequence of LCDR2 is the sequence shown in SEQ ID NO. 60 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

59. The sequence shown in SEQ ID NO. 60 has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 61, and the sequence of LCDR3 is the sequence shown in SEQ ID NO. 61 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 61; or (9) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 64 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 64; the sequence of HCDR2 is the sequence shown in SEQ ID NO. 65 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 65; the sequence of HCDR3 is the sequence shown in SEQ ID NO. 34 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 34; the sequence of LCDR1 is the sequence shown in SEQ ID NO. 35 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 35; and the sequence of LCDR2 is the sequence shown in SEQ ID NO. 36 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

64. The sequence shown in NO.36 has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.66, and the sequence of LCDR3 is the sequence shown in SEQ ID NO.66 or has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

66. The anti-FRα antibody or its antigen-binding fragment according to claim 1, wherein the anti-FRα antibody or its antigen-binding fragment is a murine, chimeric, or humanized antibody or its antigen-binding fragment, preferably, the anti-FRα antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment. The anti-FRα antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain variable region, wherein: (1) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 69 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 69; (2) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.71 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.71; (3) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.73 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.73; (4) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.75 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.75; (5) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.77 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.77; (6) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.79 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.79; (7) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.81 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.81; (8) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.7 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.7; (9) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.15 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.15; (10) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.23 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.23; (11) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.30 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.30; (12) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.38 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.38; (13) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.46 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.46; (14) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.54 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.54; (15) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 62 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 62; or (16) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.67 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.

67. The anti-FRα antibody or its antigen-binding fragment according to any one of claims 1-3, wherein the anti-FRα antibody or its antigen-binding fragment comprises a light chain variable region, wherein: (1) The light chain variable region sequence is the sequence shown in SEQ ID NO.70 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.70; (2) The light chain variable region sequence is the sequence shown in SEQ ID NO.72 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.72; (3) The light chain variable region sequence is the sequence shown in SEQ ID NO.74 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.74; (4) The light chain variable region sequence is the sequence shown in SEQ ID NO.76 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.76; (5) The light chain variable region sequence is the sequence shown in SEQ ID NO.78 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.78; (6) The light chain variable region sequence is the sequence shown in SEQ ID NO.80 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.80; (7) The light chain variable region sequence is the sequence shown in SEQ ID NO.82 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.82; (8) The light chain variable region sequence is the sequence shown in SEQ ID NO.8 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.8; (9) The light chain variable region sequence is the sequence shown in SEQ ID NO.16 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.16; (10) The light chain variable region sequence is the sequence shown in SEQ ID NO.24 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.24; (11) The light chain variable region sequence is the sequence shown in SEQ ID NO.31 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.31; (12) The light chain variable region sequence is the sequence shown in SEQ ID NO.39 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.39; (13) The light chain variable region sequence is the sequence shown in SEQ ID NO.47 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.47; (14) The light chain variable region sequence is the sequence shown in SEQ ID NO.55 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.55; (15) The light chain variable region sequence is the sequence shown in SEQ ID NO. 63 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 63; or (16) The light chain variable region sequence is the sequence shown in SEQ ID NO.68 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.

68. The anti-FRα antibody or its antigen-binding fragment according to any one of claims 1-4, wherein the anti-FRα antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein: (1) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 69 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 69, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 70 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 70; (2) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.71 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.71, and the light chain variable region sequence is the sequence shown in SEQ ID NO.72 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.72; (3) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.73 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.73, and the light chain variable region sequence is the sequence shown in SEQ ID NO.74 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.74; (4) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.75 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.75, and the light chain variable region sequence is the sequence shown in SEQ ID NO.76 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.76; (5) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.77 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.77, and the light chain variable region sequence is the sequence shown in SEQ ID NO.78 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.78; (6) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.79 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.79, and the light chain variable region sequence is the sequence shown in SEQ ID NO.80 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.80; (7) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.81 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.81, and the light chain variable region sequence is the sequence shown in SEQ ID NO.82 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.82; (8) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.7 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.7, and the light chain variable region sequence is the sequence shown in SEQ ID NO.8 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.8; (9) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.15 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.15, and the light chain variable region sequence is the sequence shown in SEQ ID NO.16 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.16; (10) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.23 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.23, and the light chain variable region sequence is the sequence shown in SEQ ID NO.24 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.24; (11) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.30 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.30, and the light chain variable region sequence is the sequence shown in SEQ ID NO.31 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.31; (12) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.38 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.38, and the light chain variable region sequence is the sequence shown in SEQ ID NO.39 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.39; (13) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.46 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.46, and the light chain variable region sequence is the sequence shown in SEQ ID NO.47 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.47; (14) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.54 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.54, and the light chain variable region sequence is the sequence shown in SEQ ID NO.55 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.55; (15) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 62 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 62; the light chain variable region sequence is the sequence shown in SEQ ID NO. 63 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 63; or (16) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 67 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 67, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 68 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.

68. The anti-FRα antibody or its antigen-binding fragment according to any one of claims 1-5, wherein the antigen-binding fragment is Fv, VHH, scFv, Fab, Fab' or (Fab')2. The anti-FRα antibody or its antigen-binding fragment according to any one of claims 1-6, wherein the anti-FRα antibody is a full-length antibody. The anti-FRα antibody or its antigen-binding fragment according to claim 7, wherein the anti-FRα antibody is an IgG antibody, preferably, the anti-FRα antibody is an IgG1 antibody. The anti-FRα antibody of claim 8 or its antigen-binding fragment thereof, wherein the Fc region of the anti-FRα antibody contains an amino acid mutation that reduces ADCC, CDC and / or ADCP effects. The anti-FRα antibody of claim 9 or its antigen-binding fragment thereof, wherein the amino acid mutation is one or more amino acid mutations selected from the group consisting of L234A, L235A, P329A, P326G and P331S. Nucleic acid molecules encoding the anti-FRα antibody or its antigen-binding fragment as described in any one of claims 1-10. An expression vector comprising the nucleic acid molecule of claim 11. Host cells containing the expression vector of claim 12. A bispecific antibody having a first antigen-binding site and a second antigen-binding site, wherein one antigen-binding site comprises the anti-FRα antibody or its antigen-binding fragment as described in any one of claims 1-10. A bispecific antibody having a first antigen-binding site and a second antigen-binding site, wherein one antigen-binding site comprises an anti-FRα antibody or an antigen-binding fragment thereof as described in any one of claims 1-10, and the other antigen-binding site comprises another anti-FRα antibody or an antigen-binding fragment thereof as described in any one of claims 1-10. The bispecific antibody of claim 15, wherein the first antigen-binding site and the second antigen-binding site bind to different epitopes of FRα. The bispecific antibody of claim 16, wherein, (1) The first antigen binding site includes a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO.1 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.1; the HCDR2 sequence is the sequence shown in SEQ ID NO.2 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.2; and the HCDR3 sequence is the sequence shown in SEQ ID NO.3 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

3. In the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO.4 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.4; and the LCDR2 sequence is the sequence shown in SEQ ID NO.5 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

4. The sequence shown in SEQ ID NO. 5 has at least 85%, 90%, 95%, 98%, or 99% identity, and the LCDR3 sequence is the sequence shown in SEQ ID NO. 6 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 6; the second antigen binding site includes a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO. 9 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 9, the HCDR2 sequence is the sequence shown in SEQ ID NO. 10 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 10, and the HCDR3 sequence is the sequence shown in SEQ ID NO. 11 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

10. The sequence shown in NO.11 has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence. In the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO.12 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.12; the LCDR2 sequence is the sequence shown in SEQ ID NO.13 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.13; and the LCDR3 sequence is the sequence shown in SEQ ID NO.14 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

14. (2) The first antigen binding site includes a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO.40 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.40; the HCDR2 sequence is the sequence shown in SEQ ID NO.41 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.41; and the HCDR3 sequence is the sequence shown in SEQ ID NO.42 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

42. In the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO.43 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.43; and the LCDR2 sequence is the sequence shown in SEQ ID NO.44 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

43. The sequence shown in SEQ ID NO. 44 has at least 85%, 90%, 95%, 98%, or 99% identity, and the LCDR3 sequence is the sequence shown in SEQ ID NO. 45 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 45; the second antigen binding site includes a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO. 32 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 32, the HCDR2 sequence is the sequence shown in SEQ ID NO. 33 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 33, and the HCDR3 sequence is the sequence shown in SEQ ID NO. 34 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 33, and the LCDR3 sequence is the sequence shown in SEQ ID NO. 34 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 45; The sequence shown in NO. 34 has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

35. In the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO. 35 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 35; the LCDR2 sequence is the sequence shown in SEQ ID NO. 36 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 36; and the LCDR3 sequence is the sequence shown in SEQ ID NO. 37 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

37. (3) The first antigen binding site includes a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO. 64 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 64; the HCDR2 sequence is the sequence shown in SEQ ID NO. 65 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 65; and the HCDR3 sequence is the sequence shown in SEQ ID NO. 34 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

34. In the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO. 35 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 35; and the LCDR2 sequence is the sequence shown in SEQ ID NO. 36 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

64. The sequence shown in SEQ ID NO. 36 has at least 85%, 90%, 95%, 98%, or 99% identity, and the LCDR3 sequence is the sequence shown in SEQ ID NO. 66 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 66; the second antigen binding site includes a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO. 40 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 40, the HCDR2 sequence is the sequence shown in SEQ ID NO. 41 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 41, and the HCDR3 sequence is the sequence shown in SEQ ID NO. 42 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

41. The sequence shown in NO.42 has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence. In the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO.43 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.43; the LCDR2 sequence is the sequence shown in SEQ ID NO.44 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.44; and the LCDR3 sequence is the sequence shown in SEQ ID NO.45 or has at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

45. The bispecific antibody of claim 17, wherein, (1) The first antigen binding site includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 69 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 69, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 70 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 70; the second antigen binding site includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 71 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 71, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 72 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 72; (2) The first antigen-binding site includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 77 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 77, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 78 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 78; the second antigen-binding site includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 75 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 75, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 76 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 76; or (3) The first antigen binding site includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 81 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 81, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 82 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 82; the second antigen binding site includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 77 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO. 77, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 78 or a sequence having at least 85%, 90%, 95%, 98%, or 99% identity with the sequence shown in SEQ ID NO.

78. The bispecific antibody according to any one of claims 14-18, wherein the bispecific antibody is an IgG-like bispecific antibody, wherein the left arm is a first antigen binding site and the right arm is a second antigen binding site; preferably, the first antigen binding site comprises a first heavy chain and a first light chain, and the second antigen binding site comprises a second heavy chain and a second light chain; or the first antigen binding site comprises a first heavy chain and a first light chain, and the second antigen binding site comprises an scFv-Fc chain. The bispecific antibody according to any one of claims 14-18, wherein the bispecific antibody is a fusion protein of an IgG-like antibody with an N-terminus or C-terminus linked to an scFv fragment, the IgG-like antibody targeting a first antigen, and the N-terminus or C-terminus linked to the scFv fragment targeting a second antigen; preferably, the scFv fragment is linked to the IgG-like antibody via a linker; more preferably, the linker is a G... n S m , where G is glycine, S is serine, n is an integer selected from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and m is an integer selected from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The bispecific antibody according to any one of claims 14-20, wherein, The Fc region of the bispecific antibody contains amino acid mutations that reduce ADCC, CDC, and / or ADCP effects, and / or amino acid mutations that facilitate the purification of the bispecific antibody to form a pestle and / or mortar; preferably, the amino acid mutations that reduce ADCC, CDC, and / or ADCP effects are selected from one or more mutations of L234A, L235A, P329A, P329G, or P331S, and the amino acid mutations that facilitate the purification of the bispecific antibody to form a pestle and / or mortar include mutations that form a mortar such as Y349C, T366S, L368A, and Y407V, and mutations that form a pestle such as S354C and T366W. The bispecific antibody according to any one of claims 14-21, wherein a CH1-CL chain exchange is carried between the first heavy chain and the first light chain or between the second heavy chain and the second light chain. The bispecific antibody according to any one of claims 14-21, wherein the heavy chain variable region and / or light chain variable region of the scFv fragment contains a mutation to cysteine ​​(Cys); preferably, the mutation to Cys is located at position 44 of the heavy chain variable region and / or position 100 or 101 of the light chain variable region; more preferably, the mutation to Cys in the heavy chain variable region is G44C, and / or the mutation to Cys in the light chain variable region is Q100C or Q101C. A bispecific antibody, wherein: (1) The bispecific antibody comprises a first heavy chain, a second heavy chain, a first light chain and a second light chain, wherein the amino acid sequence of the first heavy chain is shown in SEQ ID NO.98, the amino acid sequence of the second heavy chain is shown in SEQ ID NO.100, the amino acid sequence of the first light chain is shown in SEQ ID NO.96, and the amino acid sequence of the second light chain is shown in SEQ ID NO.

102. (2) The bispecific antibody is a fusion protein of an IgG-like antibody with an N-terminus or C-terminus linked to an scFv fragment, comprising two identical heavy chains linked to the scFv fragment and two identical light chains, wherein the amino acid sequence of the heavy chain linked to the scFv fragment is as shown in SEQ ID NO. 92 or 97, and the amino acid sequence of the light chain is as shown in SEQ ID NO. 96; or (3) The first antigen-binding site of the bispecific antibody includes one heavy chain and one light chain, and the second antigen-binding site of the bispecific antibody is an scFv-Fc chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO.105, the amino acid sequence of the light chain is shown in SEQ ID NO.96, and the amino acid sequence of the scFv-Fc chain is shown in SEQ ID NO.104; or the amino acid sequence of the heavy chain is shown in SEQ ID NO.88, the amino acid sequence of the light chain is shown in SEQ ID NO.90, and the amino acid sequence of the scFv-Fc chain is shown in SEQ ID NO.83; or the amino acid sequence of the heavy chain is shown in SEQ ID NO.110, the amino acid sequence of the light chain is shown in SEQ ID NO.112, and the amino acid sequence of the scFv-Fc chain is shown in SEQ ID NO.

106. Nucleic acid molecules encoding the bispecific antibodies of any one of claims 14-24. An expression vector comprising the nucleic acid molecule of claim 25. Host cells comprising the expression vector of claim 26. Antibody-drug conjugates comprising the anti-FRα antibody or its antigen-binding fragment as described in any one of claims 1-10 or the bispecific antibody as described in any one of claims 14-24, and their tautomers, stereoisomers or pharmaceutically acceptable salts thereof. The antibody-drug conjugate of claim 28 and its tautomers, stereoisomers or pharmaceutically acceptable salts thereof, wherein, The antibody-drug conjugate is obtained by conjugating a therapeutic agent to the anti-FRα antibody or its antigen-binding fragment as described in any one of claims 1-10 or the bispecific antibody as described in any one of claims 14-24. The antibody-drug conjugate of claim 29 and its tautomers, stereoisomers or pharmaceutically acceptable salts thereof, wherein, The therapeutic agent is selected from the group consisting of cytotoxic drugs, immune enhancers, or radioisotopes; preferably, the therapeutic agent is a cytotoxic drug selected from microtubule inhibitors, DNA topoisomerase inhibitors, DNA damaging agents, antimetabolites, or antitumor antibiotics; more preferably, the therapeutic agent is selected from DXD, SN38, irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, 22-hydroxyeclipticine, topotecan, letopotecan, belotetan, eczetine, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylanoylamino)phenyl]- DNA topoisomerase inhibitors of 4(3H)-quinazolinone, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-acrylamide, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxyphenylpropyl)-(E)-2-acrylamide, 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acrylamide dihydrochloride, or N-[2-(dimethylamino)ethyl]-4-acrylamide. The antibody-drug conjugate of claim 30 and its tautomers, stereoisomers or pharmaceutically acceptable salts thereof, wherein, The antibody or its antigen-binding fragment is conjugated to the therapeutic agent via a linker, which may be a cleavable linker or a non-cleavable linker. The antibody-drug conjugate and its tautomers, stereoisomers or pharmaceutically acceptable salts according to any one of claims 29-31, having the structure shown in formula (I): A-(L-D) d (I) Wherein A is the anti-FRα antibody or its antigen-binding fragment as described in any one of claims 1-10, or the bispecific antibody as described in any one of claims 14-24; L is the connecting sub-part, with one end connected to A and the other end connected to the therapeutic agent D; d represents the average molar ratio of the therapeutic agent to A (also known as DAR, or drug-antibody conjugate ratio), selected from an integer or decimal of 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12); when d is a decimal, it refers to the average number of linkers-therapeutic agents (LDs) for each A conjugate. The antibody-drug conjugate of claim 32 and its tautomers, stereoisomers or pharmaceutically acceptable salts thereof, wherein, A is selected from antibodies Cab-48C6C6H5, Cab-51F8C10C10, Cab-52E3B5G2, Cab-60E8C3F5, Cab-66D5GSE6, Cab-66H5D4E6, Cab-68G7A4B2, Cab-71F11D9D9, Cab-82C11E8F4, Hab48C6C6H5, and Hab51F8C10C.

10. Hab52E3B5G2, Hab66D5GSE6, Hab66H5D4E6, Hab71F11D9D9, Hab82C11E8F4, 66H5+66D5( S1), 51F8+48C6(S4), 51F8+48C6(S5), 51F8+48C6(S6), 51F8+48C6(S7) and 82C11+66H5(S8). The antibody-drug conjugate and its tautomers, stereoisomers or pharmaceutically acceptable salts as described in claim 32 or 33, wherein, -L- is selected from -L1-L2-L3-L4-, where L1 is a covalent linking unit that is covalently linked to A, L2 is an extension unit, L3 is selected from peptide residues consisting of 2-8 amino acids, and L4 is a bond or self-cleaving fragment. The antibody-drug conjugate of claim 34 and its tautomers, stereoisomers or pharmaceutically acceptable salts thereof, wherein, L1 is selected from (or its open-loop form) )、 Where * indicates a connection to A; L2 is selected from -L 2a -、-L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2b Each of the following is independently selected from -C1-C8 alkylene-, -C1-C8 alkylene-C3-C8 cycloalkylene-, -C1-C8 ynylene-, -C6-C18 arylene-, -5 to 12-membered heteroarylene-, -C6-C18 arylene-C1-8 alkylene-, -5 to 12-membered heteroarylene-C1-8 alkylene-, and straight-chain or branched heteroalkylenes with 1 to 50 (preferably 9 to 30, more preferably 9 to 26) carbon atoms, wherein the alkylene, cycloalkylene, arylene, heteroarylene, and heteroalkylene are each optionally selected as independent The L2 is substituted by one or more substituents selected from C1-C6 alkyl, heteroalkyl with 1-6 carbon atoms, C1-C6 alkoxy, hydroxy, amino, carboxyl or C3-C8 cycloalkyl, wherein the heteroalkyl or heteroalkylene contains 1-12 (preferably 1-8, more preferably 3-8) heteroatoms, and the heteroatoms of the heteroalkyl, heteroalkylene or heteroaryl are selected from one or more of N, O or S (preferably O), and L2 is optionally substituted by R1, wherein R1 is a polar hydrophilic group, and the hydrophilic polar group is a group containing polyethylene glycol, preferably. m is selected from an integer between 3 and 50, preferably an integer between 8 and 24; Preferably, L2 is selected from -L 2a -、-L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2b Each is independently selected from -C1-C6 alkylene-, -C1-C3 alkylene-C3-C6 cycloalkylene-, -ethynyl-C1-C6 alkylene-, and -(CH2CH2O). n -、-(CH2CH2O) n C 1-3 Alkylene -, -C1-C3 alkylene (CH2CH2O) n -C1-C3 alkylene-, -C1-C3 alkylene-O-C1-C3 alkylene, phenyl, -phenyl-C1-C3 alkylene-, wherein n is selected from an integer of 1-12 (preferably 3-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12); L3 is selected from peptide residues consisting of 2-8 amino acids, wherein the amino acids are selected from phenylalanine, valine, alanine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, and glycine. L4 is selected from: bond, -*NHCH2-, R1 is selected from hydrogen, *The location is connected to L3; R2 is selected from: Among them, r, s, t, and u are each independently selected from integers from 1 to 50. The antibody-drug conjugate and its tautomers, stereoisomers or pharmaceutically acceptable salts as described in claim 34 or 35, wherein, L2 is selected from: (CH2CH2O) n CH2C(O)-、-(CH2CH2O) n CH2CH2-、 The * position is connected to L1, and n is defined as in claim 35. The antibody-drug conjugate and its tautomers, stereoisomers or pharmaceutically acceptable salts as described in claim 35 or 36, wherein, L3 is selected from: The * position is connected to L2. The antibody-drug conjugate and its tautomers, stereoisomers or pharmaceutically acceptable salts according to any one of claims 34-37, wherein, L4 is selected from: -*NHCH2-、 Where r is selected from an integer between 1 and 50 (preferably 8-24). The antibody-drug conjugate of claim 36 and its tautomers, stereoisomers or pharmaceutically acceptable salts thereof, wherein L is selected from: The asterisk (*) indicates a connection to A. The antibody-drug conjugate and its tautomers, stereoisomers or pharmaceutically acceptable salts according to any one of claims 34-39, wherein D is selected from DNA topoisomerase inhibitors, preferably camptothecin analogs or DNA topoisomerase I inhibitors and their derivatives, such as DXD, SN38, irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, 22-hydroxyeclipticine, topotecan, letopotecan, belotetan, eczetine, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylanoylamino)phenyl] -4(3H)-quinazolinone, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-acrylamide, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxyphenylpropyl)-(E)-2-acrylamide, 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acrylamide dihydrochloride, N-[2-(dimethylamino)ethyl]-4-acrylamide; More preferably, D is selected from: Where X is selected from key, R3 and R4 are each independently selected from H, deuterium, and C. 1-3 Alkyl (preferably methyl, ethyl, n-propyl, isopropyl), 3-6 membered cycloalkyl (preferably cyclopropane, cyclobutane, cyclopentane, cyclohexane), or R3 and R4 together with one or more carbon atoms connected thereto form a 3-6 membered cycloalkyl (preferably cyclopropane, cyclobutane, cyclopentane, cyclohexane), where n' is selected from integers from 1 to 6. The location is connected to L4. The antibody-drug conjugate and its tautomers, stereoisomers or pharmaceutically acceptable salts according to any one of claims 34-40, wherein D is selected from: The antibody-drug conjugate and its tautomers, stereoisomers or pharmaceutically acceptable salts according to any one of claims 28-41 are selected from the following structures: in, A, d, and R2 are as defined in any one of claims 28-41. A fusion protein comprising the anti-FRα antibody or its antigen-binding fragment as described in any one of claims 1-10. The chimeric antigen receptor polypeptide (CAR) comprising the anti-FRα antibody or its antigen-binding fragment as described in any one of claims 1-10, or the bispecific antibody as described in any one of claims 14-24. A genetically modified cell, in which, The cells express the chimeric antigen receptor (CAR) polypeptide of claim 44, and the cells are selected from autologous or allogeneic T cells, B cells, NK cells, macrophages, monocytes, dendritic cells, neutrophils, basophils, eosinophils, mast cells, NK-T cells, MAIT cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, and erythrocytes. A pharmaceutical composition comprising an anti-FRα antibody or its antigen-binding fragment as described in any one of claims 1-10, a bispecific antibody as described in any one of claims 14-24, an antibody-drug conjugate or its tautomer, stereoisomer or pharmaceutically acceptable salt as described in any one of claims 28-42, a fusion protein as described in claim 43, a chimeric antigen receptor polypeptide or nucleic acid molecule expressing the same as described in claim 44, or a genetically modified cell as described in claim 45. The use of the anti-FRα antibody or its antigen-binding fragment according to any one of claims 1-10, the bispecific antibody according to any one of claims 14-24, the antibody-drug conjugate and its tautomers, stereoisomers or pharmaceutically acceptable salts according to any one of claims 28-42, the fusion protein according to claim 43, the chimeric antigen receptor polypeptide or nucleic acid molecule expressing the same according to claim 44, the genetically modified cell according to claim 45, or the pharmaceutical composition according to claim 46 in the preparation of a medicament for treating an individual tumor; preferably, the tumor is a tumor whose tumor cells express FRα (FRα+); more preferably, the tumor is a solid tumor or hematologic malignancy associated with high FRα expression; even more preferably, the tumor is ovarian cancer or oral cancer; more preferably, the tumor is ovarian cancer.

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