MSLN antibody-drug conjugates

Abstract

Provided is an antibody-drug conjugate that is a compound represented by formula (I), or a tautomer or pharmaceutically acceptable salt thereof: Ab-[SX-(Y) n -ZED] q (I), wherein Ab is an antibody or antigen-binding fragment thereof that specifically binds to mesothelin (MSLN), and D is a biologically active molecule drug or a derivative thereof. Provided is a pharmaceutical composition comprising the antibody-drug conjugate represented by formula (I). Provided is use of the antibody-drug conjugate represented by formula (I) and its pharmaceutical composition in the preparation of a drug for treating cancer.

Description

[Technical Field]

[0001] The present invention relates to the field of targeted therapy, and more particularly to the field of antibody-drug conjugates. [Background technology]

[0002] Mesothelin (MSLN) is a cell surface molecule expressed as a 71 kD precursor protein, which is further processed to a 40 kD glycoprotein anchored to the cell surface by glycosylphosphatidylinositol (GPI). Expression of MSLN in normal tissues is highly restricted. MSLN is expressed in mesothelial cells lining the pleura, pericardium, and peritoneum, and appears to play a role in cell adhesion (Chang et al. (1996), PNAS, 93:136-140). Soluble truncated MSLN has also been suggested to play a role in megakaryocyte stimulation, but its biological function remains unclear because knockout of soluble truncated MSLN in mice showed no developmental defects (Yamaguchi et al. (1994), Journal of Biological Chemistry, 269(2):805-8; Bera et al. (2000), Molecular and Cellular Biology, 20(8):2902-6).

[0003] In contrast, MSLN is highly expressed in certain human cancers, including nearly all mesotheliomas and pancreatic cancers, as well as in approximately 70% of ovarian cancers and 50% of lung adenocarcinomas (Hassan and Ho (2008), European Journal of Cancer, 44:46-53; Miettinen and Sarlomo-Rikala (2003), The American Journal of Surgical Pathology, 27:150-8; Ordonez (2003), The American Journal of Surgical Pathology, 27:1418-1428; Ho et al. (2007), Clinical Cancer Research, 13:1571-5). Because MSLN plays an important role in promoting tumor growth and invasion, high MSLN expression levels make it an attractive candidate for targeted therapy (Servais et al. (2012), Clinical Cancer Research, 18(9):2478-2489).

[0004] MSLN interacts with MUC16, mediates cell adhesion, plays an important role in peritoneal implantation of ovarian cancer cells, and increases the motility and invasiveness of pancreatic cancer cells (Rump et al. (2004), Journal of Biological Chemistry, 279(10):9190-8; Gubbels et al. (2006), Molecular Cancer, 5(1):50; Coehlo et al., Expert Review of Anticancer Therapy, 18(2):177-186; Chen et al. (2013), Scientific Reports, 3:1870). In particular, pancreatic tumors often progress too rapidly for patients to notice symptoms of the disease, resulting in a very short average survival time. These tumors typically cannot be (completely) removed by surgical resection and have already metastasized, resulting in a poor prognosis. Furthermore, chemotherapy cannot significantly improve the survival time or cure rate of these tumors. Despite multiple attempts by scientists, antibodies targeting MSLN alone still show deficiencies in efficacy and are not a good targeted therapeutic tool. Currently, amatuximab, the fastest-developing and best-known anti-mesothelin monoclonal antibody, has still shown disappointing results in clinical trials (Baldo and Cecco (2017), Onco. Targets Ther. 10:5337-5353; Nicolaides et al. (2018), Cancer Biology & Therapy, 19(7):622-630).

[0005] To further address this issue, antibody-drug conjugates (ADCs) have been introduced into MSLN-targeted therapy. Compared with conventional antibodies, MSLN-targeted ADCs offer superior tumor-killing efficacy due to the combination of antibody targeting ability and small molecule drug efficacy. Currently, the most advanced ADC in this field is anetumab ravtansine, developed by Bayer. Its phase I clinical data showed good safety and efficacy. It is currently being tested in multiple tumor types with high MSLN expression. Therefore, there is an urgent need for targeted therapy against MSLN in this field. However, in recent years, the development of ADC drugs has accelerated, and better linker-toxin systems are continuously emerging. However, both the conventional non-degradable linker and DM4 toxin used in anetumab ravtansine have problems such as high drug toxicity, a narrow therapeutic window, and a low effective drug release rate in target organs (WO 2010 / 124797 A1). In addition, MSLN itself is cleaved by various proteases at juxtamembrane sites to generate soluble MSLN (sMSLN) (Molecular Cancer Therapeutics; 15(7), July 2016). Such soluble MSLN has a sequence highly similar to that of membrane-anchored MSLN and is therefore recognized by most therapeutic antibodies targeting MSLN, including anetumab and amatuximab. Experiments have demonstrated that soluble MSLN competitively binds to MSLN antibodies, thereby impairing the efficacy of MSLN-targeted therapy.

[0006] Therefore, there is currently an urgent need in the field to develop superior ADCs targeting MSLN that, on the one hand, exhibit enhanced antitumor efficacy and improved plasma stability, and, on the other hand, are less affected by soluble MSLN in terms of their efficacy in inhibiting tumor growth. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides antibody-drug conjugates that target MSLN and their use for treating cancer. [Means for solving the problem]

[0008] In a first aspect, the present invention provides an antibody-drug conjugate that is a compound of formula (I), or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof: Ab-[SX-(Y) n -ZED] q (I), During the ceremony: Ab is an antibody that specifically binds to mesothelin (MSLN) or an antigen-binding fragment of MSLN; S is a sulfur bond connecting Ab and X; X is a 3- to 10-membered cycloalkyl, a 5- to 10-membered heterocyclic group, C 6~10 aryl, or 5-10 membered heteroaryl group; X may optionally be R * is substituted with 1, 2, 3, 4, or 5 instances of; R * H, deuterium, halogen, CN, NO2, OH, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenyl, or C 2~6 alkynyl; Y is -Y1-Y2-Y3-, where Y is linked to X through Y1 and to Z through Y3; During the ceremony, Y1 is -C(O)-, -C(O)O-, -OC(O)-, -C(O)NH-, C 2~6 Alkenyl, C 2~6 Alkynyl, 3- to 10-membered cycloalkyl, 5- to 10-membered heterocyclic group, C 6~10 aryl, or 5-10 membered heteroaryl group; Y2 is a bond or C 1~6 is alkylene; Y3 is selected from -C(O)-, -OC(O)-, or -NHC(O)-; Y is optionally substituted with 1, 2, 3, 4, or 5 R# substituents; R# is H, deuterium, halogen, CN, NO2, OH, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenyl, or C 2~6 alkynyl; Z is a peptide containing 2 to 8 amino acids, preferably a dipeptide, tripeptide, or tetrapeptide; E is absent or optionally a spacer unit connecting Z and D; D is a biologically active molecular drug or a derivative thereof; n is selected from 0, 1, 2, 3, 4, or 5, preferably 0, 1, 2, or 3; q is an integer selected from 1 to 10, and is preferably 8.

[0009] In some embodiments, the antibody-drug conjugate comprises a compound of formula (II):

[0010] [ka]

[0011] or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof; During the ceremony, Ab is anti-MSLN antibody; R1 and R2 are independently C 1~6 Alkyl, preferably C 1~4 alkyl; D is

[0012] [ka]

[0013] and; q is an integer selected from 1 to 10, for example, 4, 5, 6, 7, 8, 9, or 10, and preferably 8.

[0014] In some embodiments, the antibody-drug conjugate comprises the following compound:

[0015] [ka]

[0016] or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof; Ab is anti-MSLN antibody; q is an integer selected from 1 to 10, for example, 4, 5, 6, 7, 8, 9, or 10, and preferably 8.

[0017] In some embodiments, the antibody-drug conjugate comprises the following compound:

[0018] [ka]

[0019] or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof; Ab is anti-MSLN antibody; q is an integer selected from 1 to 10, for example, 4, 5, 6, 7, 8, 9, or 10, and preferably 8.

[0020] In some embodiments, the antibody-drug conjugate comprises the following compound:

[0021] [ka]

[0022] or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof; Ab is anti-MSLN antibody; q is an integer selected from 1 to 10, for example, 4, 5, 6, 7, 8, 9, or 10, and preferably 8.

[0023] In some embodiments, the antibody-drug conjugate comprises the following compound:

[0024] [ka]

[0025] or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof; Ab is anti-MSLN antibody; q is an integer selected from 1 to 10, for example, 4, 5, 6, 7, 8, 9, or 10, and preferably 8.

[0026] In some embodiments, the antibody-drug conjugate comprises the following compound:

[0027] [ka]

[0028] or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof; where Ab is an anti-MSLN antibody; q is an integer selected from 1 to 10, for example, 4, 5, 6, 7, 8, 9, or 10, and preferably 8.

[0029] In some embodiments, the antibody Ab in the antibody-drug conjugate of the invention is an antibody that specifically binds to mesothelin (MSLN) or an antigen-binding fragment thereof, and the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), (1) the VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 27; (2) the VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 25, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 28; (3) the VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 26, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 27; (4) the VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 29; (5) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 30; (6) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 31; (7) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 32; (8) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 33; (9) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 25, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 29; (10) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 63, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (11) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 64, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (12) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 65, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (13) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 65, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 68; (14) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 63, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 68; (15) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 66, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (16) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 64, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 69; (17) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 66, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 69; (18) The VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 63, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 69; or (19) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 65, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 69.

[0030] In some embodiments, the antibody Ab in the antibody-drug conjugate of the invention is an antibody that specifically binds to mesothelin (MSLN) or an antigen-binding fragment thereof, and the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), (1) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13; (2) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 15, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 16, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18; (3) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 15, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13; (4) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 19; (5) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 20; (6) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 21; (7) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 22; (8) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23; (9) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 15, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 16, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 19; (10) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 45, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 52, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (11) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 52, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (12) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 59, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 52, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (13) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 59, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 61, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (14) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 45, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 61, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (15) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 60, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 52, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (16) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 62, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (17) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 60, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 62, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (18) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 45, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 62, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; or (19) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 59, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 62, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56.

[0031] In some embodiments, the antibody Ab in the antibody-drug conjugate of the invention is an antibody that specifically binds to mesothelin (MSLN) or an antigen-binding fragment thereof, and the antibody comprises a VH and a VL, (1) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:27; (2) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:25, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:28; (3) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 27; (4) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29; (5) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30; (6) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31; (7) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32; (8) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33; (9) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 25, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29; (10) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (11) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (12) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 65, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (13) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 65, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68; (14) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68; (15) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 66, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (16) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; (17) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 66, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; (18) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; or (19) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 65, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69.

[0032] In some embodiments, the antibody Ab in the antibody-drug conjugate of the invention is an antibody that specifically binds to mesothelin (MSLN) or an antigen-binding fragment thereof, the antibody comprising a heavy chain (HC) and a light chain (LC), (1) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37; (2) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38; (3) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 36, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37; (4) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39; (5) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 40; (6) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 41; (7) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42; (8) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43; (9) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39; (10) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (11) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (12) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (13) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 75; (14) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 75; (15) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (16) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76; (17) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76; (18) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76; or (19) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76.

[0033] In some embodiments, the antibody-drug conjugate of the invention comprises an antibody that specifically binds to mesothelin (MSLN), wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0034] In some embodiments, the antibody-drug conjugate of the invention comprises an antigen-binding fragment that specifically binds to mesothelin, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, and ds-scFv.

[0035] In some embodiments, the antibody-drug conjugate of the invention comprises an antibody or antigen-binding fragment thereof that specifically binds to mesothelin, wherein the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0036] In a second aspect, the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate of the present invention and, optionally, a pharmaceutically acceptable carrier or excipient.

[0037] In some embodiments, the composition of the present invention further comprises a second therapeutic agent, wherein the second therapeutic agent is selected from a chemotherapeutic agent, a monoclonal antibody drug, a bispecific / multispecific antibody drug, a recombinant protein drug, a nucleotide drug (siRNA, antisense oligonucleotide), a small molecule drug, an immunomodulatory drug, and a cell therapy drug.

[0038] In a third aspect, the present invention provides a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody-drug conjugate or pharmaceutical composition of the present invention. In some embodiments, the cancer is selected from colon cancer, mesothelioma, ovarian cancer, lung cancer, breast cancer, pancreatic cancer, gastric cancer, prostate cancer, squamous cell carcinoma, and cholangiocarcinoma. In some embodiments, the method of the present invention further comprises administering to the subject a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from a chemotherapeutic agent, a monoclonal antibody drug, a bispecific / multispecific antibody drug, a recombinant protein drug, a nucleotide drug (siRNA, antisense oligonucleotide), a small molecule drug, an immunomodulatory drug, and a cell therapy drug.

[0039] In a fourth aspect, the present invention provides an antibody-drug conjugate or pharmaceutical composition of the present invention for use in treating cancer. In some embodiments, the cancer is selected from colon cancer, mesothelioma, ovarian cancer, lung cancer, breast cancer, pancreatic cancer, gastric cancer, prostate cancer, squamous cell carcinoma, and cholangiocarcinoma. In some embodiments, the antibody or pharmaceutical composition of the present invention is used in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from a chemotherapeutic agent, a monoclonal antibody drug, a bispecific / multispecific antibody drug, a recombinant protein drug, a nucleotide drug (siRNA, antisense oligonucleotide), a small molecule drug, an immunomodulatory drug, and a cell therapy drug.

[0040] In a fifth aspect, the present invention provides use of the antibody-drug conjugate or pharmaceutical composition of the present invention in preparing a medicament for treating cancer. In some embodiments, the cancer is selected from colon cancer, mesothelioma, ovarian cancer, lung cancer, breast cancer, pancreatic cancer, gastric cancer, prostate cancer, squamous cell carcinoma, and cholangiocarcinoma. In some embodiments, the drug is used in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from a chemotherapeutic agent, a monoclonal antibody drug, a bispecific / multispecific antibody drug, a recombinant protein drug, a nucleotide drug (siRNA, antisense oligonucleotide), a small molecule drug, an immunomodulatory drug, and a cell therapy drug. [Brief explanation of the drawings]

[0041] [Figure 1] Binding of antibody PR300186 and its PTM variants PR300186-2, PR300186-3, PR300186-4, PR300186-5, PR300186-6, PR300186-7, PR300186-8, PR300186-9, and PR300186-10 to human MSLN protein. [Figure 2]Binding of antibody PR300186 and its PTM variants PR300186-2, PR300186-3, PR300186-4, PR300186-5, PR300186-6, PR300186-7, PR300186-8, PR300186-9, and PR300186-10 to cynomolgus monkey MSLN protein. [Figure 3] Binding of PR300159 and its PTM variants PR300159-1, PR300159-3, PR300159-5, PR300159-6, PR300159-8, and PR300159-9 to COV644 cells. [Figure 4] Binding of PR300186 and its PTM variants PR300186-2, PR300186-3, PR300186-4, PR300186-5, PR300186-6, PR300186-7, PR300186-8, PR300186-9 (Figure 4A), and PR300186-10 (Figure 4B) to COV644 cells. [Figure 5] Internalization of PR300159 and its PTM variants PR300159-1, PR300159-3, PR300159-4, PR300159-5, PR300159-6, PR300159-7, PR300159-8, and PR300159-9 in COV644 cells. [Figure 6] Internalization of PR300186 and its PTM variants PR300186-3, PR300186-9, and PR300186-10 in COV644 cells. [Figure 7] Internalization of PR300159-8 and PR300186-10 in COV644 cells. [Figure 8] Internalization of PR300159-8 and PR300186-10 in NCI-H226 cells. [Figure 9] Internalization of PR300159-8 and PR300186-10 in HPAC cells. [Figure 10] MSLN shedding in the HT29-huMSLN wild-type 2C3 cell line and the HT29-huMSLN mutant 1A11 cell line. [Figure 11] Cytotoxicity of antibody-drug conjugate PR300159-8-B81 against huMSLN-expressing HT29-huMSLN cells. [Figure 12] Stability results of PR300159-8-B81 in human, cynomolgus monkey, SD rat, and ICR mouse plasma, and in phosphate-buffered saline (PBS). [Figure 13] Stability results of PR300186-10-B81 in human, cynomolgus monkey, SD rat, and ICR mouse plasma, and in phosphate-buffered saline (PBS). [Figure 14] Tumor volume in mice treated with various doses of PR300159-8-B81 and PR300186-10-B81 in the HT29-huMSLN wild-type 2C3 model. [Figure 15] Body weights of mice treated with different doses of PR300159-8-B81 and PR300186-10-B81 in the HT29-huMSLN wild-type 2C3 model. [Figure 16] Tumor volume in mice treated with various doses of PR300159-8-B81 and PR300186-10-B81 in the HT29-huMSLN mutant 1A11 model. [Figure 17] Body weights of mice treated with different doses of PR300159-8-B81 and PR300186-10-B81 in the HT29-huMSLN mutant 1A11 model. [Figure 18] Tumor volume in mice treated with different doses of PR300159-8-B81 and PR300186-10-B81 in the COV644 model. [Figure 19] Body weight of mice treated with different doses of PR300159-8-B81 and PR300186-10-B81 in the COV644 model. [Figure 20] Tumor volume in mice treated with various doses of PR300159-8-DXD, PR300159-8-B81, and PR300186-10-B81 in the OVCAR3 model. [Figure 21] Body weights of mice treated with various doses of PR300159-8-DXD, PR300159-8-B81, and PR300186-10-B81 in the OVCAR3 model. [Figure 22] Tumor volume in mice treated with different doses of PR300159-8-B81 and PR300186-10-B81 in the SKOV3 model. [Figure 23] Body weight of mice treated with different doses of PR300159-8-B81 and PR300186-10-B81 in the SKOV3 model. [Figure 24] Tumor volume in mice treated with 1 mg / kg PR300159-8-DXD, BAY-949343, PR300159-8-B81, and PR300186-10-B81 in the NCI-H226 model. [Figure 25] Tumor volume in mice treated with 5 mg / kg PR300159-8-DXD, BAY-949343, PR300159-8-B81, and PR300186-10-B81 in the NCI-H226 model. [Figure 26] Body weight changes in mice treated with 1 mg / kg PR300159-8-DXD, BAY-949343, PR300159-8-B81, and PR300186-10-B81 in the NCI-H226 model. [Figure 27] Body weight changes in mice treated with 5 mg / kg PR300159-8-DXD, BAY-949343, PR300159-8-B81, and PR300186-10-B81 in the NCI-H226 model. [Figure 28] Binding of PR300159-B71, PR300159, PR300186-B71, and PR300186 to CHOK-huMSLN cells. [Figure 29] Binding of PR300159-B81, PR300159, PR300186-B81, and PR300186 to COV644 cells. [Figure 30]Internalization of PR300159, PR300159-B71, PR300186, and PR300186-B71 in COV644 cells. [Figure 31] Cytotoxicity of PR300159 and PR300159-B71 antibody-drug conjugates against CHOK1-huMSLN cells expressing huMSLN (Figure 31A); and cytotoxicity of PR300186 and PR300186-B71 antibody-drug conjugates against CHOK1-huMSLN cells expressing huMSLN (Figure 31B). [Figure 32] Tumor volume (Figure 32A) and body weight (Figure 32B) of mice treated with various doses of PR300159-B71, PR300159-B81, and PR300186-B81 in the COV644 mouse tumor model. DETAILED DESCRIPTION OF THE INVENTION

[0042] The following detailed description of the following embodiments, taken in conjunction with the accompanying drawings, will provide a clearer understanding of the above-noted and other features and advantages of the present invention.

[0043] The embodiments described herein with reference to the accompanying drawings are exemplary and explanatory and are intended to provide a general understanding of the present invention, and should not be construed as limiting the scope of the present invention.

[0044] definition chemical definition The following sections provide more detailed descriptions of specific functional groups and chemical terminology.

[0045] When numerical ranges are provided, all values and subranges within the stated range are included. For example, "C 1~6 "Alkyl" refers to C1, C2, C3, C4, C5, C6, C 1~6 , C 1~5 , C 1~4 , C 1~3 , C 1~2 , C2~6 , C 2~5 , C 2~4 , C 2~3 , C 3~6 , C 3~5 , C 3~4 , C 4~6 , C 4~5 , and C 5~6 Contains alkyl.

[0046] "C 1~6 "Alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 6 carbon atoms. In some embodiments, C 1~4 Alkyl and C 1~2 Alkyl is preferred. 1~6 Examples of alkyl include: methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). 1~6 "Alkyl" includes heteroalkyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, or phosphorus). The alkyl group may be substituted with one or more substituents, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Common alkyl abbreviations include: Me(-CH), Et(-CHCH), iPr(-CH(CH)), nPr(-CHCHCH), n-Bu(-CHCHCHCHCH), or i-Bu(-CHCH(CH)).

[0047] "C 2~6 "Alkenyl" refers to a straight or branched chain hydrocarbon group having 2 to 6 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 2~4 Alkenyl is preferred. 2~6Examples of alkenyl include: vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. 2~6 "Alkenyl" includes heteroalkenyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, or phosphorus). The alkenyl group may be substituted with one or more substituents, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0048] "C 2~6 "Alkynyl" refers to a straight or branched chain hydrocarbon group having 2 to 6 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 2~4 Alkynyl is preferred. 2~6 Examples of alkynyl include, but are not limited to: ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentynyl (C5), hexynyl (C6), and the like. 2~6 "Alkynyl" includes heteroalkynyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, or phosphorus). The alkynyl group may be substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0049] "C 0~6 Alkylene is a chemical bond or "C 1~6 It refers to alkylene.

[0050] "C 1~6 "Alkylene" is C 1~6 It refers to a divalent group formed by removing another hydrogen from an alkyl, which may be substituted or unsubstituted. 1~4Alkylene, C 2~4 Alkylene, and C 1~3 Alkylene is preferred. Unsubstituted alkylene includes, but is not limited to: methylene (-CH-), ethylene (-CHCH-), propylene (-CHCHCH-), butylene (-CHCHCHCHCH-), pentylene (-CHCHCHCHCHCH-), hexylene (-CHCHCHCHCHCHCH-), and the like. Examples of substituted alkylenes, e.g., substituted with one or more alkyls (e.g., methyl), include, but are not limited to, substituted methylene (-CH(CH3)-, -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, -CH2C(CH3)2-), and substituted propylene (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), and the like.

[0051] "Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0052] Therefore, "C 1~6 Haloalkyl refers to any of the above "C" groups substituted with one or more halogen groups. 1~6 In some embodiments, C 1~4 Haloalkyl is particularly preferred, C 1~2 Haloalkyl is more preferred. Exemplary haloalkyl include, but are not limited to: -CF, -CHF, -CHFCHF, -CHCHF, -CFCF, -CCl, -CHCl, -CHCl, 2,2,2-trifluoro-1,1-dimethyl-ethyl, and the like. Haloalkyl groups can be substituted with, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent at any available point of attachment.

[0053] "C 3~10"Cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and no heteroatoms. 3~10 "Cycloalkyl" is a saturated ring. 3~10 A "cycloalkyl" may be an unsaturated ring containing one, two, or three or more unsaturated bonds, such as, for example, alkenyl or alkynyl bonds. In some embodiments, C 3~7 Cycloalkyl and C 3~6 Cycloalkyl is particularly preferred, C 5~6 Cycloalkyl is even more preferred. Cycloalkyl also includes ring systems in which the cycloalkyl ring is fused to one or more aryl or heteroaryl groups, and the point of attachment is on the cycloalkyl ring; in such cases, the number of carbons continues to refer to the number of carbons in the cycloalkyl system. Exemplary cycloalkyls include, but are not limited to: cyclopropyl (C), cyclopropenyl (C), cyclobutyl (C), cyclobutenyl (C), cyclopentyl (C), cyclopentenyl (C), cyclohexyl (C), cyclohexenyl (C), cyclohexadienyl (C), cycloheptyl (C), cycloheptenyl (C), cycloheptadienyl (C), cycloheptatrienyl (C), and the like. The cycloalkyl group may be substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0054] A "3- to 10-membered heterocyclic group" refers to a 3- to 10-membered non-aromatic ring group having ring carbon atoms and 1 to 5 ring heteroatoms, each heteroatom independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. In heterocyclic groups containing one or more nitrogen atoms, the point of attachment can be either a carbon atom or a nitrogen atom, as valence permits. In some embodiments, 5- to 10-membered heterocyclic groups are preferred, which are 3- to 10-membered non-aromatic ring systems having ring carbon atoms and 1 to 5 ring heteroatoms. In some embodiments, 3- to 7-membered heterocyclic groups are preferred, which are 3- to 7-membered non-aromatic ring systems having ring carbon atoms and 1 to 4 ring heteroatoms; 3- to 6-membered heterocyclic groups are preferred, which are 3- to 6-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; 4- to 8-membered heterocyclic groups are preferred, which are 4- to 8-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; and 5- to 6-membered heterocyclic groups are more preferred, which are 5- to 6-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms. Heterocyclic groups also include ring systems in which the heterocyclic ring is fused to one or more cycloalkyls and the point of attachment is on the cycloalkyl ring, or the heterocyclic ring is fused to one or more aryl or heteroaryl groups and the point of attachment is on the heterocyclic ring; in such cases, the number of ring members continues to refer to the number of ring members in the heterocyclic system. Examples of 3-membered heterocyclic groups having one heteroatom include, but are not limited to, aziridinyl, oxiranyl, and thiiranyl (thiorenyl). Examples of 4-membered heterocyclic groups having one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Examples of 5-membered heterocyclic groups having one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrole-2,5-dionyl. Examples of 5-membered heterocyclic groups having two heteroatoms include, but are not limited to, dioxolanyl, oxasulfuranyl, disulfanyl, and oxazolidin-2-one. Examples of 5-membered heterocyclic groups having three heteroatoms include, but are not limited to, triazolanyl, oxadiazolanyl, and thiadiazolanyl.Examples of 6-membered heterocyclic groups having one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Examples of 6-membered heterocyclic groups having two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithiocyclohexanyl, and dioxanyl. Examples of 6-membered heterocyclic groups having three heteroatoms include, but are not limited to, triazinanyl. Examples of 7-membered heterocyclic groups having one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Examples of 5-membered heterocyclic groups fused with a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclic groups) include, but are not limited to, dihydroindolyl, isodihydroindolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, benzoxazolinonyl, etc. Examples of 6-membered heterocyclic groups fused with a C6 aryl ring (also referred to herein as 6,6-bicyclic heterocyclic groups) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, etc. The heterocyclic group may be substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0055] "C 6~10 "Aryl" refers to a group having a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system having 6 to 10 ring carbon atoms and 0 heteroatoms (e.g., having 6 or 10 pi electrons shared in a cyclic arrangement). In some embodiments, an aryl has 6 ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, an aryl has 10 ring carbon atoms ("C 10 Aryl"; e.g., naphthyl, such as 1-naphthyl and 2-naphthyl). Aryl also includes fused ring systems in which an aryl ring is fused to one or more cycloalkyl or heterocyclic groups, and the point of attachment is on the aryl ring; in such cases, the number of carbons continues to refer to the number of carbons in the aryl ring system. Aryl groups may be substituted with one or more substituents, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0056] A "5- to 10-membered heteroaryl group" refers to a group having a 5- to 10-membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms (e.g., having 6 or 10 π-electrons shared in a cyclic arrangement), each heteroatom independently selected from nitrogen, oxygen, and sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be either a carbon atom or a nitrogen atom, as valence permits. Bicyclic heteroaryl groups can contain one or more heteroatoms in one or both rings. Heteroaryl groups also include ring systems in which the heteroaryl ring is fused to one or more cycloalkyl or heterocyclic groups, and the point of attachment is on the heteroaryl ring; in such cases, the number of carbon atoms continues to represent the number of carbon atoms in the heteroaryl ring system. In some embodiments, a "5- to 10-membered heteroaryl group" is a saturated ring. In other embodiments, the "5- to 10-membered heteroaryl group" may be, for example, an unsaturated ring containing one, two, or three or more unsaturated bonds, such as alkenyl or alkynyl bonds. In some embodiments, 5- to 6-membered heteroaryl groups that are 5- to 6-membered monocyclic or bicyclic 4n+2 aromatic ring systems having ring carbon atoms and 1 to 4 ring heteroatoms are particularly preferred. Examples of 5-membered heteroaryl groups having one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Examples of 5-membered heteroaryl groups having two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Examples of 5-membered heteroaryl groups having three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl), and thiadiazolyl. Examples of 5-membered heteroaryl groups having four heteroatoms include, but are not limited to, tetrazolyl. Examples of 6-membered heteroaryl groups having one heteroatom include, but are not limited to, pyridinyl. Examples of 6-membered heteroaryl groups having two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl.Examples of 6-membered heteroaryl groups each having 3 or 4 heteroatoms include, but are not limited to, triazinyl and tetrazinyl. Examples of 7-membered heteroaryl groups each having 1 heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Examples of 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiazolyl, isobenzothizolyl, benzofuranyl, isobenzofuranyl, benzimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indazinyl, and purinyl. Examples of 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. The heteroaryl group may be substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0057] Divalent groups formed by removing another hydrogen atom from groups such as alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic groups, etc., as defined above, aryl and heteroaryl groups are collectively referred to as "alkylene." Cyclic groups such as cycloalkyl groups, heterocyclic groups, aryl and heteroaryl groups are collectively referred to as "cyclic groups."

[0058] Alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, etc. groups as defined herein are optionally substituted groups.

[0059] Examples of substituents on carbon atoms are: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa , -ON(R bb )2, -N(R bb )2, -N(R bb )3 + X - , -N(ORcc )R bb 、-SH、-SR aa 、-SSR cc 、-C(=O)R aa 、-CO2H、-CHO、-C(OR cc )2、-CO2R aa 、-OC(=O)R aa 、-OCO2R aa 、-C(=O)N(R bb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa 、-NR bb CO2R aa 、-NR bb C(=O)N(R bb )2、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-C(=NR bb )N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2、-C(=O)NR bb SO2R aa 、-NR bb SO2R aa 、-SO2N(R bb )2、-SO2R aa 、-SO2OR aa 、-OSO2R aa 、-S(=O)R aa 、-OS(=O)R aa 、-Si(R aa )3、-OSi(R aa )3、-C(=S)N(R bb )2、-C(=O)SR aa 、-C(=S)SR aa 、-SC(=S)SR aa 、-SC(=O)SR aa 、-OC(=O)SR aa, -SC(=O)OR aa , -SC(=O)R aa , -P(=O)2R aa , -OP(=O)2R aa , -P(=O)(R aa )2, -OP(=O)(R aa )2, -OP(=O)(OR cc )2, -P(=O)2N(R bb )2, -OP(=O)2N(R bb )2, -P(=O)(NR bb )2, -OP(=O)(NR bb )2, -NR bb P(=O)(OR cc )2, -NR bb P(=O)(NR bb )2, -P(R cc )2, -P(R cc )3, -OP(R cc )2, -OP(R cc )3, -B(R aa )2, -B(OR cc )2, -BR aa (OR cc ), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups each independently have 0, 1, 2, 3, 4, or 5 R dd substituted with a group; In addition, the two geminal hydrogens on a carbon atom can be =O, =S, or =NN(R bb )2, =NNR bb C(=O)R aa , =NNR bb C(=O)OR aa , =NNR bb S(=O)2R aa , =NR bb , or =NOR cc and the like; R aaare each independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups, or two R aa The groups may combine to form a heterocyclic or heteroaryl ring, where each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group independently has 0, 1, 2, 3, 4, or 5 R dd is substituted with a group.

[0060] R bb are each independently hydrogen, -OH, or -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR cc ) OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , -P(=O)2R aa , -P(=O)(R aa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups, or two R bb The groups may combine to form a heterocyclic or heteroaryl ring, where each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group independently has 0, 1, 2, 3, 4, or 5 R dd is substituted with a group.

[0061] R ccare each independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups, or two R cc The groups may combine to form a heterocyclic or heteroaryl ring, where each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group independently has 0, 1, 2, 3, 4, or 5 R dd is substituted with a group.

[0062] R dd are each independently halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee , -ON(R ff )2, -N(R ff )2, -N(R ff )3 + X - , -N(OR ee )R ff , -SH, -SR ee , -SSR ee , -C(=O)R ee , -CO2H, -CO2R ee , -OC(=O)R ee , -OCO2R ee , -C(=O)N(R ff )2, -OC(=O)N(R ff )2, -NR ff C(=O)R ee , -NR ff CO2R ee , -NR ff C(=O)N(R ff )2, -C(=NR ff ) OR ee , -OC(=NR ff )R ee , -OC(=NR ff ) OR ee , -C(=NR ff )N(R ff )2, -OC(=NR ff )N(R ff )2, -NR ff C(=NR ff )N(R ff )2, -NRff SO2R ee , -SO2N(R ff )2, -SO2R ee , -SO2OR ee , -OSO2R ee , -S(=O)R ee , -Si(R ee )3, -OSi(R ee )3, -C(=S)N(R ff )2, -C(=O)SR ee , -C(=S)SR ee , -SC(=S)SR ee , -P(=O)2R ee , -P(=O)(R ee )2, -OP(=O)(R ee )2, -OP(=O)(OR ee )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups each independently have 0, 1, 2, 3, 4, or 5 R gg or two geminal R dd The substituents may combine to form =O or =S.

[0063] R ee are each independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, and heteroaryl groups, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups each independently have 0, 1, 2, 3, 4, or 5 R gg is substituted with a group.

[0064] R ff are each independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups, or two R ffThe groups may combine to form a heterocyclic or heteroaryl ring, where each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group independently has 0, 1, 2, 3, 4, or 5 R gg is substituted with a group.

[0065] R gg are each independently halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1~6 Alkyl, -ON(C 1~6 alkyl)2, -N(C 1~6 alkyl)2, -N(C 1~6 alkyl)3 + X - , -NH(C 1~6 alkyl)2 + X - , -NH2(C 1~6 alkyl) + X - , -NH3 + X - , -N(OC 1~6 Alkyl)(C 1~6 alkyl), -N(OH)(C 1~6 alkyl), -NH(OH), -SH, -SC 1~6 Alkyl, -SS(C 1~6 alkyl), -C(=O)(C 1~6 alkyl), -CO2H, -CO2(C 1~6 alkyl), -OC(=O)(C 1~6 alkyl), -OCO2(C 1~6 alkyl), -C(=O)NH2, -C(=O)N(C 1~6 alkyl)2, -OC(=O)NH(C 1~6 alkyl), -NHC(=O)(C 1~6 alkyl), -N(C 1~6 alkyl)C(=O)(C 1~6 alkyl), -NHCO2(C 1~6 alkyl), -NHC(=O)N(C 1~6 alkyl)2, -NHC(=O)NH(C 1~6 alkyl), -NHC(=O)NH2, -C(=NH)O(C 1~6alkyl), -OC(=NH)(C 1~6 alkyl), -OC(=NH)OC 1~6 Alkyl, -C(=NH)N(C 1~6 alkyl)2, -C(=NH)NH(C 1~6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1~6 alkyl)2, -OC(NH)NH(C 1~6 alkyl), -OC(NH)NH2, -NHC(NH)N(C 1~6 alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1~6 alkyl), -SO2N(C 1~6 alkyl)2, -SO2NH(C 1~6 alkyl), -SO2NH2, -SO2C 1~6 Alkyl, -SO2OC 1~6 Alkyl, -OSO2C 1~6 Alkyl, -SOC 1~6 Alkyl, -Si(C 1~6 alkyl)3, -OSi(C 1~6 alkyl)3, -C(=S)N(C 1~6 alkyl)2, C(=S)NH(C 1~6 alkyl), C(=S)NH2, -C(=O)S(C 1~6 alkyl), -C(=S)SC 1~6 Alkyl, -SC(=S)SC 1~6 Alkyl, -P(=O)2(C 1~6 alkyl), -P(=O)(C 1~6 alkyl)2, -OP(=O)(C 1~6 alkyl)2, -OP(=O)(OC 1~6 Alkyl)2, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~ C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C6-C 10 Aryl, C3-C7 heterocyclic groups, and C5-C 10 a heteroaryl group or two geminal R gg The substituents may combine to form =O or =S, and X - is the counterion.

[0066] Exemplary substituents on a nitrogen atom include: hydrogen, —OH, —OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR bb )R aa , -C(=NR CC ) OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R CC , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , -P(=O)2R aa , -P(=O)(R aa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc ) 2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups, or two R bonded to a nitrogen atom. cc The groups combine to form a heterocyclic or heteroaryl ring, where the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups each independently contain 0, 1, 2, 3, 4, or 5 R dd group, where R aa , R bb , R cc , and R dd is as defined above.

[0067] Other definitions In the description herein, reference to the terms "embodiments," "some embodiments," "examples," "specific examples," or "some examples" means that the specific features, structures, materials, or characteristics described in connection with that embodiment or example are included in at least one embodiment or example of the present invention. References to the above terms in this specification do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples, as appropriate. Furthermore, where no inconsistency exists, a person skilled in the art may combine and integrate the various embodiments or examples described herein and the features of the various embodiments or examples.

[0068] Unless otherwise specified or clearly contradicted by context, the articles "a," "a type," and "the," as used herein, are intended to include "at least one" or "one or more." Thus, as used herein, an article refers to one or more (i.e., at least one) object. For example, a "component" refers to one or more components, meaning that multiple components may be considered in making or using the described embodiments.

[0069] Unless otherwise indicated or defined, the term "comprise" and variations thereof (such as "include" and "have") should be understood to include a particular element or step or group of elements or steps, but not to exclude any other elements, steps, or other groups of elements or steps. The term "comprise" encompasses the meanings of "comprise" and "consist of." For example, a composition "comprising" X may consist solely of X, or may include additional components, e.g., the composition may consist of X+Y.

[0070] The term "approximately" in reference to a numerical value x is optional and refers, for example, to x+10% or x±5%.

[0071] "Stereoisomers" refer to compounds that have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometric isomers (cis / trans isomers), atropisomers, etc.

[0072] "Chirality" refers to a molecule that cannot be superimposed on its mirror image, while "achirality" refers to a molecule that can be superimposed on its mirror image.

[0073] "Enantiomers" refer to two isomers of a compound that are non-superimposable mirror images of one another.

[0074] "Diastereomer" refers to a stereoisomer with two or more chiral centers and whose molecules are not mirror images of one another. Diastereomers exhibit different physical properties, such as melting points, boiling points, spectral properties, and reactivities. Diastereomeric mixtures can be separated using high-resolution analytical techniques such as electrophoresis and chromatography, e.g., HPLC.

[0075] The stereochemical definitions and rules used in the present invention generally follow those outlined in S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York, and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds," John Wiley & Sons, Inc., New York, 1994.

[0076] Many organic compounds exist in optically active forms, meaning they have the ability to rotate the plane of plane-polarized light. When describing optically active compounds, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its one or more chiral centers. The prefixes d and l or (+) and (-) are symbols used to indicate the rotation of plane-polarized light induced by the compound, with (-) or l indicating that the compound is levorotatory. Compounds with the prefix (+) or d are dextrorotatory. Specific types of stereoisomers are enantiomers, and a mixture of enantiomers is called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur when there is no stereoselection or stereospecificity in a chemical reaction or process.

[0077] The present invention discloses that any asymmetric atom (e.g., carbon) in the compound can be present in a racemic or enantiomerically enriched form, such as the (R)-, (S)-, or (R,S)-configuration. In certain embodiments, each asymmetric atom exhibits at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess with respect to the (R)- or (S)-configuration.

[0078] Depending on the selection of starting materials and methods, the compounds of the present invention may exist as one of the possible isomers or mixtures thereof, such as racemates and diastereomeric mixtures (depending on the number of asymmetric carbon atoms). Optically active (R)- or (S)-isomers can be prepared using chiral auxiliaries or chiral reagents or separated using conventional techniques. If the compound contains a double bond, the substituents may exhibit E- or Z-configuration; if the compound contains a disubstituted cycloalkyl, the substituents on the cycloalkyl may have cis- or trans-configuration.

[0079] Any resulting mixture of stereoisomers can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on the differences in the physicochemical properties of their components, for example, by chromatography and / or stepwise crystallization methods.

[0080] Any racemic mixture of final products or intermediates obtained can be resolved into their optical enantiomers using methods known to those skilled in the art, such as by separating the resulting diastereomeric salts thereof. Racemic products can also be separated using chiral chromatography, such as high performance liquid chromatography (HPLC) using a chiral adsorbent. Specifically, enantiomers can be separated using methods described, for example, in Jacques, et al., Enantiomers, Racemates, and Resolutions (Wiley Interscience, New York, 1981); Principles of Asymmetric Synthesis (2 nd Ed., Robert E. Gawley, Jeffrey Aube, Elsevier, Oxford, UK, 2012); Eliel, EL Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, SH, Tables of Resolving Agents and Optical Resolutions, p. 268 (EL Eliel, Ed., University of Notre Dame Press, Notre Dame, IN 1972); and Chiral Separation Techniques: A Practical Approach (Subramanian, G. Ed., Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2007).

[0081] The term "tautomer" or "tautomeric form" refers to structural isomers differing in energy that can be interconverted via a low energy barrier. If tautomerism is possible (e.g., in solution), a chemical equilibrium between tautomers can be established. For example, proton tautomers (also called prototropic tautomers) include interconversions via proton transfer, such as keto-enol tautomerism and imine-enamine tautomerism. Valence tautomers include interconversions via rearrangement of some bond electrons. A specific example of keto-enol tautomerism is the interconversion between the tautomers of pentane-2,4-dione and 4-hydroxy-pent-3-en-2-one. Another example of tautomerism is phenol-ketone tautomerism. A specific example of phenol-ketone tautomerism is the interconversion between the tautomers of pyridin-4-ol and pyridin-4(1H)-one. Unless otherwise specified, all tautomeric forms of the compounds of the present invention are within the scope of the present invention.

[0082] The term "pharmaceutically acceptable salts" as used herein refers to both organic and inorganic salts of the compounds described herein. Pharmaceutically acceptable salts are well known in the art, as detailed in S.M. Berge et al., Journal of Pharmaceutical Sciences, 1977, 66:1-19. Salts formed with non-toxic pharmaceutically acceptable acids include, but are not limited to, inorganic acid salts formed by reaction with an amino group, such as hydrochloride, hydrobromide, phosphate, sulfate, and perchlorate, and organic acid salts thus formed, such as acetate, oxalate, maleate, tartrate, citrate, succinate, and malonate, or salts obtained by other methods described in books and literature, such as ion exchange methods. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, gluceptate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydrogen phosphate, and the like. Salts obtained with suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N-methyl-N ... +(C1-C4 alkyl)4 salts are also included. The present invention also promotes quaternary ammonium salts formed with compounds of any N-containing group. Water- or oil-soluble or dispersible products can be obtained by quaternization. Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Pharmaceutically acceptable salts further include suitable non-toxic ammonium salts, quaternary ammonium salts, and ammonium cations formed with counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C1-8 sulfonates, and aromatic sulfonates.

[0083] Unless otherwise indicated or defined, all terms used herein have meanings that are commonly understood in the art and that are apparent to those of ordinary skill in the art. For example, Leuenberger, HGW, Nagel, B., and K1bl, H. eds., “A Multilingual Glossary of Biotechnological Terms: (IUPAC Recommendations),” Helvetica Chimica Acta (1995), CH-4010 Basel, Switzerland; Sambrook et al., “Molecular Cloning: A Laboratory Manual” (2nd Ed.), Vols.1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al., eds., “Current Protocols in Molecular Biology,” Green Publishing and Wiley InterScience, New York (1987); Roitt et al., “Immunology” (6th Ed.), Mosby / Elsevier, Edinburgh (2001); and Janeway et al. al., “Immunobiology” (6th Ed.), Garland Science Publishing / Churchill Livingstone, New Standard manuals such as York (2005) can be consulted.

[0084] The term "subject" as used herein refers to an animal. Typically, an animal is a mammal. For example, a subject may refer to a primate (e.g., a human male or female), cow, sheep, goat, horse, dog, cat, rabbit, rat, mouse, fish, bird, etc. In certain embodiments, the subject is a primate. In other embodiments, the subject is a human.

[0085] As used herein, the term "patient" refers to a human (including adults and children) or other animal. In some embodiments, the term "patient" refers to a human.

[0086] The terms "antibody-drug conjugate," "antibody conjugate," "conjugate," "immunoconjugate," and "ADC" are used interchangeably to refer to an antibody or antigen-binding fragment thereof covalently attached to a drug. The drug is typically a small molecule drug, toxin, cytotoxic agent, therapeutic agent, etc.

[0087] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a particular antigen. Antibodies typically comprise a heavy chain and a light chain; the heavy chain comprises a heavy chain variable region and a heavy chain constant region, and the light chain comprises a light chain variable region and a light chain constant region. The variable regions of the heavy and light chains of an antibody contain binding domains that interact with an antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors. Host tissues or factors include various cells of the immune system (e.g., effector cells) and components of the complement system (e.g., C1q). Most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL), which together form the portion of the antibody that binds to the antigen.

[0088] Both the "light chain variable region" (VL) and the "heavy chain variable region" (VH) contain four "framework" regions separated by three "complementarity-determining regions" or "CDRs." The framework regions align the CDRs to enable specific binding of the CDRs to an antigen epitope. The CDRs contain the amino acid residues primarily responsible for antigen binding. From the amino to carboxyl terminus, both the VL and VH domains contain the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDR1, CDR2, and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; CDR1, CDR2, and CDR3 of the VH domain are referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0089] The assignment of amino acids to each VL and VH domain follows any conventional definition of a CDR, including the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)) or the Chothia definition (Chothia & Lesk, Journal of Molecular Biology, 196:901-917, 1987; Chothia et al., Nature, 342:878-883, 1989); Chothia-Kabat CDR complexes (also called combined Chothia and Kabat CDRs) (each CDR is a complex of a Chothia CDR and a Kabat CDR); the AbM definition used by Oxford Molecular antibody modeling software; and the contact definition by Martin et al. (See the World Wide Web at bioinfo.org.uk / abs for the aforementioned conventional CDR definition systems). Kabat provides a widely used numbering system (the Kabat numbering system) in which corresponding residues between different heavy chains or different light chains are assigned the same number.

[0090] Although the present invention encompasses CDRs defined according to any of these numbering systems, preferred embodiments encompass CDRs defined by the combined Chothia and Kabat definitions.

[0091] [Table 1]

[0092] In Table 1, Laa to Lbb can refer to the amino acid sequence from position aa (according to the Chothia numbering system) to position bb (according to the Chothia numbering system) starting from the N-terminus of the antibody light chain; Haa to Hbb can refer to the amino acid sequence from position aa (according to the Chothia numbering system) to position bb (according to the Chothia numbering system) starting from the N-terminus of the antibody heavy chain. For example, L24 to L34 can refer to the amino acid sequence from positions 24 to 34 (according to the Chothia numbering system) starting from the N-terminus of the antibody light chain; H26 to H32 can refer to the amino acid sequence from positions 26 to 32 (according to the Chothia numbering system) starting from the N-terminus of the antibody heavy chain.

[0093] The term "antibody" as used herein should be understood in its broadest sense and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two different antigen-binding regions. Antibodies may contain additional modifications such as unnatural amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing antibody antigenic determinants, and any other immunoglobulin molecules with modifications to the antigen recognition site, so long as the antibody exhibits the desired biological activity.

[0094] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., MSLN). It has been demonstrated that fragments of full-length antibodies can perform the antigen-binding function of an antibody.

[0095] Examples of antigen-binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment composed of the VL, VH, CL, and CH1 structural domains; (ii) a F(ab')2 fragment, a bivalent fragment composed of two Fab fragments linked by disulfide bonds in the hinge region; (iii) a Fab' fragment, which is essentially a Fab with a partial hinge region (see FUNDAMENTAL IMMUNOLOGY (Paul ed., 3rd ed., 1993)); (iv) a Fd fragment composed of the VH and CH1 structural domains; (v) a Fd' fragment composed of the VH and CH1 structural domains along with one or more cysteine residues at the C-terminus of the CH1 structural domain; (vi) a Fv fragment composed of the VL and VH structural domains of a single antibody arm; (vii) a dAb fragment composed of the VH structural domain (Ward et al. (viii) isolated complementarity-determining regions (CDRs); and (ix) nanobodies comprising a single heavy chain region having a variable region and two constant regions. In addition, the two structural domains of an Fv fragment, VL and VH, are encoded by separate genes but can be linked via a synthetic linker using recombinant techniques, allowing the VL and VH regions to pair and form a single protein chain to create a monovalent molecule (referred to as single-chain Fv (scFv)) (see, for example, Bird et al. (1988), Science, 242:423-426; and Huston et al. (1988), Proceedings of the National Academy of Sciences, USA, 85:5879-5883). Such single-chain antibodies are also encompassed by the term "antigen-binding fragment" of an antibody. Furthermore, the term also includes "linear antibodies" which comprise a pair of tandem Fd fragments (VH-CH1-VH-CH1) which form an antigen-binding region together with complementary light chain polypeptides, and any modified form of such fragments which retain antigen-binding activity.

[0096] These antigen-binding fragments can be obtained using conventional techniques known to those with skill in the art and can be screened for use in the same manner as intact antibodies.

[0097] As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as the non-random binding reaction between an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and / or avidity. Affinity is determined by the equilibrium dissociation constant (K D ) and is a measure of the binding strength between an antigenic determinant (epitope) and the antigen-binding site on an antibody: K D The smaller the value, the stronger the binding between the antigenic determinant (epitope) and the antibody. D It can also be expressed as an affinity constant (KA), where

[0098] Avidity is a measure of the binding strength between an antibody and its cognate antigen. Avidity is related to the affinity between an antigenic determinant (epitope) and its antigen-binding site on the antibody, and the number of cognate binding sites present on the antibody. Typically, antibodies have a binding affinity of 10 -5 ~10 -12 M or less, preferably 10 -7 ~10 -12 M or less, preferably 10 -8 ~10 -12 K within M D and / or at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , e.g. at least 10 12 M -1 It binds with a binding affinity of 10 -4 Any K exceeding DThe value is generally considered to be non-specific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined using any suitable known detection method, including, for example, Scatchard analysis, which is known in the art, and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA), biolayer interferometry (BLI), and sandwich competition assays, and various modifications thereof.

[0099] The term "k" on " or "k a " refers to the association rate constant of an antibody that associates with an antigen to form an antibody / antigen complex. This rate can be determined using standard assays such as Biacore or ELISA.

[0100] The term "k" off " or "k d " refers to the dissociation rate constant of an antibody dissociating from the antibody / antigen complex. This rate can be determined using standard assays such as Biacore or ELISA.

[0101] The term “K D " refers to the equilibrium dissociation constant of a specific antibody-antigen interaction. D is k a / k d The rate can be measured using standard assays such as Biacore or ELISA. The antibody or antigen-binding fragment thereof in the conjugate of the present application has a K D It can bind to the target antigen with an equilibrium dissociation constant of ≦1 mM, ≦100 nM, or ≦10 nM, or any value within these ranges.

[0102] The term "sequence identity" as used herein refers to the degree to which two sequences (amino acids) have identical residues at the same positions in an alignment. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" means that an amino acid sequence has X% identity with SEQ ID NO: Y, specifically, X% of the residues in the amino acid sequence are identical to the residues in the sequence disclosed in SEQ ID NO: Y. Typically, a computer program is used for such calculations. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0103] Furthermore, when determining the degree of sequence identity between two amino acid sequences, those skilled in the art can consider so-called "conservative" amino acid substitutions, which are typically described as amino acid substitutions in which one amino acid residue is replaced with another amino acid having a similar chemical structure, but which have little or substantially no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art.

[0104] Particularly preferred conservative substitutions are: substitution of Ala with Gly or Ser; substitution of Arg with Lys; substitution of Asn with Gln or His; substitution of Asp with Glu; substitution of Cys with Ser; substitution of Gln with Asn; substitution of Glu with Asp; substitution of Gly with Ala or Pro; substitution of His with Asn or Gln; substitution of Ile with Leu or Val; substitution of Leu with Ile or Val; substitution of Lys with Arg, Gln, or Glu; substitution of Met with Leu, Tyr, or Ile; substitution of Phe with Met, Leu, or Tyr; substitution of Ser with Thr; substitution of Thr with Ser; substitution of Trp with Tyr; substitution of Tyr with Trp; and / or substitution of Phe with Val, Ile, or Leu.

[0105] The terms "MSLN antibody," "anti-MSLN antibody," or "antibody that specifically binds to MSLN" refer to any form of antibody or fragment thereof that specifically binds to MSLN, and cover monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, so long as the fragment specifically binds to MSLN.

[0106] "Internalization," as used herein with respect to an antibody or antigen-binding fragment, refers to the ability of the antibody or antigen-binding fragment, upon binding to a cell, to cross the lipid bilayer membrane of the cell and enter an internal compartment (i.e., "internalize"), preferably into a degradation compartment within the cell.

[0107] The terms "therapeutic agent," "drug," or "drug moiety" refer to an agent capable of modulating biological processes and / or exhibiting biological activity.

[0108] The term "cytotoxic agent" refers to a substance that induces cell death primarily by interfering with the expression activity and / or function of a cell. Examples of cytotoxic agents include (but are not limited to) antimitotic agents such as eribulin and auristatins (e.g., monomethylauristatin E (MMAE)).

[0109] The term "antibody:drug ratio" or "drug-to-antibody ratio" or "DAR" refers to the number of drug moieties, i.e., drug payload, conjugated to each antibody.

[0110] As used herein, the term "treatment" refers to the administration of an agent or the performance of a procedure to achieve an effect. This action can be prophylactic, to completely or partially prevent a disease or its symptoms, and / or therapeutic, to achieve a partial or complete cure of a disease and / or its symptoms. As used herein, "treatment" can include the treatment of a disease or disorder (e.g., cancer) in a mammal, particularly a human: (a) preventing the onset of a disease or disease symptom in a subject who is predisposed to the disease (e.g., including a disease that may be associated with or caused by a primary disease) but has not yet been diagnosed with the disease; (b) suppressing the disease, i.e., preventing the progression of the disease; and (c) palliating the disease, i.e., causing regression of the disease. Treatment can refer to any measure of success in treating, ameliorating, or preventing cancer, including any objective or subjective parameter, such as remission, reduction of symptoms, or improvement in the patient's ability to tolerate disease symptoms. Thus, the term "treatment" includes administration of an ADC or composition disclosed herein to prevent, delay, alleviate, arrest, or inhibit the onset of signs or symptoms associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, disease symptoms, or side effects of the disease in a subject.

[0111] The term "cancer" refers to a medical condition characterized by unregulated cell growth in a population of cells within a mammal.

[0112] "Tumor-associated antigen" refers to an antigen that is differentially expressed in cancer cells compared to normal cells. Thus, tumor-associated antigens can be used to target cancer cells.

[0113] A "pharmaceutical composition" refers to a formulation that allows for administration and subsequently provides the desired biological activity of the active ingredient and / or achieves a therapeutic effect without containing other ingredients whose toxicity levels are unacceptable for administration to a subject. The pharmaceutical composition can be sterile.

[0114] The term "pharmaceutical excipient" includes substances such as adjuvants, carriers, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and preservatives.

[0115] The term "pharmaceutically acceptable" refers to a material that is, within the scope of sound medical judgment, suitable for contact with human tissue and lower animal tissue without excessive toxicity, irritation, allergic response, or other adverse effect, and is commensurate with a reasonable risk-to-benefit ratio.

[0116] As disclosed herein, an "effective amount" of an ADC is an amount sufficient to produce a therapeutic effect after administration, e.g., to achieve a specifically described purpose, such as reducing tumor growth rate or tumor volume, alleviating cancer symptoms, or some other sign of therapeutic efficacy. The effective amount can be determined by conventional methods with respect to the described purpose. The term "therapeutically effective amount" refers to an amount of an ADC effective to treat a disease or condition of a subject. In the case of cancer, a therapeutically effective amount of an ADC may reduce the number of cancer cells, reduce tumor size, inhibit (e.g., slow or stop) tumor metastasis, inhibit (e.g., slow or stop) tumor growth, and / or alleviate one or more symptoms. A "prophylactically effective amount" refers to the amount necessary to effectively achieve a prophylactic outcome at the required dosage and for the required period of time. Typically, because a prophylactic dose is used in a subject prior to the onset of disease or at an early stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0117] Some embodiments of the present invention will now be described in more detail, examples of which are illustrated by the accompanying structural and chemical formulae. The present invention is intended to encompass all alternatives, modifications, and equivalent technical solutions, all of which are included within the scope of the present invention as defined by the claims. Those skilled in the art will recognize that the present invention can be practiced using numerous methods and materials similar to or equivalent to those described herein. The present invention is in no way limited to the methods and materials described herein. In the event that portions of one or more incorporated documents, patents, or similar materials differ or contradict this application (including, but not limited to, definitions of terms, term usage, described techniques, etc.), this application shall prevail.

[0118] It is to be further understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, can also be implemented in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, can also be implemented separately or in any suitable subcombination.

[0119] Unless otherwise specified, all technical terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. All patents and publications referenced in this application are incorporated herein by reference in their entirety.

[0120] Unless otherwise specified, the following definitions shall apply when used herein. For purposes of the present invention, the chemical elements are in accordance with the CAS version of the Periodic Table of the Elements and the Handbook of Chemistry and Physics, 75th Edition, 1994. In addition, for general principles of organic chemistry, reference may be made to "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry" by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, the contents of which are incorporated herein by reference in their entirety.

[0121] Specific Embodiments The following embodiments are provided to further illustrate the present invention. It should be understood that these embodiments are provided only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0122] In one embodiment, the present invention relates to an MSLN antibody-drug conjugate that is a compound of formula (I), or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof: Ab-[SX-(Y) n -ZED] q (I), During the ceremony, Ab is an antibody that specifically binds to mesothelin (MSLN) or an antigen-binding fragment thereof; S is a sulfur bond connecting Ab and X; X is C 3~10 Cycloalkyl, 5-10 membered heterocyclic group, C 6~10 aryl, or 5-10 membered heteroaryl group; X may optionally be R * is substituted with 1, 2, 3, 4, or 5 instances of; R *H, deuterium, halogen, CN, NO2, OH, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenyl, or C 2~6 alkynyl; Y is -Y1-Y2-Y3-, where Y is linked to X through Y1 and to Z through Y3; During the ceremony, Y1 is -C(O)-, -C(O)O-, -OC(O)-, -C(O)NH-, C 2~6 Alkenyl, C 2~6 Alkynyl, 3- to 10-membered cycloalkyl, 5- to 10-membered heterocyclic group, C 6~10 aryl, or 5-10 membered heteroaryl group; Y2 is a bond or C 1~6 is alkylene; Y3 is selected from -C(O)-, -OC(O)-, or -NHC(O)-; Y is optionally substituted with 1, 2, 3, 4, or 5 instances of R#; R# is H, deuterium, halogen, CN, NO2, OH, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenyl, or C 2~6 alkynyl; Z is a peptide containing 2 to 8 amino acids, preferably a dipeptide, tripeptide, or tetrapeptide; E is absent or optionally a spacer unit connecting Z and D; D is a biologically active molecular drug or a derivative thereof; n is selected from 0, 1, 2, 3, 4, or 5, preferably 0, 1, 2, or 3; q is an integer selected from 1 to 10, and is preferably 8.

[0123] Ab In one specific embodiment, the Ab is an MSLN antibody. In another specific embodiment, the Ab is an MSLN antigen-binding fragment.

[0124] X In one particular embodiment, X is C 3~10 In another particular embodiment, X is a 5- to 10-membered heterocyclic group; in another particular embodiment, X is C 6~10 In another particular embodiment, X is a 5- to 10-membered heteroaryl group; in another particular embodiment, X is a 5- to 6-membered heteroaryl group.

[0125] In one particular embodiment, X is pyrimidinyl, e.g.,

[0126] [ka]

[0127] is.

[0128] In one particular embodiment, X is unsubstituted; in another particular embodiment, X is R * In another particular embodiment, X is substituted with one example of R * In another particular embodiment, X is substituted with two instances of R * In another particular embodiment, X is substituted with three instances of R * In another particular embodiment, X is substituted with four instances of R * It has been replaced by five examples of

[0129] Y In one particular embodiment, Y is Y1-Y2-Y3-, where Y is linked to X via Y1 and to Z via Y3.

[0130] In another particular embodiment, Y is

[0131] [ka]

[0132] is.

[0133] In one particular embodiment, Y is unsubstituted; in another particular embodiment, Y is substituted with one instance of R#; in another particular embodiment, Y is substituted with two instances of R#; in another particular embodiment, Y is substituted with three instances of R#; in another particular embodiment, Y is substituted with four instances of R#; in another particular embodiment, Y is substituted with five instances of R#.

[0134] Y 1 In one particular embodiment, Y is -C(O)-; in another particular embodiment, Y is -C(O)O-; in another particular embodiment, Y is -OC(O)-; in another particular embodiment, Y is -C(O)NH-; 2~6 alkenyl; in another particular embodiment, Y is C 2~4 alkenyl; in another particular embodiment, Y is C 2~6 alkynyl; in another particular embodiment, Y is C 2~4 alkynyl; in another particular embodiment, Y is C 3~10 In another particular embodiment, Y is a 5- to 10-membered heterocyclic group; in another particular embodiment, Y is C 6~10 aryl; in another particular embodiment, Y1 is a 5-10 membered heteroaryl group.

[0135] Y 2 In one particular embodiment, Y2 is a bond; in another particular embodiment, Y2 is C 1~6 alkylene; in another particular embodiment, Y2 is C 1~4 It is alkylene.

[0136] Y 3 In one particular embodiment, Y3 is -C(O)-; in another particular embodiment, Y3 is -OC(O)-; in another particular embodiment, Y3 is -NHC(O)-.

[0137] Z In one particular embodiment, Z is a linker peptide containing a peptide of 2 to 8 amino acids; preferably a dipeptide, tripeptide, or tetrapeptide.

[0138] In some embodiments, Z is a cleavable peptide. In some embodiments, the cleavable peptide can be cleaved by an enzyme. In some embodiments, the cleavable peptide can be cleaved by a tumor-associated protease. In some embodiments, the cleavable peptide can be cleaved by a lysosomal enzyme. In some embodiments, the cleavable peptide can be cleaved by a cathepsin. In some embodiments, the cleavable peptide can be cleaved by a lysosomal cysteine cathepsin (e.g., cathepsin B, C, F, H, K, L, O, S, V, X, or W). In some embodiments, the cleavable peptide can be cleaved by cathepsin B. An exemplary dipeptide cleavable by cathepsin B is valine-citrulline (Val-Cit) (Dubowchik et al., (2002), Bioconjugate Chemistry, 13:855-69).

[0139] In a particular embodiment, Z is selected from val-cit, val-cit-gly, gly-gly, gly-gly-gly, gly-gly-gly-gly, val-gly-gly, val-gln-gly, val-glu-gly, phe-lys-gly, leu-lys-gly, gly-val-lys-gly, val-lys-gly-gly, val-lys-gly, val-lys-ala, val-lys-leu, leu-leu-gly, gly-gly-phe-gly, gly-gly-phe-gly-gly, val-gly, val-lys-β-ala, or the foregoing dipeptides, tripeptides, and tetrapeptides after modification by substitution.

[0140] In one particular embodiment, Z is

[0141] [ka]

[0142] and In the formula, R1 and R2 are independently H, deuterium, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenyl, or C 2~6 alkynyl.

[0143] In one particular embodiment, Z is

[0144] [ka]

[0145] and wherein R1 and R2 are independently H, deuterium, halogen, or C 1~6 Alkyl, preferably H or C 1~4 alkyl.

[0146] E E is absent or optionally a spacer unit connecting Z and D; E is absent or -NH-C 1~6 Alkylene- or -OC 1~6 alkylene- selected from -NH-C 1~6 Alkylene- and -OC 1~6 Alkylene- is H, halogen, C 1~6 Alkyl, and C 1~6 It may be substituted with 1, 2, or 3 substituents selected from haloalkyl.

[0147] Preferably, E is absent or -NH-C 1~4 alkylene-, where -NH-C 1~4 Alkylene- is H, halogen, C 1~4 Alkyl, and C 1~4 It may be substituted with 1, 2, or 3 substituents selected from haloalkyl.

[0148] More preferably, E is -NH-(CH) 1~4 -, for example -NH-CH2-.

[0149] In some embodiments, E can function as a spacer unit linking Z and D. The spacer can be a functional group that facilitates attachment of XYZ to the camptothecin derivative, or it can provide an additional structural moiety to further facilitate release of the camptothecin derivative from the conjugate.

[0150] D In one particular embodiment, D is a biologically active molecular drug or a derivative thereof.

[0151] In a particular embodiment, D is selected from: metal complexes; glycopeptide antibiotics; DNA topoisomerase inhibitors; drugs that interfere with DNA synthesis; drugs that target structural proteins; tumor signaling pathway inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor angiogenesis inhibitors; cyclin inhibitors; serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors, or histidine kinase inhibitors; and other active substances that inhibit tumor cell proliferation or promote apoptosis or induce necrosis of tumor cells.

[0152] In a particular embodiment, D is: oxaliplatin; bleomycin or pingjiangmycin; camptothecin, camptothecin derivatives (e.g., hydroxycamptothecin, 9-aminocamptothecin, etc.), SN-38, irinotecan, topotecan, belotecan, or rubitecan; actinomycin D, doxorubicin, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide; methotrexate. , 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, or nelarabine; vinca alkaloids, vincristine, vinblastine, paclitaxel, docetaxel, or cabazitaxel; maytansinoid derivatives; calicheamicin derivatives; auristatin derivatives; pyrrolobenzodiazepine dimer derivatives; melphalan; mitomycin C; or chlorambucil.

[0153] In one particular embodiment, D is

[0154] [ka]

[0155] and During the ceremony, R D is absent or selected from -NH- or -O-; L D -C0~6 Alkylene-OC 0~6 Alkylene- or -C 0~6 Alkylene-OC 0~6 Alkylene-C(O)NH-CHR D4 -Selected from; R D1 and R D2 are independently H, halogen, C 1~6 Alkyl, C 1~6 Haloalkyl or C 1~6 alkoxy; or R D1 and R D2 together with the carbon atoms to which they are attached, form C 3~7 forming a cycloalkyl or a 3- to 7-membered heterocyclic group, preferably a 5- or 6-membered heterocyclic group; R D3 and R D4 are independently H, halogen, C 1~6 Alkyl, C 1~6 Haloalkyl or C 1~6 alkoxy; or R D3 and R D4 together with the carbon atoms to which they are attached, form C 5~10 Cycloalkyl or C 5~10 Heterocyclic groups, preferably C 5~6 Forms a cycloalkyl.

[0156] In certain embodiments, D is:

[0157] [ka]

[0158] is selected from.

[0159] R * In certain embodiments, R * is H; in another particular embodiment, R * is deuterium; in another particular embodiment, R *is halogen; in another particular embodiment, R * is CN; in another particular embodiment, R * is NO; in another particular embodiment, R * is OH; in another particular embodiment, R * is C 1~6 alkyl; in another particular embodiment, R * is C 1~4 alkyl; in another particular embodiment, R * is C 1~6 haloalkyl; in another particular embodiment, R * is C 1~4 haloalkyl; in another particular embodiment, R * is C 2~6 alkenyl; in another particular embodiment, R * is C 2~4 alkenyl; in another particular embodiment, R * is C 2~6 alkynyl; in another particular embodiment, R * is C 2~4 It is alkynyl.

[0160] R# In certain embodiments, R# is H; in another particular embodiment, R# is deuterium; in another particular embodiment, R# is halogen; in another particular embodiment, R# is CN; in another particular embodiment, R# is NO2; in another particular embodiment, R# is OH; in another particular embodiment, R# is C 1~6 alkyl; in another particular embodiment, R# is C 1~4 alkyl; in another particular embodiment, R# is C 1~6 haloalkyl; in another particular embodiment, R# is C 1~4 haloalkyl; in another particular embodiment, R# is C 2~6 alkenyl; in another particular embodiment, R# is C 2~4 alkenyl; in another particular embodiment, R# is C2~6 alkynyl; in another particular embodiment, R# is C 2~4 It is alkynyl.

[0161] n In certain embodiments, n is 0; in other particular embodiments, n is 1; in other particular embodiments, n is 2; in other particular embodiments, n is 3; in other particular embodiments, n is 4; in other particular embodiments, n is 5.

[0162] q In certain embodiments, q is an integer selected from 1 to 10; in other particular embodiments, q is 1; in other particular embodiments, q is 2; in other particular embodiments, q is 3; in other particular embodiments, q is 4; in other particular embodiments, q is 5; in other particular embodiments, q is 6; in other particular embodiments, q is 7; in other particular embodiments, q is 8; in other particular embodiments, q is 9; in other particular embodiments, q is 10.

[0163] Any technical solution or any combination thereof in any of the above specific embodiments can be combined with any technical solution or any combination thereof in other specific embodiments. For example, any technical solution related to Ab or any combination thereof can be combined with X, Y, Y1, Y2, Y3, Z, E, D, R * , R#, n, and q, or any combination thereof. The present invention is intended to include all such combinations of technical solutions, which for the sake of brevity are not listed individually.

[0164] In more particular embodiments, the present invention provides an MSLN antibody-drug conjugate of formula (I) above, X is C 6~10selected from aryl or 5-10 membered heteroaryl groups; X may optionally be R * is substituted with 1, 2, 3, 4, or 5 instances of; R * is H, deuterium, halogen, CN, NO2, OH, or C 1~6 alkyl; Preferably, X is a 5-6 membered heteroaryl group; X may optionally be R * is substituted with one, two, or three instances of; R * is H, deuterium, halogen, or C 1~4 alkyl; More preferably, X is pyrimidinyl, e.g.

[0165] [ka]

[0166] is.

[0167] In more particular embodiments, the present invention provides an MSLN antibody-drug conjugate of formula (I) above, Y is Y1-Y2-Y3-, where Y is linked to X through Y1 and to Z through Y3; During the ceremony, Y1 is C 2~6 Alkenyl or C 2~6 alkynyl; Y2 is a bond or C 1~6 is alkylene; Y3 is selected from -C(O)- or -NHC(O)-; Y is optionally substituted with 1, 2, 3, 4, or 5 instances of R#; R# is H, deuterium, halogen, CN, NO2, OH, or C 1~6 alkyl; Preferably, Y is Y1-Y2-Y3-, where Y is linked to X through Y1 and to Z through Y3; During the ceremony, Y1 is C 2~4 Alkenyl or C 2~4 alkynyl; Y2 is C 1~4 is alkylene; Y3 is -C(O)-; Y is optionally substituted with 1, 2, or 3 instances of R#; R# is H, deuterium, halogen, or C 1~4 alkyl; More preferably, Y is

[0168] [ka]

[0169] is.

[0170] In more particular embodiments, the present invention provides an MSLN antibody-drug conjugate of formula (I) above, Z is selected from val-cit, val-cit-gly, gly-gly, gly-gly-gly, gly-gly-gly, val-gly-gly, val-gln-gly, val-glu-gly, phe-lys-gly, leu-lys-gly, gly-val-lys-gly, val-lys-gly-gly, val-lys-gly, val-lys-ala, val-lys-leu, leu-leu-gly, gly-gly-phe-gly, gly-gly-phe-gly-gly, val-gly, val-lys-β-ala, or any of the foregoing dipeptides, tripeptides, or tetrapeptides after modification by substitution; Preferably, Z is

[0171] [ka]

[0172] and; R1 and R2 are independently H, deuterium, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenyl, or C 2~6 alkynyl; More preferably, Z is

[0173] [ka]

[0174] and; R1 and R2 are independently H, deuterium, halogen, or C 1~6 Alkyl, preferably H or C 1~4 alkyl.

[0175] In more particular embodiments, the present invention provides an MSLN antibody-drug conjugate of formula (I) above, E is absent or -NH-C 1~6 Alkylene- or -OC 1~6 alkylene- selected from -NH-C 1~6 Alkylene- and -OC 1~6 Alkylene- is H, halogen, C 1~6 Alkyl, and C 1~6 optionally substituted with 1, 2, or 3 substituents selected from haloalkyl; Preferably, E is absent or -NH-C 1~4 alkylene-, where -NH-C 1~4 Alkylene- is H, halogen, C 1~4 Alkyl, and C 1~4 optionally substituted with 1, 2, or 3 substituents selected from haloalkyl; More preferably, E is -NH-(CH) 1~4 -, for example -NH-CH2-.

[0176] In more particular embodiments, the present invention provides an MSLN antibody-drug conjugate of formula (I) above, D is selected from metal complexes; glycopeptide antibiotics; DNA topoisomerase inhibitors; drugs that interfere with DNA synthesis; drugs that target structural proteins; tumor signaling pathway inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor angiogenesis inhibitors; cyclin inhibitors; serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors, or histidine kinase inhibitors; and other active substances that inhibit tumor cell proliferation or promote tumor cell apoptosis or necrosis; Preferably, D is oxaliplatin; bleomycin or pingjiangmycin; camptothecin, camptothecin derivatives (e.g., hydroxycamptothecin, 9-aminocamptothecin, etc.), SN-38, irinotecan, topotecan, belotecan, or rubitecan; actinomycin D, doxorubicin, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide; methotrexate, 5-fluoxetine, or 5-fluoxetine. selected from louracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, or nelarabine; vinca alkaloids, vincristine, vinblastine, paclitaxel, docetaxel, or cabazitaxel; maytansinoid derivatives; calicheamicin derivatives; auristatin derivatives; pyrrolobenzodiazepine dimer derivatives; melphalan; mitomycin C; or chlorambucil; More preferably, D is

[0177] [ka]

[0178] and During the ceremony, R D is absent or selected from -NH- or -O-; L D -C0~6 Alkylene-OC 0~6 Alkylene- or -C 0~6 Alkylene-OC 0~6 Alkylene-C(O)NH-CHR D4 -Selected from; R D1 and R D2 are independently H, halogen, C 1~6 Alkyl, C 1~6 Haloalkyl or C 1~6 alkoxy; or R D1 and R D2 together with the carbon atoms to which they are attached, form C 3~7 forming a cycloalkyl or a 3- to 7-membered heterocyclic group, preferably a 5- or 6-membered heterocyclic group; R D3 and R D4 are independently H, halogen, C 1~6 Alkyl, C 1~6 Haloalkyl or C 1~6 alkoxy; or R D3 and R D4 together with the carbon atoms to which they are attached, form C 5~10 Cycloalkyl or C 5~10 Heterocycle, preferably C 5~6 Forming a cycloalkyl; More preferably, D is:

[0179] [ka]

[0180] is selected from.

[0181] In more particular embodiments, the present invention provides an MSLN antibody-drug conjugate of formula (I) above, X is C 6~10 selected from aryl or 5-10 membered heteroaryl groups; X may optionally be R * is substituted with 1, 2, 3, 4, or 5 instances of; R * is H, deuterium, halogen, CN, NO2, OH, or C 1~6 alkyl; Y is -Y1-Y2-Y3-, where Y is linked to X through Y1 and to Z through Y3; During the ceremony, Y1 is C 2~6 Alkenyl or C 2~6 alkynyl; Y2 is a bond or C 1~6 is alkylene; Y3 is selected from -C(O)- or -NHC(O)-; Y is optionally substituted with 1, 2, 3, 4, or 5 instances of R#; R# is H, deuterium, halogen, CN, NO2, OH, or C 1~6 alkyl; Z is selected from val-cit, val-cit-gly, gly-gly, gly-gly-gly, gly-gly-gly, val-gly-gly, val-gln-gly, val-glu-gly, phe-lys-gly, leu-lys-gly, gly-val-lys-gly, val-lys-gly-gly, val-lys-gly, val-lys-ala, val-lys-leu, leu-leu-gly, gly-gly-phe-gly, gly-gly-phe-gly-gly, val-gly, val-lys-β-ala, or any of the foregoing dipeptides, tripeptides, or tetrapeptides after modification by substitution; E is absent or -NH-C 1~6 Alkylene- or -OC 1~6 alkylene- selected from -NH-C 1~6 Alkylene- and -OC 1~6 Alkylene- is H, halogen, C 1~6 Alkyl, and C 1~6 optionally substituted with 1, 2, or 3 substituents selected from haloalkyl; D is selected from metal complexes; glycopeptide antibiotics; DNA topoisomerase inhibitors; drugs that interfere with DNA synthesis; drugs that target structural proteins; tumor signaling pathway inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor angiogenesis inhibitors; cyclin inhibitors; serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors, or histidine kinase inhibitors; and other active substances that inhibit tumor cell proliferation or promote tumor cell apoptosis or necrosis.

[0182] In more particular embodiments, the present invention provides an MSLN antibody-drug conjugate of formula (I) above, X is a 5-6 membered heteroaryl group; X may optionally be R * is substituted with one, two, or three instances of; R * is H, deuterium, halogen, or C 1~4 alkyl; Y is Y1-Y2-Y3-, where Y is linked to X through Y1 and to Z through Y3; During the ceremony, Y1 is C 2~4 Alkenyl or C 2~4 alkynyl; Y2 is C 1~4 is alkylene; Y3 is -C(O)-; Y is optionally substituted with 1, 2, or 3 instances of R#; R# is H, deuterium, halogen, or C 1~4 alkyl; Z is

[0183] [ka]

[0184] and; E is absent or -NH-C 1~4 alkylene-, where -NH-C1~4 Alkylene- is H, halogen, C 1~4 Alkyl, and C 1~4 optionally substituted with 1, 2, or 3 substituents selected from haloalkyl; R1 and R2 are independently H, deuterium, halogen, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenyl, or C 2~6 alkynyl; D is oxaliplatin; bleomycin or pingjiangmycin; camptothecin, camptothecin derivatives (e.g., hydroxycamptothecin, 9-aminocamptothecin, etc.), SN-38, irinotecan, topotecan, belotecan, or rubitecan; actinomycin D, doxorubicin, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide; methotrexate, 5-fluoxetine, or 5-fluoxetine. selected from louracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, or nelarabine; vinca alkaloids, vincristine, vinblastine, paclitaxel, docetaxel, or cabazitaxel; maytansinoid derivatives; calicheamicin derivatives; auristatin derivatives; pyrrolobenzodiazepine dimer derivatives; melphalan; mitomycin C; or chlorambucil; Preferably, D is

[0185] [ka]

[0186] and During the ceremony, R D is absent or selected from -NH- or -O-; L D -C 0~6 Alkylene-OC 0~6 Alkylene- or -C 0~6 Alkylene-OC 0~6Alkylidene-C(O)NH-CHR D4 -Selected from; R D1 and R D2 are independently H, halogen, C 1~6 Alkyl, C 1~6 Haloalkyl or C 1~6 alkoxy; or R D1 and R D2 together with the carbon atoms to which they are attached, form C 3~7 forming a cycloalkyl or a 3- to 7-membered heterocyclic group, preferably a 5- or 6-membered heterocyclic group; R D3 and R D4 are independently H, halogen, C 1~6 Alkyl, C 1~6 Haloalkyl or C 1~6 alkoxy; or R D3 and R D4 together with the carbon atoms to which they are attached, form C 5~10 Cycloalkyl or C 5~10 Heterocyclic groups, preferably C 5~6 Forms a cycloalkyl.

[0187] In more particular embodiments, the present invention provides an MSLN antibody-drug conjugate of formula (I) above, X is pyrimidinyl, preferably

[0188] [ka]

[0189] and; Y is

[0190] [ka]

[0191] and; Z is

[0192] [ka]

[0193] and: R1 and R2 are independently H, deuterium, halogen, C 1~6 Alkyl, preferably H or C 1~4 alkyl; E is -NH-CH2-; D is:

[0194] [ka]

[0195] Selected from; n is selected from 1, 2, or 3, preferably 1; q is selected from 4, 5, 6, 7, 8, 9, or 10, and is preferably 8.

[0196] In more particular embodiments, the present invention provides an MSLN antibody-drug conjugate, the MSLN antibody-drug conjugate comprising a compound of formula (II):

[0197] [ka]

[0198] or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof; During the ceremony, Ab is anti-MSLN antibody; R1 and R2 are independently C 1~6 Alkyl, preferably C 1~4 alkyl; D is:

[0199] [ka]

[0200] Selected from; q is an integer selected from 1 to 10, preferably 4, 5, 6, 7, 8, 9, or 10, and most preferably 8.

[0201] Anti-MSLN antibody The antibody that specifically binds to MSLN in the antibody-drug conjugate of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), (1) the VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 27; (2) the VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 25, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 28; (3) the VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 26, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 27; (4) the VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 29; (5) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 30; (6) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 31; (7) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 32; (8) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 33; (9) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 25, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 29; (10) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 63, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (11) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 64, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (12) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 65, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (13) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 65, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 68; (14) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 63, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 68; (15) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 66, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (16) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 64, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 69; (17) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 66, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 69; (18) The VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 63, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 69; or (19) VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 65, and VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 69.

[0202] In some embodiments, LCDRs 1-3 and HCDRs 1-3 are defined according to the EU Kabat definition / numbering system. In some embodiments, LCDRs 1-3 and HCDRs 1-3 are defined according to the Chothia definition / numbering system. In some embodiments, LCDRs 1-3 and HCDRs 1-3 are defined according to the AbM definition / numbering system. In some preferred embodiments, LCDRs 1-3 and HCDRs 1-3 are defined according to the combined Kabat and Chothia numbering system.

[0203] In some embodiments, the Ab in the antibody-drug conjugate of the invention is an antibody that specifically binds to mesothelin (MSLN) or an antigen-binding fragment thereof, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), (1) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13; (2) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 15, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 16, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18; (3) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 15, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13; (4) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 19; (5) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 20; (6) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 21; (7) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 22; (8) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23; (9) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 15, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 16, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 19; (10) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 45, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 52, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (11) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 52, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (12) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 59, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 52, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (13) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 59, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 61, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (14) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 45, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 61, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (15) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 60, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 52, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (16) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 62, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (17) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 60, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 62, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (18) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 45, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 62, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; or (19) VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 59, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 62, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56.

[0204] In some embodiments, the CDRs are determined using the combinatorial Kabat Chothia system.

[0205] In some embodiments, the Ab in the antibody-drug conjugate of the invention is an antibody that specifically binds to mesothelin (MSLN) or an antigen-binding fragment thereof, wherein the antibody comprises a VL and a VH, (1) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:27; (2) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:25, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:28; (3) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 27; (4) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29; (5) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30; (6) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31; (7) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32; (8) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33; (9) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 25, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29; (10) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (11) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (12) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 65, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (13) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 65, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68; (14) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68; (15) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 66, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (16) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; (17) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 66, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; (18) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; or (19) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 65, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69.

[0206] In some embodiments, the VL comprises a functional variant of any one of the amino acid sequences set forth in SEQ ID NOS: 27 to 33 and 67 to 69, formed by inserting, deleting, and / or substituting one or more amino acids within the amino acid sequences, provided that antibodies containing a VL having a functional variant retain the ability to bind to MSLN. In some embodiments, the VH comprises a functional variant of any one of the amino acid sequences set forth in SEQ ID NOS: 24 to 26 and 63 to 66, formed by inserting, deleting, and / or substituting one or more amino acids within the amino acid sequences, provided that antibodies containing a VH having a functional variant retain the ability to bind to MSLN.

[0207] A functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the amino acid sequence of the parent polypeptide. For example, a functional variant of any one of SEQ ID NOs: 27 to 33 and 67 to 69 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NOs: 27 to 33 and 67 to 69, respectively. A functional variant of any one of SEQ ID NOs: 24 to 26 and 63 to 66 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NOs: 24 to 26 and 63 to 66, respectively.

[0208] In the context of functional variants, the number of inserted, deleted, and / or substituted amino acids preferably does not exceed 40%, more preferably does not exceed 35%, more preferably does not exceed 1-33%, more preferably does not exceed 5-30%, more preferably does not exceed 10-25%, and more preferably does not exceed 15-20% of the total number of amino acids in the parent amino acid sequence. For example, the number of inserted, deleted, and / or substituted amino acids can be 1-20, preferably 1-10, more preferably 1-7, more preferably 1-5, and most preferably 1-2. In a preferred embodiment, the number of inserted, deleted, and / or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

[0209] In some embodiments, insertions, deletions, and / or substitutions may be made in framework (FR) regions, such as FR1, FR2, FR3, and / or FR4.

[0210] In a preferred embodiment, the VL comprises the amino acid sequence set forth in SEQ ID NO:27 and the VH comprises the amino acid sequence set forth in SEQ ID NO:24; the VL comprises the amino acid sequence set forth in SEQ ID NO:28 and the VH comprises the amino acid sequence set forth in SEQ ID NO:25; the VL comprises the amino acid sequence set forth in SEQ ID NO:27 and the VH comprises the amino acid sequence set forth in SEQ ID NO:26; the VL comprises the amino acid sequence set forth in SEQ ID NO:29; the VH comprises the amino acid sequence set forth in SEQ ID NO:24; the VL comprises the amino acid sequence set forth in SEQ ID NO:30; or the VL comprises the amino acid sequence set forth in SEQ ID NO: 31 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 24; or the VL comprises the amino acid sequence set forth in SEQ ID NO: 32 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 24; or the VL comprises the amino acid sequence set forth in SEQ ID NO: 33 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 24; or the VL comprises the amino acid sequence set forth in SEQ ID NO: 29 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 25; or the VL comprises the amino acid sequence set forth in SEQ ID NO: 67 or the VL comprises the amino acid sequence set forth in SEQ ID NO: 67 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 64; or the VL comprises the amino acid sequence set forth in SEQ ID NO: 67 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 65; or the VL comprises the amino acid sequence set forth in SEQ ID NO: 68 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 65; or the VL comprises the amino acid sequence set forth in SEQ ID NO: 68 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 63; or the VL comprises the amino acid sequence set forth in SEQ ID NO: 67 wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 66; wherein the VL comprises the amino acid sequence set forth in SEQ ID NO: 69 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 64; wherein the VL comprises the amino acid sequence set forth in SEQ ID NO: 69 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 66; wherein the VL comprises the amino acid sequence set forth in SEQ ID NO: 69 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 63; or wherein the VL comprises the amino acid sequence set forth in SEQ ID NO: 69 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 65.

[0211] In some embodiments, the Ab in the antibody-drug conjugate of the invention is an antibody that specifically binds to mesothelin (MSLN) or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), (1) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37; (2) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38; (3) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 36, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37; (4) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39; (5) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 40; (6) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 41; (7) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42; (8) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43; (9) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39; (10) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (11) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (12) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (13) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 75; (14) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 75; (15) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (16) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76; (17) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76; (18) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76; or (19) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76.

[0212] In some embodiments, the light chain comprises a functional variant of any one of the amino acid sequences set forth in SEQ ID NOs: 37-43 and 74-76, formed by inserting, deleting, and / or substituting one or more amino acids within the amino acid sequences, provided that an antibody containing a light chain having such a functional variant retains the ability to bind to MSLN. In some embodiments, the heavy chain comprises a functional variant of any one of the amino acid sequences set forth in SEQ ID NOs: 34-36 and 70-73, formed by inserting, deleting, and / or substituting one or more amino acids within the amino acid sequences, provided that an antibody containing a VH having such a functional variant retains the ability to bind to MSLN.

[0213] For example, a functional variant of any one of SEQ ID NOs: 37 to 43 and 74 to 76 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NOs: 37 to 43 and 74 to 76. For example, a functional variant of any one of SEQ ID NOs: 34 to 36 and 70 to 73 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NOs: 34 to 36 and 70 to 73.

[0214] In some embodiments, the number of inserted, deleted, and / or substituted amino acids preferably does not exceed 40%, more preferably does not exceed 35%, more preferably does not exceed 1-33%, more preferably does not exceed 5-30%, even more preferably does not exceed 10-25%, and more preferably does not exceed 15-20% of the total number of amino acids in the parent amino acid sequence. For example, the number of inserted, deleted, and / or substituted amino acids can be 1-50, preferably 1-20, more preferably 1-10, and even more preferably 1-5. In preferred embodiments, the number of inserted, deleted, and / or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

[0215] In some embodiments, insertions, deletions, and / or substitutions may be made in framework (FR) regions, e.g., FR1, FR2, FR3, and / or FR4; and / or constant regions, e.g., CL, CH1, CH2, and / or CH3.

[0216] In some embodiments, the substitution of one or more amino acids may include one or more conservative amino acid substitutions. Examples of conservative substitutions are described above.

[0217] In preferred embodiments, the light chain comprises the amino acid sequence set forth in SEQ ID NO: 37 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34; the light chain comprises the amino acid sequence set forth in SEQ ID NO: 38 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 35; the light chain comprises the amino acid sequence set forth in SEQ ID NO: 37 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 36; the light chain comprises the amino acid sequence set forth in SEQ ID NO: 39; the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34; the light chain comprises the amino acid sequence set forth in SEQ ID NO: 40; the heavy chain or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 41 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 42 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 43 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 39 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 35; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 74 or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 70; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 74 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 71; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 74 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 72; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 75 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 72; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 75 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 70; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 74 or wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 73; or wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO: 76 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 71; or wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO: 76 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 73; or wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO: 76 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 70; or wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO: 76 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 72.

[0218] In some embodiments, the antibody comprises an Fc region. In some embodiments, the Fc region may be of any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and may contain one or more mutations or modifications. In one embodiment, the Fc region is of or derived from the IgG1 isotype, optionally with one or more mutations or modifications. In one embodiment, the Fc region is a human IgG1 Fc.

[0219] In one embodiment, the Fc region lacks effector function. For example, the Fc region can be of an IgG1 or non-IgG1 isotype, such as IgG2, IgG3, or IgG4, mutated to reduce or eliminate its ability to mediate effector function (e.g., ADCC). See, e.g., Dall'Acqua WF et al., The Journal of Immunology, 177(2):1129-1138 (2006) and Hezareh M, Journal of Virology, 75(24):12161-12168 (2001). In some embodiments, the Fc region of the antibody comprises a wild-type IgG1 Fc with L234A, L235A, and G237A mutations.

[0220] In some embodiments, the antibody is mutated at one or more post-translational modification sites, hi one embodiment, the Fc region contains mutations to eliminate Asn-linked glycosylation acceptor sites or has been engineered to eliminate antibody effector functions.

[0221] Post-translational modifications (PTMs) are widespread in proteins expressed by mammalian cells. In addition to conserved PTM sites in antibodies, such as the conserved N-glycosylation site in the CH2 structural domain of IgG1 antibodies, other PTM sites present in the antigen-binding region (i.e., CDR region) of an antibody can reduce antigen-binding activity or chemical stability. For example, deamidation or isomerization can lead to molecular instability and heterogeneity. To reduce sequence sensitivity, PTM motifs can be removed by mutation. The VH or VL sequence can be scanned for the presence of PTM motifs, such as isomerization motifs (e.g., DG). "Hot spot" residues (e.g., D or G in the DG motif) can then be mutated to the corresponding residue in the germline sequence or to other residues with similar biophysical properties.

[0222] In some embodiments, the antibody-drug conjugate of the present invention comprises an antibody that specifically binds to mesothelin (MSLN), wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody-drug conjugate of the present invention comprises an antibody that specifically binds to mesothelin (MSLN), wherein the antibody is of an isotype selected from IgG, IgA, IgM, IgE, and IgD. In some embodiments, the antibody-drug conjugate of the present invention comprises an antibody that specifically binds to mesothelin (MSLN), wherein the antibody is of a subclass selected from IgG1, IgG2, IgG3, and IgG4.

[0223] In some embodiments, the antibody-drug conjugate of the invention comprises an antigen-binding fragment that specifically binds to mesothelin, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, ds-scFv, and dAb.

[0224] In some embodiments, the antibody-drug conjugate of the invention comprises an antibody or antigen-binding fragment thereof that specifically binds to mesothelin, wherein the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0225] In some embodiments, an antibody-drug conjugate of the invention comprises an antibody or antigen-binding fragment thereof that specifically binds to mesothelin, wherein the antibody is monovalent, bivalent, or multivalent.

[0226] In some embodiments, the antibody-drug conjugate of the invention comprises an antibody or antigen-binding fragment thereof that specifically binds to mesothelin, wherein the antibody or antigen-binding fragment is conjugated to a fluorescent label, a radioactive label, or a cytotoxic agent.

[0227] In some embodiments, the antibody-drug conjugate of the invention comprises an antibody or antigen-binding fragment thereof that specifically binds to mesothelin, wherein the antibody is a bispecific antibody, and the bispecific antibody further comprises a second antigen-binding region that specifically binds to a tumor-associated antigen, an immune cell antigen, or an immune checkpoint molecule.

[0228] In some embodiments, the antibody-drug conjugate of the invention comprises the following compound:

[0229] [ka]

[0230] or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof; Ab is anti-MSLN antibody; q is an integer selected from 1 to 10, for example, 4, 5, 6, 7, 8, 9, or 10, and preferably 8.

[0231] In some embodiments, the antibody-drug conjugate of the invention comprises the following compound:

[0232] [ka]

[0233] or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof; Ab is anti-MSLN antibody; q is an integer selected from 1 to 10, for example, 4, 5, 6, 7, 8, 9, or 10, and preferably 8.

[0234] In some embodiments, the antibody-drug conjugate of the invention comprises the following compound:

[0235] [ka]

[0236] or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof; Ab is anti-MSLN antibody; q is an integer selected from 1 to 10, for example, 4, 5, 6, 7, 8, 9, or 10, and preferably 8.

[0237] In some embodiments, the antibody-drug conjugate of the invention comprises the compound:

[0238] [ka]

[0239] or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof; Ab is anti-MSLN antibody; q is an integer selected from 1 to 10, for example, 4, 5, 6, 7, 8, 9, or 10, and preferably 8.

[0240] In some embodiments, the antibody-drug conjugate of the invention comprises the following compound:

[0241] [ka]

[0242] or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof; where Ab is an anti-MSLN antibody; q is an integer selected from 1 to 10, for example, 4, 5, 6, 7, 8, 9, or 10, and preferably 8.

[0243] In some preferred embodiments, the MSLN antibody comprises a heavy chain (HC) and a light chain (LC), wherein: (1) HC comprises the amino acid sequence set forth in SEQ ID NO: 34, and LC comprises the amino acid sequence set forth in SEQ ID NO: 37; (2) the HC comprises the amino acid sequence set forth in SEQ ID NO: 34 and the LC comprises the amino acid sequence set forth in SEQ ID NO: 43; (3) HC comprises the amino acid sequence set forth in SEQ ID NO: 70, and LC comprises the amino acid sequence set forth in SEQ ID NO: 74; or (4) HC comprises the amino acid sequence shown in SEQ ID NO: 72, and LC comprises the amino acid sequence shown in SEQ ID NO: 76.

[0244] bispecific antibody In some embodiments, the antibody in the antibody-drug conjugate of the present invention may be a bispecific antibody or a multispecific antibody. In some embodiments, the antibody in the antibody-drug conjugate of the present invention is a bispecific antibody that comprises an antibody that specifically binds to MSLN or an antigen-binding fragment thereof and further comprises a second antigen-binding domain that binds to a second antigen. In some embodiments, the second antigen may be a tumor-associated antigen, an immune checkpoint molecule, or an immune cell antigen.

[0245] Many tumor-associated antigens associated with specific cancers have been identified in the art. In some embodiments, tumor-associated antigens are antigens capable of stimulating significant tumor-specific immune responses. Some of these antigens are encoded by normal cells but are not necessarily expressed in normal cells. These antigens can typically be described as antigens that are silenced (i.e., not expressed) in normal cells, antigens that are expressed only at specific differentiation stages, or antigens that are expressed sequentially, such as embryonic and fetal antigens. Other cancer antigens are encoded by mutated cellular genes, e.g., oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutated p53), and fusion proteins result from internal deletions or chromosomal translocations. Additional cancer antigens can be encoded by viral genes, such as RNA- and DNA-bearing tumor viruses. Many other tumor-associated antigens and antibodies thereto are known and / or commercially available, or can be produced by those skilled in the art.

[0246] In some embodiments, a bispecific antibody comprises a single polypeptide chain containing a first antigen-binding region, a second antigen-binding region, and optionally an Fc region.

[0247] Pharmaceutical Composition In a second aspect, the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate according to the first aspect of the invention, and optionally a pharmaceutically acceptable carrier or excipient.

[0248] The present invention provides pharmaceutical compositions comprising the antibody-drug conjugates of the present invention. In some embodiments, the pharmaceutical compositions further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or local administration (e.g., by injection or infusion). For example, in some embodiments, compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffer.

[0249] The pharmaceutical composition of the present invention is formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous injection, intradermal administration, subcutaneous administration, and oral administration (e.g., inhalation), transdermal administration (i.e., topical administration), transmucosal administration, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile diluent, such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid (EDTA); a buffer, such as acetate buffer, citrate buffer, or phosphate buffer; and a tonicity adjuster, such as sodium chloride or glucose. The pH can be adjusted using an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be packaged in glass or plastic ampoules, disposable syringes, or multiple dose vials.

[0250] Treatment method In a third aspect, the present invention provides a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of the antibody-drug conjugate described in the first aspect of the invention or the pharmaceutical composition described in the second aspect of the invention. The antibody-drug conjugates provided herein can be used to delay or inhibit the progression of mesothelin-positive cancers or to inhibit metastasis of mesothelin-positive cancers. In these applications, a therapeutically effective amount of the composition is administered to the subject in an amount sufficient to inhibit cancer cell growth, replication, or metastasis, or to suppress symptoms or signs of cancer. Suitable subjects may include those diagnosed with mesothelin-expressing cancers, such as colon cancer, mesothelioma, ovarian cancer, lung cancer, breast cancer, pancreatic cancer, gastric cancer, prostate cancer, squamous cell carcinoma, and cholangiocarcinoma.

[0251] The antibody-drug conjugates disclosed herein may also be administered in combination with other anti-cancer agents or therapeutic treatments (e.g., surgical resection of tumors). Any suitable anti-cancer agent may be administered in combination with the antibody-drug conjugates disclosed herein. Examples of anti-cancer agents include, but are not limited to, chemotherapeutic agents, such as antimitotic agents, alkylating agents, antimetabolites, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormonal agents (e.g., anti-androgens), and anti-angiogenic agents. Other anti-cancer treatments include radiation therapy and other antibodies that target cancer cells.

[0252] Another common treatment for certain cancer types is surgery, e.g., surgical resection of metastatic tumors. Another example of a treatment is radiation therapy, which involves the application of radioactive material or energy to the tumor site to help eradicate or shrink the tumor before surgical removal.

[0253] In a fourth aspect, the present invention provides an antibody-drug conjugate or pharmaceutical composition of the present invention for use in treating cancer. In some embodiments, the cancer is selected from colon cancer, mesothelioma, ovarian cancer, lung cancer, breast cancer, pancreatic cancer, gastric cancer, prostate cancer, squamous cell carcinoma, and cholangiocarcinoma. In some embodiments, the antibody or pharmaceutical composition of the present invention is used in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, an siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

[0254] In a fifth aspect, the present invention provides use of the antibody-drug conjugate or pharmaceutical composition of the present invention in preparing a medicament for treating cancer. In some embodiments, the cancer is selected from colon cancer, mesothelioma, ovarian cancer, lung cancer, breast cancer, pancreatic cancer, gastric cancer, prostate cancer, squamous cell carcinoma, and cholangiocarcinoma. In some embodiments, the medicament is used in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, an siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug. [Example]

[0255] Example 1. Preparation of initial antibodies and variants To obtain MSLN-specific antibodies, we immunized Harbour H2L2 transgenic mice (https: / / harbourantibodies.com / ) using various methods. Immunization resulted in the production of numerous antibodies that bind to the MSLN extracellular domain (ECD) protein and MSLN-expressing cells, such as COV644 cells (a mucinous epithelial ovarian cancer cell line that expresses MSLN).

[0256] Specifically, recombinant human MSLN ECD His-tagged protein (Acro Biosystem, catalog #MSN-H5223) or recombinant cynomolgus macaque MSLN ECD His-tagged protein (having the amino acid sequence shown in SEQ ID NO: 77) was used as an immunogen to immunize Harbour H2L2 transgenic mice.

[0257] Using the Beacon® Optofluidic System, single B cell screening was performed to obtain antibody heavy and light chain sequences from single plasma cells. The general procedure involved extracting and purifying total RNA from single plasma cell lysates, performing reverse transcription to synthesize cDNA, amplifying and purifying the cDNA, amplifying DNA sequences encoding the antibody heavy and light chains, cloning and transfection, and Sanger sequencing. The obtained sequences were subjected to uniqueness analysis and clustering analysis, followed by synthesis of DNA sequences encoding the paired antibody heavy and light chains. Initial antibodies PR300159 and PR300186 were obtained.

[0258] The VH and VL sequences of the anti-MSLN antibodies PR300159 and PR300186 were further optimized by a PTM (post-translational modification) removal procedure. The designed variants obtained by PTM removal from PR300159 and PR300186 are shown in Table 2. The amino acid sequences of these anti-MSLN antibodies are shown in Tables 3 to 6. PR300159-1, PR300159-3, PR300159-4, PR300159-5, PR300159-6, PR300159-7, PR300159-8, and PR300159-9 are PTM-removed antibodies derived from PR300159. PR300186-2, PR300186-3, PR300186-4, PR300186-5, PR300186-6, PR300186-7, PR300186-8, PR300186-9, and PR300186-10 are PTM-removed antibodies derived from PR300186.

[0259] [Table 2]

[0260] [Table 3-1]

[0261]

Table 3-2

[0262]

Table 3-3

[0263]

Table 3-4

[0264]

Table 3-5

[0265]

Table 3-6

[0266]

Table 4-1

[0267]

Table 4-2

[0268]

Table 5-1

[0269]

Table 5-2

[0270]

Table 5-3

[0271]

Table 5-4

[0272]

Table 6-1

[0273]

Table 6-2

[0274]

Table 6-3

[0275] Example 2. Binding activity of antibodies to human and cynomolgus monkey MSLN proteins Human MSLN (Acro Biosystems, catalog #: MSN-H5223) or cynomolgus monkey MSLN protein (Harbour BioMed, batch #: 2019072202) was diluted to a concentration of 1 μg / mL in PBS. 100 μL of diluted human or cynomolgus monkey MSLN was added to each well of an ELISA microplate and incubated overnight at 4°C. The plate was blocked with ELISA blocking solution (containing 2% w / v BSA, 0.05% (v / v) Tween-20, and PBS buffer at pH 7.4) for 1 hour at 37°C. The plates were then washed and incubated with diluted (15 μg / mL) anti-MSLN antibodies (PR300186, PR300186-2, PR300186-3, PR300186-4, PR300186-5, PR300186-6, PR300186-7, PR300186-8, PR300186-9, and PR300186-10) (100 nM, 10-fold dilution, 8-point dilution) for 1 hour at 37° C. The plates were then washed and incubated with HRP-conjugated goat anti-human IgG (H+L) antibody (Jackson, catalog: 109-035-088) for 1 hour at 37° C. Then, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Biopanda, catalog: TMB-S-003) was added, and the plate was incubated at room temperature for 15 minutes. The reaction was stopped by adding 100 μL of ELISA stop solution (Solarbio, catalog #: C1058), and the optical density at 450 nm (OD450nm) was measured using an ELISA plate reader (Molecular Devices, SpectraMax 384 Plus).

[0276] The binding activity results of PR300186, PR300186-2, PR300186-3, PR300186-4, PR300186-5, PR300186-6, PR300186-7, PR300186-8, PR300186-9, and PR300186-10 for human MSLN protein are shown in Figure 1 and Table 7, and the binding activity results for cynomolgus monkey MSLN protein are shown in Figure 2 and Table 7. The results show that all of these antibodies exhibit good binding activity for both human and cynomolgus monkey MSLN protein.

[0277] [Table 7]

[0278] Example 3. Binding activity of MSLN antibodies to MSLN-expressing cells Binding of recombinant anti-MSLN antibodies (PR300159, PR300159-1, PR300159-3, PR300159-5, PR300159-6, PR300159-8, PR300159-9, PR300186, PR300186-2, PR300186-3, PR300186-4, PR300186-5, PR300186-6, PR300186-7, PR300186-8, PR300186-9, or PR300186-10) to the COV644 cell line (ECACC, Cat#: 07071908) was detected by flow cytometry. Briefly, anti-MSLN antibodies were serially diluted in staining buffer (PBS containing 2% FBS). Add 50 μL of diluted antibody solution to 1-2 × 10 5 The solution was added to 50 μL of cell suspension containing 100 μL of 100 μL of 1:1000 diluted fluorescently labeled anti-human IgG antibody (Alexa Fluor® 488 AffiniPure Goat Anti-Human IgG (H+L), Jackson ImmunoResearch, Cat#: 109-545-088) per well. After incubation at 4° C. for 1 hour, the cells were washed twice with staining buffer and subjected to flow cytometry.

[0279] As shown in Figure 3 and Table 8, PR300159, PR300159-1, PR300159-3, PR300159-5, PR300159-6, PR300159-8, and PR300159-9 all showed high affinity for COV644 cells.

[0280] [Table 8]

[0281] As shown in Figure 4, PR300186, PR300186-2, PR300186-3, PR300186-4, PR300186-5, PR300186-6, PR300186-7, PR300186-8, PR300186-9, and PR300186-10 all showed high affinity for COV644 cells.

[0282] Example 4. Determination of antibody internalization by MSLN-expressing cells In this example, pHAb Amine Reactive Dye (Promega, catalog # G9841) is used to determine the internalization efficiency of an anti-MSLN antibody in various tumor cells. pHAb dye is a pH sensor dye that exhibits very low fluorescence at pH values above 7 and significantly increases fluorescence as the pH value decreases. When pHAb dye-labeled antibodies bind to the outer cell membrane under neutral pH conditions, little or no fluorescence is detected. After pHAb dye-labeled antibodies are internalized into cells, strong fluorescence is detected in the low pH environment of the cell's endosomes and lysosomes.

[0283] The antibody is labeled with pHAb dye, and the DAR value is calculated according to the instructions in the reagent kit. The pHAb dye-labeled antibody is incubated with COV644 cells at 4°C (a temperature with low antibody internalization activity, used as a background control) or 37°C for 24 hours. Fluorescence is then measured at an excitation value (Ex) of 532 nm and an emission value (Em) of 560 nm. The antibody internalization result is calculated by dividing the fluorescence intensity at 37°C minus the background fluorescence intensity at 4°C by the DAR value of the antibody-conjugated dye. A higher fluorescence value indicates a higher antibody internalization efficiency in the cells.

[0284] As shown by the results in Figures 5 and 6 and Table 9, PR300159, PR300159-1, PR300159-3, PR300159-4, PR300159-5, PR300159-6, PR300159-7, PR300159-8, PR300159-9, PR300186, PR300186-3, PR300186-9, and PR300186-10 all showed good internalization activity.

[0285] [Table 9]

[0286] Example 5. Binding activity of PR300159-8 and PR300186-10 to various tumor cells The binding activity of antibodies PR300159-8 and PR300186-10 against the following tumor cells was assessed using flow cytometry: breast cancer cell lines COV644 (ECACC, 07071908), OVCAR3 (ATCC, HTB-161), and SKOV3 (ATCC, HTB77); lung cancer cell lines NCI-H226 (ATCC, CRL5826) and HCl-H1568 (ATCC, CRL5876); pancreatic cancer cell lines HPAC (ATCC, CRL2199) and CAPAN2 (ATCC, HTB80); and gastric cancer cell line MKN45 (CCTCC, GDC0220).

[0287] The PR300159-8 and PR300186-10 antibodies were serially diluted in staining buffer (PBS with 2% FBS). 50 μL of diluted antibody solution was added to 1-2 × 10 5 The cells were added to 50 μL of a cell suspension containing 100 μL of cells and incubated for 1 hour at 4° C. The cells were washed twice with buffer (PBS with 2% fetal bovine serum), and 100 μL of fluorescently labeled anti-human IgG antibody (Alexa Fluor® 488 AffiniPure Goat Anti-Human IgG (H+L), Jackson ImmunoResearch, catalog 109-545-088) was added to each well. After incubation for 1 hour at 4° C., the cells were washed twice with buffer and analyzed using a flow cytometer.

[0288] The experimental results are shown in Table 10. The experimental results show that antibody PR300159-8 exhibited strong binding activity across a variety of tumor cell lines. Antibody PR300159-8 exhibited higher binding activity than PR300186-10.

[0289] [Table 10]

[0290] Example 6. Internalization activity of PR300159-8 antibody and PR300186-10 antibody against various tumor cells The internalization efficiency of antibodies PR300159-8 and PR300186-10 against various tumor cells was assessed using flow cytometry: breast cancer cell line COV644 (ECACC, 07071908); lung cancer cell line NCI-H226 (ATCC, CRL5826); and pancreatic cancer cell line HPAC (ATCC, CRL2199).

[0291] In this example, pHAb Amine Reactive Dye (Promega, catalog #G9841) was used to determine the internalization efficiency of anti-MSLN antibodies into various tumor cells. pHAb dye is a pH sensor dye that exhibits very low fluorescence at pH values above 7, and its fluorescence increases significantly as the pH value of the solution decreases. When pHAb dye-labeled antibodies bind to the outer cell membrane under neutral pH conditions, little or no fluorescence is detected. After pHAb dye-labeled antibodies are internalized into cells, strong fluorescence is detected in the low pH environment of the cell's endosomes and lysosomes.

[0292] The antibody was labeled with pHAb dye, and the DAR value was calculated according to the instructions in the reagent kit. The pHAb dye-labeled antibody was incubated with COV644 cells at 4°C (a temperature with low antibody internalization activity, used as a background control) or 37°C for 24 hours. Fluorescence was then measured at an excitation value (Ex) of 532 nm and an emission value (Em) of 560 nm. The antibody internalization result was calculated as follows: the fluorescence intensity at 37°C minus the background fluorescence intensity at 4°C divided by the DAR value of the antibody-conjugated dye. A higher fluorescence value indicates a higher antibody internalization efficiency into the cells.

[0293] The results shown in Figures 7, 8, and 9 indicate that both PR300159-8 and PR300186-10 exhibited good internalization activity in COV644, NCI-H226, and HPAC cells. The cell internalization activity of antibody PR300159-8 was higher than that of PR300186-10.

[0294] Example 7. Synthesis of biologically active molecules and intermediates used in the process of synthesizing "drug-linker compounds" Example 7.1: Synthesis of (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13 dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1)

[0295] [ka]

[0296] Step 1: Synthesis of (S)-14-(3-chloropropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1B)

[0297] [ka]

[0298] In an ice bath, ferrous sulfate heptahydrate (570 mg dissolved in 1 mL of water) and 4,4-dimethoxychlorobutane (3.89 g) were added to a 75% sulfuric acid solution (5 mL) of the compound (S)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1A, 500 mg). The reaction mixture was stirred for 3 minutes, and then hydrogen peroxide (29%, 2.5 mL) was added dropwise. The reaction mixture was stirred and reacted at 0°C for 5 minutes, then warmed to room temperature and stirred for 3 hours. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (80 mL × 2). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the crude product, which was further purified using a C18 column (acetonitrile / 0.05% formic acid aqueous solution: 5% to 60%) to give the target compound A1B (yellow solid, 400 mg, yield: 67%). LCMS(ESI)[M+H] + :468.9; 1H NMR(400MHz,DMSO-d6)δ 7.65(s,1H),7.51(s,1H),7.24(s,1H),6.50(s,1H),6.30(s,2H),5.42(s,2H),5.26(s ,2H),3.81(d,J=5.9Hz,2H),3.22(s,2H),1.98(d,J=6.7Hz,4H),0.88(t,J=7.2Hz,3H).

[0299] Step 2: Synthesis of (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13 dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1)

[0300] [ka]

[0301] The compound (S)-14-(3-chloropropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1B, 100 mg, 0.213 mmol) was dissolved in a solution of 10% sulfuric acid (5 mL) and reacted at 110° C. for 48 hours. Saturated sodium bicarbonate solution (mL) was added to the reaction mixture, and extraction was performed using dichloromethane (10 mL × 5), followed by drying over anhydrous sodium sulfate, filtration, and concentration under reduced pressure to obtain a crude product. The crude product was purified using preparative high-performance liquid chromatography (acetonitrile / water containing 0.05% formic acid) to give (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13 dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1, 1.78 mg). LCMS(ESI)[M+H] + :451.0; 1H NMR(400MHz,DMSO-d6)δ 7.63(s,1H),7.50(s,1H),7.24(s,1H),6.48(s,1H),6.28(s,2H),5.47-5.37(m,2H),5.32 -5.19(m,2H),3.51-3.46(m,2H),3.17-3.13(m,2H),1.92-1.76(m,4H),0.90-0.84(m,3H).

[0302] Example 7.2: Synthesis of (S)-4-ethyl-8-fluoro-4-hydroxy-11-(3-hydroxypropyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A2)

[0303] [ka]

[0304] Step 1: At 0°C, 1 mol / L boron trichloride (96 mL) and 4-chlorobutyronitrile (9.9 g) were added dropwise to a solution of compound A2A (10 g) in 1,2-dichloroethane (200 mL). The mixture was stirred and reacted at 80°C for 2 hours. After the reaction mixture was cooled to room temperature, 2 mol / L hydrochloric acid (90 mL) was added and the mixture was refluxed and stirred at 80°C for 0.5 hours. The reaction mixture was cooled to room temperature, diluted with a small amount of water, and extracted with dichloromethane (200 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain the target compound A2B (4 g). LCMS(ESI)[M+H] + :230.0.

[0305] Step 2: To compound A2B (50 mg) were added (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolo-3,6,10(4H)-trione (A2C, 35 mg), p-toluenesulfonic acid monohydrate (41.4 mg), and dichloromethane (30 mL). After clarification and mixing, the solution was concentrated under reduced pressure and vacuumed using an oil pump. The reaction was carried out under vacuum at 120 °C for 3 hours. LCMS showed that the reaction was complete. After the reaction mixture was cooled to room temperature, water (20 mL) was added, and the mixture was extracted with dichloromethane (20 mL × 3). The combined organic phases were successively dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified by column chromatography (dichloromethane:methanol=20:1) to give the target compound A2D (80 mg) as a white solid. LCMS(ESI)[M+H] + :457.0.

[0306] Step 3: Compound A2D (75 mg) was dissolved in hexamethylphosphoramide, purified water (0.8 mL) was added, and the reaction mixture was stirred at 100° C. for 72 hours. LCMS showed the completion of the reaction. The crude product was purified by preparative chromatography (0.01% TFA in water, MeCN) to give target compound A2 (10 mg). LCMS(ESI)[M+H] + :439.2; 1 H NMR(400MHz,DMSO-d6)δ 8.22(d,J=8.4Hz,1H),7.87(d,J=10.9Hz,1H),7.31(s,1H),6.50(s,1H),5.43(s,2H),5.30(s,2H),4.67(t, J=4.9Hz,1H),3.55-3.47(m,2H),3.28-3.20(m,2H),2.51(s,3H),1.93-1.81(m,4H),0.88(t,J=7.3Hz,3H).

[0307] Example 7.3: Synthesis of (S)-2-amino-N-((3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13 tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)propoxy)methyl)acetamide (B1)

[0308] [ka]

[0309] Step 1: Compound B1A (368 mg), Compound A1 (440 mg), and pyridinium p-toluenesulfonate (PPTS, 25 mg) were refluxed in dichloromethane (20 mL) for 20 hours. The reaction mixture was then washed with aqueous sodium bicarbonate and hydrochloric acid, respectively. The organic solvent was removed under reduced pressure to obtain the crude product, which was separated and purified by column chromatography (dichloromethane:methanol=10:1) to obtain the target compound B1B (240 mg). LCMS(ESI)[M+H] + :759.5.

[0310] Step 2: Compound B1B (240 mg) was dissolved in DMF (5 mL) and piperidine (1 mL) was added. The compound was stirred for 20 minutes and dissolved under reduced pressure to remove low-boiling components. The residue was used directly in the next step of synthesis. A small amount of the crude product was purified by reverse phase chromatography (acetonitrile / 0.05% FA aqueous solution: 5% to 50%) to obtain target compound B1. ESI-MS(m / z):537.4[M+H] + ; 1H NMR(400MHz,DMSO-d6)δ 9.13(t,1H),8.04(br,2H),7.58(s,1H),7.51(s,1H),7.25(s,1H),6.29(s,2H),5.43(S,2H) ,5.21(s,2H),4.65(d,2H),3.63(m,2H),3.53(m,2H),3.11(m,2H),1.87(m,4H),0.88(t,3H).

[0311] Example 7.4: Synthesis of (S)-2-amino-N-((3-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)propoxy)methyl)acetamide (B2)

[0312] [ka]

[0313] Step 1: Compound A2 (160 mg, 0.365 mmol) was dissolved in N,N-dimethylformamide (DMF, 3 mL), and (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (B2A, 672 mg, 1.83 mmol) was added. HCl-ethyl acetate (0.073 mL, 3 M) was added to the reaction mixture, which was then stirred at room temperature overnight. Completion of the reaction was detected by LCMS. The reaction mixture was directly purified by reverse-phase chromatography (acetonitrile / 0.05% FA in water: 5% to 50%) to give the target compound B2B as a white solid (80 mg, 29.0% yield). LCMS(ESI)[M+H] + =747.4; 1H NMR(400MHz,DMSO-d6)δ 8.74(t,J=6.4Hz,1H),8.28-8.17(m,1H),7.99-7.88(m,3H),7.73(d,J=7.3Hz,2H),7.63(t,J= 5.7Hz,1H),7.44(t,J=7.4Hz,2H),7.35(t,J=7.2Hz,3H),6.58(s,1H),5.48(s,2H),5.29(s,2H) ),4.66(d,J=6.3Hz,2H),4.32(d,J=6.9Hz,2H),4.26(d,J=6.1Hz,1H),3.71(d,J=5.8Hz,2H),3 .57(t,J=5.7Hz,2H),3.29-3.20(m,2H),2.55(s,3H),2.00-1.86(m,4H),0.93(t,J=7.2Hz,3H).

[0314] Step 2: B2B (240 mg) was dissolved in DMF (5 mL) and piperidine (1 mL) was added. The compound was stirred for 20 minutes and dissolved under reduced pressure to remove low boiling components. The residue was used directly in the next step of the synthesis. ESI-MS(m / z):525.2[M+H] + .

[0315] A small amount of the crude product was purified by reverse phase chromatography (acetonitrile / 0.05% FA in water: 5% to 50%) to give the target compound B2. ESI-MS(m / z):525.1[M+H] + ; 1 H NMR(400MHz,DMSO)δ 9.13(t,J=6.6Hz,1H),8.21(d,J=8.1Hz,1H),8.02(brs,2H),7.89(d,J=10.8Hz,1H),7.32(s,1H),6.54(s,1H),5.44(s,2H),5.28(s,2H) ),4.66(d,J=6.5Hz,2H),3.64(s,2H),3.53(t,J=6.1Hz,2H),3.25-3.18(m,2H),2.52(s,3H),1.98-1.84(m,4H),0.88(t,J=7.3Hz,3H).

[0316] Example 7.5: N 6 ,N 6 -Dimethyl-N 2 Synthesis of -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valine)-L-lysine (C1)

[0317] [ka]

[0318] 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (268 mg), compound C1A (328 mg), and triethylamine (322 mg) were dissolved in N,N-dimethylformamide (5 mL). 1-Hydroxybenzotriazole (HOBT, 162 mg) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 229 mg) were then added. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was directly purified using a C18 reverse-phase column (acetonitrile and 0.05% formic acid aqueous system) to give the target compound N. 6 ,N 6 -Dimethyl-N 2 -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valine)-L-lysine (C1, white solid, 327 mg) was obtained. LCMS(ESI)[M+H] + :524.4. 1 H NMR(400MHz,)δ 9.13(s,2H),7.95(t,J=8.8Hz,2H),4.21(dd,J=8.8,6.9Hz,1H),4.08-4 .03(m,1H),3.41(s,3H),2.55(t,J=7.0Hz,2H),2.42-2.32(m,4H),2.27 (s,6H),1.98(dd,J=13.6,6.8Hz,1H),1.86-1.77(m,2H),1.74-1.55(m, 2H),1.47-1.37(m,2H),1.31-1.23(m,2H),0.85(dd,J=12.8,6.8Hz,6H).

[0319] Example 7.6: N 6 ,N 6 -Diethyl-N 2 Synthesis of -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valine)-L-lysine (C2)

[0320] [ka]

[0321] Step 1: Compound C2A (5.0 g, 12.05 mmol) was dissolved in dichloromethane (100 mL). Acetaldehyde (3.2 g, 72.3 mmol) was added to the reaction mixture, and the mixture was stirred and reacted at room temperature for 10 minutes. Sodium triacetoxyborohydride (12.8 g, 60.25 mmol) was then added to the reaction mixture, and the mixture was stirred and reacted at room temperature for 1 hour. LCMS showed that the reaction was complete. Saturated aqueous ammonium chloride solution was added to the reaction mixture, and the mixture was stirred for 1 hour. The mixture was dried by centrifugation and filtered. The filtrate was separated and purified using a C18 reverse-phase column (acetonitrile and 0.05% formic acid in water: 5% to 55%) to obtain the target compound C2B (4.57 g, 82% yield) as a white solid. LCMS(ESI)[M+H] + =436.4; 1 H NMR (400 MHz, DMSO-d6) δ 7.70(d,J=7.0Hz,1H),7.41(d,J=9.0Hz,1H),7.38-7.26(m,5H),5.08-4.99( m,2H),4.00(dd,J=12.6,6.5Hz,1H),3.86(dd,J=8.6,6.8Hz,1H),2.74(dd,J= 14.0,6.9Hz,4H),2.64-2.54(m,2H),2.05-1.94(m,1H),1.72-1.52(m,2H),1. 52-1.38(m,2H),1.38-1.18(m,2H),1.04(t,J=7.1Hz,6H),0.87-0.81(m,6H).

[0322] Step 2: Compound C2B (1.6 g, 3.68 mmol) was dissolved in methanol (80 mL) at room temperature, and then Pd / C (0.16 g) was added to the reaction mixture, which was stirred under hydrogen gas at room temperature for 12 hours. LCMS showed that the reaction was complete. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give the target compound C2C (900 mg, 82% yield) as an off-white solid. 1 H NMR(400MHz,DMSO-d6)δ 8.04(br s,1H),4.02 - 3.99(m,1H),3.10(d,J=4.5Hz,1H),2.65(q,J=7.1Hz,4H),2.55-2.51(m,2H),2.06-1.93(m,1H),1.73-1.54(m, 2H),1.47-1.38(m,2H),1.30-1.21(m,2H),1.01(t,J=7.1Hz,6H),0.89(d,J=6.9Hz,3H),0.79(d,J=6.8Hz,3H).

[0323] Step 3: Compound 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (268 mg, 1 mmol), HATU (380 mg, 1 mmol), and triethylamine (322 mg, 2.5 mmol) were added sequentially in DMF (8 mL) and stirred at room temperature for 20 minutes. Compound C2C (301 mg, 1 mmol) was then added and stirring was continued at room temperature for 30 minutes. After confirming the completion of the reaction by LCMS, the reaction mixture was directly purified using a C18 reverse-phase column (acetonitrile and 0.05% formic acid aqueous system) to obtain the target compound C2 (280 mg, 51% yield) as a white solid. LCMS(ESI)[M+H] + =552.3; 1H NMR(400MHz,DMSO-d6)δ 9.13(s,2H),7.95(d,J=8.9Hz,1H),7.86(d,J=7.2Hz,1H),4.18(dd,J=8.8,6.8Hz ,1H),4.02(dd,J=12.8,7.2Hz,1H),3.41(s,3H),2.74-2.69(m,4H),2.62-2.52(m ,4H),2.44-2.29(m,2H),2.04-1.94(m,1H),1.86-1.77(m,2H),1.72-1.54(m,2H) ,1.51-1.39(m,2H),1.33-1.23(m,2H),1.02(t,J=7.2Hz,6H),0.87-0.82(m,6H).

[0324] Example 7.7: N 6 ,N 6 -Dipropyl-N 2 Synthesis of -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valine)-L-lysine (C3)

[0325] [ka]

[0326] Step 1: Compound C2A (5.0 g, 12 mmol) was dissolved in dichloromethane (100 mL). n-Propionaldehyde (4.2 g, 72.3 mmol) was added to the reaction mixture, and the mixture was stirred and reacted at room temperature for 10 minutes. Sodium triacetoxyborohydride (12.8 g, 60.25 mmol) was then added to the reaction mixture, and the mixture was stirred and reacted at room temperature for 1 hour. LCMS showed that the reaction was complete. Saturated aqueous ammonium chloride solution was added to the reaction mixture, and the mixture was stirred for 1 hour. The mixture was dried by centrifugation and filtered. The filtrate was separated and purified using a C18 reverse-phase column (acetonitrile and 0.05% formic acid aqueous solution: 5% to 55%) to obtain the target compound C3A (4.57 g, 82.0% yield) as a white solid. LCMS(ESI)[M+H] + =464.0; 1H NMR(400MHz,DMSO-d6)δ 7.81(d,J=7.3Hz,1H),7.38-7.28(m,5H),5.04(d,J=1.7Hz,2H),4.12-4.02(m,1H),3.94-3.82(m,1H),2.65-2.52(m,6 H),2.06-1.94(m,1H),1.76-1.64(m,1H),1.64-1.53(m,1H),1.52-1.40(m,6H),1.34-1.18(m,2H),0.92-0.80(m,12H).

[0327] Step 2: Compound C3A (2.0 g, 4.32 mmol) was dissolved in methanol (80 mL) at room temperature. Then, Pd / C (0.16 g) was added to the reaction mixture, and the mixture was stirred and reacted under hydrogen gas at room temperature for 12 hours. LCMS showed that the reaction was complete. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give the target compound C3B (1.2 g, 85.5% yield) as a white solid.

[0328] Step 3: Compound 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (100 mg, 0.373 mmol) was dissolved in N,N-dimethylformamide (1 mL). HATU (142 mg, 0.373 mmol) and N,N-diisopropylethylamine (120 mg, 0.93 mmol) were then added, and the system was stirred for 30 minutes. Compound C3B (122 mg, 0.371 mmol) was then added, and the reaction mixture was stirred at room temperature for 1 hour. After LCMS detected the completion of the reaction, the reaction mixture was directly purified using a C18 reverse-phase column (acetonitrile and 0.05% formic acid aqueous system) to give the target compound N. 6 ,N 6 -Dipropyl-N 2 -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valine)-L-lysine (C3, 50 mg, 28% yield) was obtained as a pale yellow solid. LCMS(ESI)[M+H] + =580.0; 1H NMR(400MHz,DMSO-d6)δ 8.24(s,2H),7.98-7.93(m,2H),4.24-4.16(m,1H),4.10(d,J=5.2Hz,1 H),3.41(s,3H),2.79-2.64(m,6H),2.55(t,J=7.1Hz,2H),2.45-2.26( m,2H),2.06-1.91(m,1H),1.89-1.78(m,2H),1.76-1.66(m,1H),1.64- 1.57(m,1H),1.57-1.42(m,6H),1.37-1.24(m,2H),0.93-0.78(m,12H).

[0329] Example 7.8: N 6 ,N 6 -Dibutyl-N 2 Synthesis of -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valine)-L-lysine (C4)

[0330] [ka]

[0331] Step 1: Compound C2A (10.0 g) was dissolved in dichloromethane (200 mL). n-Butylaldehyde (10.4 g) was added to the reaction mixture, and the mixture was stirred and reacted at room temperature for 10 minutes. Sodium triacetoxyborohydride (25.6 g) was then added to the reaction mixture in portions, and the mixture was stirred and reacted at room temperature for 1 hour. LCMS showed that the reaction was complete. Saturated aqueous ammonium chloride solution was added to the reaction mixture, and the mixture was stirred for 1 hour. The mixture was spun dry and filtered. The filtrate was separated and purified using a C18 reverse-phase column (acetonitrile and 0.05% formic acid aqueous solution, 5% to 55%) to obtain the target compound C4A (4.9 g) as a white solid. LCMS(ESI)[M+H] + =492.7; 1H NMR(400MHz,CDCl3)δ 7.35-7.30(m,5H),5.14-5.08(m,2H),4.33-4.30(m,1H),4.14-4.12(m,1H),2.94-2.80(m,6H), 2.13-2.11(m,1H),1.85-1.82(m,2H),1.58-1.55(m,4H),1.40-1.22(m,8H),0.98-0.87(m,12H).

[0332] Step 2: Compound C4A (5.0 g) was dissolved in a methanol solution (100 mL) at room temperature. Then, Pd / C (10%, 1 g) was added to the reaction mixture, and the mixture was stirred and reacted under hydrogen gas for 12 hours. LCMS showed that the reaction was complete. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give target compound C4B (3.68 g) as a white solid. LCMS(ESI)[M+H] + =358.3.

[0333] Step 3: Compound 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (1.0 g) was dissolved in N,N-dimethylformamide (15 mL). Then, N,N-diisopropylethylamine (1.2 g) and HATU (1.4 g) were added sequentially, and the reaction was stirred for 30 minutes. Compound C4B (1.3 g) was then added, and the reaction mixture was stirred at room temperature for 1 hour. After LCMS detected the completion of the reaction, the reaction mixture was directly purified using preparative chromatography (0.01% aqueous trifluoroacetic acid and acetonitrile) to obtain the target compound N. 6 ,N 6 -Dibutyl-N 2 -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valine)-L-lysine (C4, 500 mg) was obtained as a yellow solid. LCMS(ESI)[M+H] + =608.7; 1H NMR(400MHz,DMSO)δ 9.37(s,1H),9.13(s,2H),8.19(d,J=7.3Hz,1H),7.92(d,J=8.8Hz,1H),4. 25-4.20(m,1H),4.20-4.13(m,1H),3.42(s,3H),3.06-2.99(m,6H),2.57- 2.53(m,2H),2.41-2.30(m,2H),1.99-1.95(m,2H),1.87-1.79(m,2H),1.7 8-1.71(m,1H),1.62-1.56(m,4H),1.34-1.32(m,8H),0.94-0.85(m,12H).

[0334] Example 8. Synthesis of drug-linker compounds Example 8.1: N-((11S,14S)-11-(4-(di-n-propylamino)butyl)-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecan-14-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoylamide (DL-01)

[0335] [ka]

[0336] Compound C3 (43 mg, 0.074 mmol) and compound B1 (40 mg, 0.075 mmol) were dissolved in N,N-dimethylformamide (1 mL). Then, HBTU (28 mg, 0.075 mmol) and N,N-diisopropylethylamine (24 mg, 0.187 mmol) were added successively. The reaction mixture was stirred at room temperature for 1 hour. After LCMS detected the completion of the reaction, the reaction mixture was directly purified using preparative chromatography (0.01% aqueous trifluoroacetic acid and acetonitrile) to obtain the target compound DL-01 (8.5 mg, 10% yield) as a yellow solid. LCMS(ESI)[M+H] + =1098.6; 1 H NMR(400MHz,DMSO-d6)δ 9.10(s,2H),9.03(s,1H),8.64(t,J=6.4Hz,1H),8.19(t,J=5.9Hz,1H),8.08(d,J=7.4Hz,1H),7.92(d,J=8.5Hz,1H),7.59( s,1H),7.51(s,1H),7.24(s,1H),6.49(s,1H),6.29(s,2H),5.42(s,2H),5.24(s,2H),4.66-4.52(m,2H),4.31-4.21(m,1H), 4.19-4.10(m,1H),3.74(d,J=5.5Hz,2H),3.49-3.48(m,2H),3.40(s,3H),3.15-3.06(m,2H),3.02-2.95(m,6H),2.59-2.52 (m,3H),2.41-2.29(m,2H),2.05-1.90(m,2H),1.91-1.77(m,6H),1.63-1.57(m,6H),1.31-1.29(m,2H),0.92-0.80(m,15H).

[0337] Example 8.2: N-((11S,14S)-11-(4-(diethylamino)butyl)-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecan-14-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoylamide (DL-02)

[0338] [ka]

[0339] Compound C2 (40 mg, 0.075 mmol) and Compound B1 (41 mg, 0.075 mmol) were dissolved in DMF (1 mL), followed by the addition of HOBt (15.2 mg, 0.113 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (21.5 mg, 0.113 mmol), followed by the addition of triethylamine (23 mg, 0.225 mmol). The reaction mixture was then stirred at room temperature for 16 hours. After LCMS detected the completion of the reaction, the reaction mixture was concentrated to give the crude product, which was purified using preparative chromatography (0.01% TFA in water, MeCN) to give the target compound N-((11S,14S)-11-(4-(diethylamino)butyl)-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[ 1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecan-14-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoylamide (16 mg, 20% yield) was obtained as a yellow solid. LCMS(ESI)[M+H] + =1070.6; 1H NMR(400MHz,DMSO-d6)δ 9.13-9.08(m,2H),9.01(s,1H),8.64(t,J=6.5Hz,1H),8.20(t,J=5.7Hz,1H),8.09(d,J=7.4Hz,1H),7.92(d,J=8.4Hz,1H),7.59(s ,1H),7.51(s,1H),7.24(s,1H),6.50(s,1H),6.29(s,2H),5.43(s,2H),5.24(s,2H),4.65-4.53(m,2H),4.26(d,J=6.5Hz,1H),4.2 0-4.10(m,1H),3.74(d,J=5.5Hz,2H),3.50(t,J=5.8Hz,2H),3.41(s,3H),3.14-3.07(m,6H),2.98(s,2H),2.55(d,J=7.3Hz,2H),2 .41-2.29(m,2H),2.03-1.89(m,2H),1.89-1.77(m,7H),1.61-1.56(m,2H),1.32(s,2H),1.16(t,J=7.2Hz,6H),0.91-0.78(m,9H).

[0340] Example 8.3: N-((11S,14S)-11-(4-(dimethylamino)butyl)-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecan-14-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoylamide (DL-03)

[0341] [ka]

[0342] Compound B1 (100 mg, 0.186 mmol) and Compound N 6 ,N 6 -Dimethyl-N 2-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valine)-L-lysine (C1) (100 mg, 0.191 mmol) was dissolved in DMF (2 mL). 1-Hydroxybenzotriazole (38 mg, 0.280 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (53 mg, 0.280 mmol) were then added, followed by triethylamine (56 mg, 0.559 mmol). After the addition was complete, the reaction mixture was stirred at room temperature for 16 hours. After LCMS detected the completion of the reaction, the reaction mixture was purified by preparative chromatography (0.01% TFA in water, MeCN) to give the target compound N-((11S,14S)-11-(4-(dimethylamino)butyl)-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecan-14-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoylamide (DL-03) (20 mg, 10% yield) as a yellow solid. LCMS(ESI)[M+H] + =1042.5; 1H NMR(400MHz,DMSO-d6)δ 9.28(s,1H,TFA),9.10(s,2H),8.64(br s,1H),8.19(br s,1H),8.09(d,J=6.4Hz,1H),7.92(d,J=7.8Hz,1H),7.59(s,1H),7.51(s,1H),7.24(s,1H),6.5 0(s,1H),6.29(s,2H),5.43(s,2H),5.24(s,2H),4.67-4.52(m,2H),4.30-4.10(m,2H),3.74(br s,2H),3.50(br s,2H),3.41(s,3H),3.17-3.07(m,2H),3.04-2.91(m,2H),2.75(s,6H),2.46-2.24( m,3H),2.04-1.66(m,9H),1.63-1.46(m,3H),1.32-1.29(m,2H),0.87-0.82(m,9H).

[0343] Example 8.4: N-((11S,14S)-11-(4-(dibutylamino)butyl)-1-((S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecan-14-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoylamide (DL-04)

[0344] [ka]

[0345] Compound C4 (50 mg, 0.08 mmol) and compound B2 (43 mg, 0.08 mmol) were dissolved in N,N-dimethylformamide (1.5 mL). Then, DIPEA (26 mg, 0.21 mmol) and HBTU (31 mg, 0.08 mmol) were added sequentially. The mixture was stirred and reacted at room temperature for 1 hour. After the completion of the reaction was detected by LCMS, the reaction mixture was directly separated and purified using high-performance preparative chromatography (0.01% aqueous trifluoroacetic acid, MeCN) to obtain the target compound DL-04 (13.57 mg) as a yellow solid. LCMS(ESI)[M+H] + =1114.6; 1 H NMR(400MHz,DMSO)δ 9.09(s,2H),9.05(s,1H,TFA),8.64(t,J=7.2Hz,1H),8.23-8.16(m,2H),8.07(d,J=7.3Hz,1H), 7.93-7.85(m,2H),7.32(s,1H),6.52(s,1H),5.44(s,2H),5.29(s,2H),4.61-4.59(m,2H),4.24- 4.22(m,1H),4.19-4.10(m,1H),3.73-3.70(m,2H),3.50(s,3H),3.21-3.18(m,6H),3.00-2.98(m ,8H),2.54(s,3H),1.97-1.78(m,7H),1.56-1.52(m,8H),1.36-1.25(m,6H),0.93-0.78(m,15H).

[0346] Example 8.5: N-((10S,13S)-10-(4-(N,N-dimethylamino)butyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-14-methyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triaza-pentadecan-13-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoylamide (DL-05)

[0347] [ka]

[0348] Compound N 6 ,N 6 -Dimethyl-N 2 -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valine)-L-lysine (C1, 52.3 mg) was dissolved in N,N-dimethylformamide (2 mL), and then HBTU (38 mg, 0.10 mmol) and N,N-diisopropylethylamine (26 mg, 0.20 mmol) were added sequentially. After the addition was completed, the reaction mixture was stirred at room temperature for 30 minutes, and the compound 2-amino-N-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)acetamide (DL-05A, 63 mg, prepared according to the method described in Chinese Patent Publication No. 104755494A, pages 147-148) was added. After LCMS detected the completion of the reaction, the reaction mixture was directly purified by preparative chromatography (0.01% aqueous trifluoroacetic acid and acetonitrile) to give the target compound N-((10S,13S)-10-(4-(N,N-dimethylamino)butyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15 -Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-14-methyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazapentadecan-13-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoylamide DL-05 (8.1 mg, yield 7%) was obtained. LCMS(ESI)[M+H] + =1085.5; 1 H NMR(400MHz,DMSO-d6)δ 9.31(s,1H),9.11(s,2H),8.72(t,J=6.8Hz,1H),8.51(d,J=8.8Hz,1H),8.19(t,J=6.8Hz,1H),8.10(d,J=7.1Hz,1H),7.91(d,J=8. 6Hz,1H),7.79(d,J=10.9Hz,1H),7.32(s,1H),6.53(s,1H),5.63-5.54(m,1H),5.42(s,2H),5.20(s,2H),4.68-4.58(m,2H),4.26-4 .12(m,2H),4.01(s,2H),3.73(d,J=5.4Hz,2H),3.20-3.10(m,2H),3.05-2.95(m,2H),2.76(d,J=4.7Hz,6H),2.55-2.53(m,2H),2. 42-2.28(m,6H),2.21-2.13(m,2H),2.02-1.77(m,6H),1.76-1.62(m,2H),1.62-1.51(m,3H),1.37-1.21(m,2H),0.90-0.80(m,9H).

[0349] Example 9. Preparation of anti-MSLN antibody-drug conjugates Antibody - Preparation of PR300159-B81, PR300159-8-B81, PR300186-B81, and PR300186-10-B81

[0350]

change

[0351] Ab, anti-MSLN antibody PR300159, PR300159-8, PR300186, and PR300186-10.

[0352] 30 mg of anti-MSLN antibody PR300159-8 or PR300186-10 was diluted with diluent (20 mM PB, pH 7.5), and sodium edetate solution was added to a final concentration of 5 mM and mixed thoroughly. 5.5 molar equivalents of TCEP solution relative to the antibody were added, mixed thoroughly, and allowed to stand at room temperature for 60 minutes. 12 molar equivalents of DL-01 dissolved in dimethyl sulfoxide relative to the antibody were added to the solution, mixed thoroughly, and allowed to stand at room temperature for 2 hours to obtain a conjugate sample. After the reaction, the sample was exchanged into 20 mM histidine buffer, pH 6.0, using a 30 kDa ultrafiltration tube to remove low molecular weight substances. Finally, the sample was concentrated to obtain a solution of ADC conjugates containing anti-MSLN antibody PR300159-8 or PR300186-10, designated PR300159-B81, PR300159-8-B81, PR300186-B81, and PR300186-10-B81, respectively. Using the mass spectrometry method described in Example 10, the DAR value was determined to be 8.0.

[0353] Preparation of antibody-drug conjugate PR300159-B71 or PR300186-B71 samples

[0354] [ka]

[0355] Ab is anti-MSLN antibody PR300159 or PR300186.

[0356] 30 mg of anti-MSLN antibody PR300159 or PR300186 was diluted with diluent (20 mM PB, pH 7.5), and sodium edetate solution was added to a final concentration of 5 mM and mixed thoroughly. 5.5 molar equivalents of TCEP solution relative to the antibody were added, mixed thoroughly, and allowed to stand at room temperature for 60 minutes. 12 molar equivalents of DL-03 dissolved in dimethyl sulfoxide relative to the antibody were added to the solution, mixed thoroughly, and allowed to stand at room temperature for 2 hours to obtain a conjugate sample. After the reaction, the sample was exchanged into 20 mM histidine buffer, pH 6.0, using a 30 kDa ultrafiltration tube to remove low molecular weight substances, and then the sample was concentrated to obtain a solution of ADC conjugates containing anti-MSLN antibody PR300159 or PR300186, designated PR300159-B71 and PR300186-B71, respectively. Using the mass spectrometry method described in Example 10, the DAR value was determined to be 8.0.

[0357] Example 10. Determination of DAR values of conjugate samples using mass spectrometry Detection: The molecular weight and DAR value of PR300159-8-B81 were analyzed using LC-MS under the following conditions: Chromatography conditions: Chromatography column: Xbridge Protein BEH SEC (2.5 μm, 4.6 × 150 mm) Mobile phase A: 0.1% FA / H2O Mobile phase B: 0.1%FA / ACN Column temperature: 30℃ Sample compartment temperature: 8°C Flow rate: 0.3mL / min Injection volume: 1μL

[0358] [Table 11]

[0359] Sample preparation: 50 μg of each sample was taken, added with 2 μL of 1 M DTT, and diluted to 50 μL with ultrapure water to achieve a concentration of approximately 1.0 mg / mL. This was mixed thoroughly and reduced at room temperature for 30 minutes.

[0360] LC / MS model: UPLC (AB SCIEX), high resolution mass spectrometer (AB SCIEX).

[0361] Mass spectrometry conditions: Gas 1: 45; Gas 2: 45; CUR: 30; TEM: 450; ISVF: 5000; DP: 120; CE: 12; Mass range: 600-4000

[0362] The results are shown in Table 12.

[0363] [Table 12]

[0364] In Table 12, mAb represents an unconjugated monoclonal antibody; LC represents an antibody light chain; HC represents an antibody heavy chain; DAR1 represents a conjugate containing one toxin molecule conjugated to the light or heavy chain; DAR2 represents a conjugate containing two toxin molecules conjugated to the light or heavy chain; and DAR3 represents a conjugate containing three toxin molecules conjugated to the light or heavy chain. The theoretical molecular weight of the monoclonal antibody is calculated based on the G0F glycoform. In the following text, mAb, LC, HC, DAR1, DAR2, and DAR3 are as previously described.

[0365] The test results show that for PR300159-8-B81, the percentage of antibody light chains conjugated with 0 to 1 toxin molecules (LC, DAR1) is 0% and 100%, respectively. For heavy chains conjugated with 0 to 3 toxin molecules (mAb, DAR1, DAR2, DAR3), the percentages are 0%, 0%, 0%, and 100.0%, respectively. Based on this, the drug-to-antibody ratio (DAR value) of PR300159-8-B81 and PR300186-10-B81 is calculated to be 8.0.

[0366] Example 11. Construction of HT29 huMSLN wild-type cell line and HT29-huMSLN mutant cell line The HT29-huMSLN wild-type cell line (stably transfected wild-type human MSLN) was prepared in the lab. The specific procedures are as follows.

[0367] HT-29 cell suspensions were added to 6-well plates at a density of 5E5 / well and cultured overnight at 37°C. A plasmid encoding the human MSLN gene was added to a tube and then diluted with Opti-MEM (Gibco, catalog number 31985070). The transfection reagent Lipofectamine 3K was added to a separate tube and also diluted with Opti-MEM. The Lipofectamine 3K to DNA ratio was 3:1. The diluted Lipofectamine 3K was added to the diluted plasmid, mixed thoroughly, and incubated at room temperature for 15 minutes. This mixture was then added to the cells, mixed gently by shaking, and incubated overnight at 37°C. The supernatant was then discarded, and fresh medium containing 0.3 μg / mL of G418 was added as a replacement medium. The cells were cultured at 37°C, and the G418-containing medium was replaced every three days. Once the cells reached confluence, FACS analysis was performed. The validated cell pool was diluted to 5 cells / mL and seeded into five 96-well plates at 100 μL / well. The plates were incubated at 37°C for 14 days. Single clones were selected and validated by FACS. Validated clones were subjected to continuous expansion culture and cryopreserved.

[0368] The HT29-huMSLN mutant cell line (stably transfected with mutant human MSLN) was constructed in our laboratory using the same construction method as the HT29-huMSLN wild-type cell line, except that the mutant human MSLN gene was transfected and the YLVLDLSV sequence of the wild-type MSLN gene was mutated to GGGGS in the mutant MSLN gene.

[0369] Example 12. Detection of MSLN shedding in HT29-huMSLN wild-type 2C3 cells and HT29-huMSLN mutant 1A11 cells HT29-huMSLN wild-type 2C3 cells and HT29-huMSLN mutant 1A11 cells were each plated in a 6-well plate at 5 × 10 5 The cells were seeded at a density of 100 cells / well. The next day, the medium was replaced with 1 mL of fresh medium. Every hour, 100 μL of medium was collected and stored at −80°C for subsequent detection of intracellular MSLN shedding.

[0370] PR300159 was diluted to a concentration of 5 μg / mL and added to a 96-well plate (100 μL per well) and incubated overnight at 4°C. The 96-well plate was washed three times with PBST solution, followed by the addition of PBS containing 2% BSA and incubation at 37°C for 1 hour. Human MSLN protein (Acro Biosystems, catalog #MSN-H522) was diluted two-fold starting at 0.2 μg / mL for a total of 11 dilutions to generate a standard curve. The collected cell supernatants were added to the 96-well plate and incubated at 37°C for 1 hour. The plate was washed three times with PBST solution, and biotin-labeled anti-MSLN positive control antibody PR300175 (which binds to a different epitope than PR300159; the heavy and light chain sequences are set forth in SEQ ID NOs: 78 and 79, respectively) was added and incubated at 37°C for 1 hour. The plate was washed three times with PBST solution, and SA-HRP secondary antibody (5000x dilution) was added and incubated for 30-60 minutes at 37°C. The plate was washed three times with PBST solution, and TMB color developing solution was added for 5-15 minutes. The color development was stopped by adding stop solution.

[0371] As shown in Figure 10, the HT29-huMSLN mutant 1A11 cell line showed less MSLN shedding compared to the HT29-huMSLN wild-type 2C3 cell line.

[0372] Example 13. Killing assay of PR300159-8-B81 antibody-drug conjugate against MSLN-expressing cells HT29-huMSLN wild-type 2C3 cells were added to a 96-well plate at 2000 cells / well. Serial dilutions of PR300159-8, hIgG-B81 (human IgG control conjugated with DL-01), and PR300159-8-B81 were then added. After 6 days, cell viability was measured using the Promega CellTiter Glo assay (G7572).

[0373] As shown by the experimental results in Figure 11, PR300159-8-B81 exhibits a significant killing effect on HT29-huMSLN cells.

[0374] Example 14. Pharmacokinetic studies of PR300159-8-B81 and PR300186-10-B81 antibody-drug conjugates Six male SD rats were selected. The antibody-drug conjugate was intravenously administered at a dose of 5 mg / kg. Plasma and serum samples were collected at the following time points: before administration, 10 minutes, 2 hours, 8 hours, 24 hours (day 1), 3 days, 7 days, 10 days, 14 days, 21 days, and 28 days after administration.

[0375] Plasma sample collection: Whole blood was placed into a test tube containing K2-EDTA and centrifuged at 2-8°C for 6 minutes, and plasma samples were stored frozen at approximately -80°C until analysis.

[0376] Serum sample collection: Whole blood was allowed to clot for 30 minutes, centrifuged at 2200 g for 10 minutes at 4°C, and the separated serum samples were frozen at -80°C until analysis.

[0377] Detection of antibody-drug conjugates (bound antibodies): In this example, an ELISA method was used to quantify the concentration of antibody-drug conjugates in rat serum. Specifically, a mouse Fab monoclonal antibody against the toxin molecule A1 was coated on a 96-well plate to capture the antibody-drug conjugates in mouse serum. Then, an HRP-labeled goat anti-human Fc secondary antibody was added for detection.

[0378] Total antibody detection: In this example, the concentration of total antibody in rat serum was quantified using an ELISA method. In the ELISA method, sheep anti-human Fc monoclonal antibody was coated on a 96-well plate to capture antibodies in mouse serum. Then, HRP-labeled goat anti-human IgG secondary antibody was added for detection.

[0379] Detection of toxin molecule A1: In this example, the concentration of small molecules in serum was determined using an LC-MS / MS method. For protein precipitation, 30 μL of plasma sample was added to 100 μL of acetonitrile containing 0.5 ng / mL of internal standard (YL0010098). The mixture was vortexed for 1 minute and then centrifuged. The supernatant was taken for LC-MS / MS analysis (Waters XEVO TQ-XS).

[0380] Serum drug concentration data were analyzed using Phoenix WinNonlin software version 7.0 and a noncompartmental analysis (NCA) model to calculate pharmacokinetic parameters.

[0381] Table 13 shows the pharmacokinetic parameters of PR300159-8-B81 and PR300186-10-B81. The results show that after administration of PR300159-8-B81 and PR300186-10-B81, the half-lives of the total antibody for PR300159-8-B81 and PR300186-10-B81 in rats were 203 hours and 232 hours, respectively, while the half-lives of the antibody-drug conjugates (ADCs) for PR300159-8-B81 and PR300186-10-B81 in rats were 193 hours and 250 hours, respectively.

[0382] [Table 13]

[0383] Example 15. Stability studies of PR300159-8-B81 and PR300186-10-B81 antibody-drug conjugates in human, cynomolgus monkey, SD rat, and ICR mouse plasma The antimicrobial agent ProClin was added to plasma from various species (human, cynomolgus monkey, SD rat, and ICR mouse) and to PBS containing 1% BSA. The final concentration of ProClin was 0.1%. Sterile procedures were performed at 37°C. The antibody-drug conjugate stock solution was diluted to a working solution of 20 mg / mL using PBS at pH 7 or 1 mM EDTA buffer. Four microliters of the working solution of PR300159-8-B81 or PR300186-10-B81 was mixed with 396 μL of plasma from various species (human, cynomolgus monkey, SD rat, or ICR mouse) or PBS containing 1% BSA, respectively, to prepare plasma samples from various species containing PR300159-8-B81 or PR300186-10-B81 at a concentration of 200 μg / mL. The volume of organic solvent did not exceed 1%. The samples were incubated in a 37°C, 5% CO2 incubator, and the sample plate was sealed with film and shaken at 80 cycles / min. The incubation times were set to 0 h, 4 h, 24 h, 2 days, 3 days, 7 days, 10 days, 14 days, 17 days, 21 days, and 28 days. 20 μL of plasma sample was transferred, to which 200 μL of precipitant (containing internal standard) was added and thoroughly mixed to precipitate proteins. After all samples underwent protein precipitation, they were centrifuged, and the supernatants were collected, diluted with water, and analyzed using LC-MS / MS.

[0384] Figures 12 and 13 show the stability results of PR300159-8-B81 and PR300186-10-B81 in human, cynomolgus monkey, SD rat, and ICR mouse plasma, as well as in phosphate buffered saline (PBS).

[0385] The results in Figure 12 show that within 28 days, the percentage of cytotoxic drug released from PR300159-8-B81 was less than 0.5% in human and cynomolgus monkey plasma, less than 1.5% in SD rat plasma, and less than 2.5% in ICR mouse plasma.

[0386] The results in Figure 13 show that within 28 days, the percentage of cytotoxic drug released from PR300186-10-B81 was less than 1.0% in human and cynomolgus monkey plasma, less than 2.5% in SD rat plasma, and less than 4.0% in ICR mouse plasma.

[0387] Example 16. Colon cancer HT29-huMSLN wild-type cell line model HT29-huMSLN wild-type 2C3 cells were cultured in RPMI-1640 medium (containing 10% fetal bovine serum and 0.3 mg / mL G418) at 37°C in a 5% CO2 atmosphere. On the day of inoculation, exponentially growing HT29-huMSLN wild-type 2C3 cells were harvested. Tumor cells were resuspended in a 1:1 mixture of PBS and Matrigel and then subcutaneously inoculated. Each BALB / c nude mouse received 1 x 10 6 HT29-huMSLN wild-type 2C3 cells were inoculated subcutaneously. The mean tumor volume in each group of mice was 100 mm 3 When the tumor volume reached 100 mg / kg, the mice were divided into groups for treatment. Thirty mice were randomly divided into six groups, after which treatment was initiated. The treatment schedule was twice weekly for a total of six treatments via tail vein injection at a dose of 5 mg / kg body weight or 1 mg / kg body weight. After treatment initiation, body weight and tumor volume were measured twice weekly. Tumor volume was calculated using the following formula: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 Observation was terminated on day 38 after administration, and all mice were euthanized. Data analysis was performed using the t-test.

[0388] Figure 14 shows the results of an in vivo pharmacodynamic study of PR300159-8-B81 and PR300186-10-B81. At day 38 post-dose, the mean tumor volume in the solvent vehicle control group of mice was 1131 mm 3 In the hIgG1-B81 (5 mg / kg) control group of mice, the mean tumor volume at 38 days after administration was 934 mm 3 In the group treated with the test drug PR300159-8-B81 (1 mg / kg), the mean tumor volume on day 38 after administration was 34 mm 3The tumors in the mice treated with the test drug PR300159-8-B81 (5 mg / kg) were completely eliminated on day 38 after administration, showing a significant difference (p<0.001) compared with the vehicle control group and a tumor growth inhibition rate (TGI, %) of 100%. In the group treated with the test drug PR300186-10-B81 (1 mg / kg), the mean tumor volume on day 38 after administration was 168 mm 3 This showed a significant difference (p<0.001) compared to the vehicle control group, and a tumor growth inhibition rate (TGI, %) of 85%. In the test drug PR300186-10-B81 (5 mg / kg) treatment group, tumors in mice were completely eliminated 38 days after administration, demonstrating significant tumor inhibition (p<0.001) compared to the vehicle control group, and a tumor growth inhibition rate (TGI, %) of 100%.

[0389] Figure 15 shows the mouse weight results. Throughout the treatment and administration period, all animals tolerated the drug well, and no severe weight loss or death was observed.

[0390] Example 17. Colon cancer HT29-huMSLN mutant cell line model HT29-huMSLN mutant 1A11 cells were cultured in RPMI-1640 medium (containing 10% fetal bovine serum and 0.3 mg / mL G418) at 37°C in 5% CO 2 On the day of inoculation, HT29-huMSLN mutant 1A11 cells in the exponential growth phase were harvested. Each BALB / c nude mouse was inoculated with 1 × 10 6 HT29-huMSLN mutant 1A11 cells were inoculated subcutaneously. The tumor cells were first resuspended in a mixture of PBS and Matrigel (1:1) before inoculation. The average tumor volume in each group of mice was 100 mm 3 When the tumor volume reached 100 mm, the mice were divided into groups for treatment. 25 mice were divided into 5 groups, and then treatment was initiated. The treatment schedule was twice weekly for a total of 6 treatments via tail vein injection at a dose of 1 mg / kg body weight or 5 mg / kg body weight. After treatment initiation, body weight and tumor volume were measured twice weekly. Tumor volume was calculated using the following formula: tumor volume (mm3 ) = 0.5 × tumor long diameter × tumor short diameter 2 Observation was terminated on day 32 post-administration, after which all mice were euthanized. Data analysis was performed using the t-test.

[0391] Figure 16 shows the results of the antitumor efficacy of PR300159-8-B81 and PR300186-10-B81. Figure 17 shows the results of the body weight of the mice.

[0392] As shown in Figure 16, at 32 days post-administration, the mean tumor volume in the control group of mice was 1109 mm 3 In the group treated with the test drug PR300159-8-B81 (1 mg / kg), the mean tumor volume on day 32 after administration was 60.4 mm 3 The tumors in the mice treated with the test drug PR300159-8-B81 (5 mg / kg) were completely eliminated on day 32 after administration, showing a significant difference (p<0.001) compared to the vehicle control group and a tumor growth inhibition rate (TGI, %) of 100%. In the group treated with the test drug PR300186-10-B81 (1 mg / kg), the mean tumor volume on day 32 after administration was 59.5 mm 3 This showed a significant difference (p<0.001) compared to the vehicle control group and a tumor growth inhibition rate (TGI, %) of 95%. In the group treated with the test drug PR300186-10-B81 (5 mg / kg), tumors in mice were completely eliminated 32 days after administration, demonstrating significant tumor suppression (p<0.001) compared to the vehicle control group and a tumor growth inhibition rate (TGI, %) of 100%. These results indicate that PR300159-8-B81 and PR300186-10-B81 exhibit comparable antitumor efficacy in the HT29-huMSLN mutant cell in vivo model.

[0393] As shown in Figure 17, all animals tolerated the drug well throughout the treatment period, and no severe weight loss or animal deaths were observed.

[0394] Example 18. Ovarian cancer COV644 model COV644 cells (ECACC, catalog: 07071908) were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C under a 5% CO2 atmosphere. On the day of inoculation, COV644 cells in the exponential growth phase were harvested. Tumor cells were resuspended in a mixture of PBS and Matrigel (1:1) and then inoculated subcutaneously. 5 × 10 cells were inoculated into each BALB / c nude mouse. 6 COV644 tumor cells were inoculated subcutaneously. The mean tumor volume in each group of mice was 150 mm 3 When the tumor volume reached 100 mg / kg, the mice were divided into groups for treatment. 25 mice were divided into 5 groups, after which treatment was initiated. The treatment schedule was twice weekly for a total of 5 treatments via tail vein injection at a dose of 1 mg / kg body weight or 5 mg / kg body weight. After treatment initiation, body weight and tumor volume were measured twice weekly. Tumor volume was calculated using the following formula: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 Observation was terminated on day 38 after administration, and all mice were euthanized. Data analysis was performed using the t-test.

[0395] Figure 18 shows the in vivo pharmacodynamic results of PR300159-8-B81 and PR300186-10-B81. At day 38 post-administration, the mean tumor volume in the vehicle control group of mice was 1429 mm 3 In the group treated with the test drug PR300159-8-B81 (1 mg / kg), the mean tumor volume on day 38 after administration was 993 mm 3 The tumor growth inhibition rate (TGI) was 31%. In the group treated with the test drug PR300159-8-B81 (5 mg / kg), tumors were completely eliminated by day 38 after administration, showing a significant difference (p<0.001) compared with the vehicle control group and a tumor growth inhibition rate (TGI, %) of 100%. In the group treated with the test drug PR300186-10-B81 (1 mg / kg), the mean tumor volume on day 38 after administration was 995 mm 3The tumor growth inhibition rate (TGI, %) was 30%. In the group treated with the test drug PR300186-10-B81 (5 mg / kg), 38 days after administration, 4 mice in the group showed complete tumor elimination, while the tumor volume of one mouse was 14 mm 3 The mean tumor volume in this group of mice was 8 mm 3 and demonstrated significant tumor suppression (p<0.001) and a tumor growth inhibition rate (TGI, %) of 99% compared to the vehicle control group. PR300159-8-B81 and PR300186-10-B81 demonstrate comparable antitumor efficacy in the COV644 in vivo model.

[0396] Figure 19 shows the mouse weight results. All animals tolerated the drug well throughout the treatment period, and no severe weight loss or animal deaths were observed.

[0397] Example 19. Ovarian cancer OVCAR3 model OVCAR3 cells were cultured in RPMI-1640 medium (containing 20% fetal bovine serum and 0.01 mg / mL bovine insulin) at 37°C in a 5% CO2 atmosphere. On the day of inoculation, exponentially growing OVCAR3 cells were harvested. Tumor cells were resuspended in a 1:1 mixture of PBS and Matrigel and then subcutaneously inoculated. Each BALB / c nude mouse received 1 x 10 7 OVCAR3 tumor cells were inoculated subcutaneously. The mean tumor volume in each group of mice was 200 mm 3 When the tumor volume reached 100 mg / kg, the mice were divided into groups for treatment. Thirty-five mice were divided into seven groups, after which treatment was initiated. The treatment schedule was twice weekly for a total of five treatments via tail vein injection at a dose of 1 mg / kg body weight or 5 mg / kg body weight. After treatment initiation, body weight and tumor volume were measured twice weekly. Tumor volume was calculated using the following formula: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 Observation was terminated on day 23 post-administration, after which all mice were euthanized. Data analysis was performed using the t-test.

[0398] PR300159-8-DXD was synthesized by ChemPartner (Shanghai) according to the trastuzumab delactecan (DS-8201, Enhertu) synthesis method (WO 2015115091 A1), in which trastuzumab in DS-8201 was replaced with PR300159-8.

[0399] Figure 20 shows the in vivo pharmacodynamic results of PR300159-8-DXD, PR300159-8-B81, and PR300186-10-B81. At 23 days post-administration, the mean tumor volume in the vehicle control group of mice was 1811 mm 3 In the group treated with the test drug PR300159-8-DXD (1 mg / kg), the mean tumor volume on day 23 after administration was 51 mm 3 The mean tumor volume at 23 days after administration was 47 mm. 3 The tumor growth inhibition rate (TGI, %) was 97%, which was significantly different from the vehicle control group (p=0.001). In the test drug PR300159-8-B81 (1 mg / kg) treatment group, the mean tumor volume on day 23 after administration was 35 mm 3 The mean tumor volume on day 23 after administration in the group treated with the test drug PR300159-8-B81 (5 mg / kg) was 54 mm 3 The mean tumor volume on day 23 after administration in the group treated with the test drug PR300186-10-B81 (1 mg / kg) was 429 mm 3 The group showed a significant tumor inhibition effect (p=0.002) and a tumor growth inhibition rate (TGI, %) of 76% compared with the vehicle control group. In the group treated with the test drug PR300186-10-B81 (1 mg / kg), the mean tumor volume on day 23 after administration was 67 mm 3Compared with the vehicle control group, there was a significant tumor inhibitory effect (p=0.001) and a tumor growth inhibition rate (TGI, %) of 96%. PR300159-8-DXD, PR300159-8-B81, and PR300186-10-B81 showed significant antitumor effects at both 1 mg / kg and 5 mg / kg, and PR300186-10-B81 showed a clear dose-dependence.

[0400] Figure 21 shows the mouse weight results. All animals tolerated the drug well throughout the treatment period, and no severe weight loss or animal deaths were observed.

[0401] Example 20. Ovarian cancer SKOV3 model SKOV3 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C with 5% CO 2 On the day of inoculation, exponentially growing SKOV3 cells were harvested. The tumor cells were resuspended in a mixture of PBS and Matrigel (1:1) and then inoculated subcutaneously. Each BALB / c nude mouse received 1 × 10 7 SKOV3 tumor cells were inoculated subcutaneously. The mean tumor volume in each group of mice was 200 mm 3 When the tumor volume reached 100 mm, the mice were divided into groups for treatment. 42 mice were divided into 7 groups, after which treatment was initiated. The treatment schedule was twice weekly for a total of four treatments via tail vein injection. After treatment initiation, body weight and tumor volume were measured twice weekly. Tumor volume was calculated using the following formula: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 Observation was terminated 21 days after administration, and all mice were euthanized. Data analysis was performed using the t-test.

[0402] Figure 22 shows the in vivo pharmacodynamic results of PR300159-8-B81 and PR300186-10-B81. At 21 days post-administration, the mean tumor volume in the vehicle control group of mice was 2011 mm 3 On day 21 after administration, the mean tumor volume in the group treated with the test drug PR300159-8-B81 (1.28 mg / kg) was 657 mm 3The mean tumor volume in the group treated with the test drug PR300159-8-B81 (6.4 mg / kg) was 261 mm , which was significantly different from the vehicle control group (p<0.001) and showed a tumor growth inhibition rate (TGI, %) of 67%. On the 21st day after administration, the mean tumor volume in the group treated with the test drug PR300159-8-B81 (6.4 mg / kg) was 261 mm . 3 The mean tumor volume in the group treated with the test drug PR300159-8-B81 (12.8 mg / kg) was 122 mm , which was significantly different from the vehicle control group (p<0.001) and showed a tumor growth inhibition rate (TGI, %) of 87%. On the 21st day after administration, the mean tumor volume in the group treated with the test drug PR300159-8-B81 (12.8 mg / kg) was 122 mm . 3 The mean tumor volume in the group treated with the test drug PR300186-10-B81 (0.78 mg / kg) was 1064 mm , which was significantly different from the vehicle control group (p<0.001) and showed a tumor growth inhibition rate (TGI, %) of 94%. On the 21st day after administration, the mean tumor volume in the group treated with the test drug PR300186-10-B81 (0.78 mg / kg) was 1064 mm . 3 The tumor growth inhibition rate (TGI, %) was 47% compared with the vehicle control group (p<0.001). On the 21st day after administration, the mean tumor volume in the test drug PR300186-10-B81 (3.9 mg / kg) treatment group was 478 mm 3 The tumor growth inhibition rate (TGI, %) was 76% and showed a significant tumor inhibition effect (p<0.001) compared with the vehicle control group. On the 21st day after administration, the mean tumor volume in the test drug PR300186-10-B81 (7.8 mg / kg) treatment group was 265 mm 3 The compounds showed a significant tumor inhibition effect (p<0.001) and a tumor growth inhibition rate (TGI, 8%) of 87% compared with the vehicle control group. PR300159-8-B81 and PR300186-10-B81 demonstrated significant antitumor effects with a clear dose-dependent relationship.

[0403] Figure 23 shows the mouse weight results. All animals tolerated the drug well throughout the treatment period, and no severe weight loss or animal deaths were observed.

[0404] Example 21. NCI-H226 lung cancer model NCI-H226 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C in 5% CO 2The tumor cells were cultured under ambient conditions. On the day of inoculation, NCI-H226 cells in the exponential growth phase were harvested. The tumor cells were first resuspended in a mixture of PBS and Matrigel (1:1) and then inoculated subcutaneously. Each BALB / c nude mouse received 5 × 10 6 NCI-H226 tumor cells were inoculated subcutaneously. The mean tumor volume in each group of mice was 150 mm 3 When the tumor volume reached 100 mg / kg, the mice were divided into groups for treatment. 55 mice were divided into 11 groups, after which treatment was initiated. The treatment schedule was twice weekly for a total of five treatments via tail vein injection at a dose of 1 mg / kg body weight or 5 mg / kg body weight. After treatment initiation, body weight and tumor volume were measured twice weekly. Tumor volume was calculated using the following formula: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 Observation was terminated 28 days after administration, and all mice were euthanized. Data analysis was performed using the t-test.

[0405] BAY-949343, also known as anetumab ravtansine, is an ADC developed by Bayer. It is formed by conjugating the fully human anti-MSLN monoclonal antibody MF-T (anetumab) to the microtubule inhibitor maytansinoid DM4 via an amino bond, with the linker being N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), which has bystander killing properties (WO 2010124797 A1). BAY-949343 was synthesized by Shanghai Chempartner Co., Ltd.

[0406] Figures 24 and 25 show the in vivo pharmacodynamic results of PR300159-8-DXD, BAY-949343, PR300159-8-B81, and PR300186-10-B81. At 28 days post-dose, the mean tumor volume in the vehicle control group of mice was 1535 mm 3 In the group treated with the test drug PR300159-8-DXD (1 mg / kg), the mean tumor volume on day 28 after administration was 743 mm 3The tumor growth inhibition rate (TGI, %) was 52%, which was significantly different from the vehicle control group (p=0.05). In the test drug PR300159-8-DXD (5 mg / kg) treatment group, the mean tumor volume on day 28 after administration was 460 mm 3 The mean tumor volume at 28 days after administration in the group treated with the test drug BAY-949343 (1 mg / kg) was 497 mm 3 The mean tumor volume at 28 days after administration in the group treated with the test drug BAY-949343 (5 mg / kg) was 253 mm 3 The mean tumor volume at 28 days after administration in the group treated with the test drug PR300159-8-B81 (1 mg / kg) was 556 mm 3 The mean tumor volume at 28 days after administration was 164 mm , which was significantly different from the vehicle control group (p=0.02) and showed a tumor growth inhibition rate (TGI, %) of 64%. In the test drug PR300159-8-B81 (5 mg / kg) treatment group, the mean tumor volume at 28 days after administration was 164 mm . 3 The tumor growth inhibition rate (TGI, %) was 89%, which was significantly different from the vehicle control group (p=0.003). In the test drug PR300186-10-B81 (1 mg / kg) treatment group, the mean tumor volume on day 28 after administration was 526 mm 3 The group showed a significant tumor inhibition effect (p=0.01) and a tumor growth inhibition rate (TGI, %) of 66% compared with the vehicle control group. In the group treated with the test drug PR300186-10-B81 (5 mg / kg), the mean tumor volume on day 28 after administration was 211 mm 3 and showed a significant tumor inhibition effect (p=0.003) and a tumor growth inhibition rate (TGI, %) of 86% compared to the vehicle control group. PR300159-8-B81, PR300186-10-B81, and BAY-949343 showed comparable antitumor efficacy, and their antitumor efficacy was all superior to that of PR300159-8-DXD.

[0407] Figures 26 and 27 show the mouse weight results. All animals tolerated the drug well throughout the treatment period, and no severe weight loss or animal deaths were observed.

[0408] Example 22. Binding activity assay of PR300159, PR300159-B71, PR300186, and PR300186-B71 to CHOK1 cells overexpressing human mesothelin (CHOK1-huMSLN) Flow cytometry was used to detect the binding activity of the PR300159 and PR300186 antibodies, and the antibody-drug conjugates (ADCs) PR300159-B71 and PR300186-B71, to CHOK1-huMSLN cells.

[0409] Antibodies PR300159 and PR300186, and ADCs PR300159-B71 and PR300186-B71 were serially diluted in buffer (PBS containing 2% FBS). 50 μL of diluted antibody solution (30 μg / mL) was added to 1-2 × 10 5 The cells were added to 50 μL of cell suspension containing 100 μL of PBS containing 100 μL of 2% serum and incubated for 1 hour at 4° C. The cells were washed twice with buffer (PBS containing 2% serum), and 100 μL of fluorescently labeled anti-human IgG antibody (Alexa Fluor® 488 AffiniPure Goat Anti-Human IgG (H+L), Jackson ImmunoResearch, catalog 109-545-088) was added to each well. After incubation for 1 hour at 4° C., the cells were washed twice with buffer and analyzed by flow cytometry.

[0410] The results are shown in Table 14 and Figure 28. The results show that antibodies PR300159 and PR300186, and their corresponding ADCs PR300159-B71 and PR300186-B71, all exhibited comparable binding activity to the cells.

[0411] [Table 14]

[0412] Example 23. Experiment on the binding activity of PR300159, PR300159-B81, PR300186, and PR300186-B81 to tumor cells Flow cytometry was used to detect the binding activity of the PR300159 and PR300186 antibodies, and the antibody-drug conjugates (ADCs) PR300159-B81 and PR300186-B81, to tumor cells. The tumor cells used in this example were the breast cancer cell line COV644.

[0413] Antibodies PR300159 and PR300186, and ADCs PR300159-B81 and PR300186-B81 were serially diluted in buffer (PBS containing 2% FBS). 50 μL of diluted antibody solution was added to 1-2 × 10 5 The solution was added to 50 μL of the cell suspension containing COV644 cells and incubated for 1 hour at 4° C. The cells were washed twice with buffer (PBS containing 2% serum), and 100 μL of fluorescently labeled anti-human IgG antibody (Alexa Fluor® 488 AffiniPure Goat Anti-Human IgG (H+L), Jackson ImmunoResearch, catalog 109-545-088) was added to each well. After incubation for 1 hour at 4° C., the cells were washed twice with buffer and analyzed by flow cytometry.

[0414] The results are shown in Table 15 and Figure 29. The results show that the antibodies and their corresponding ADCs exhibited comparable binding activity to COV644 cells.

[0415] [Table 15]

[0416] Example 24. Experiment on the endocytic activity of PR300159, PR300159-B71, PR300186, and PR300186-B71 against tumor cells Flow cytometry was used to evaluate the endocytic efficacy of antibodies PR300159 and PR300186, and antibody-drug conjugates (ADCs) PR300159-B71 and PR300186-B71 against COV644 tumor cells. The tumor cells used in this example were breast cancer cells derived from the COV644 cell line.

[0417] In this example, pHAb Amine Reactive Dye (Promega, catalog #G9841) was used to determine the endocytosis efficacy of anti-MSLN antibodies against COV644 tumor cells. pHAb dye is a pH sensor dye that exhibits very low fluorescence at pH values above 7, and the fluorescence increases significantly as the pH value of the solution decreases. pHAb dye-labeled antibodies exhibit little or no fluorescence when bound to the outer cell membrane under neutral pH conditions. However, after pHAb dye-labeled antibodies are endocytosed by cells, strong fluorescence is detected in the low pH environment of the cell's endosomes and lysosomes.

[0418] The antibody was labeled with pHAb dye, and the DAR value was calculated according to the instructions in the reagent kit. The pHAb dye-labeled antibody was incubated with COV644 for 24 hours at 4°C (a temperature with low antibody internalization activity, used as a background control) or 37°C. Fluorescence was then detected at an excitation value (Ex) of 532 nm and an emission value (Em) of 560 nm. The antibody endocytosis result was calculated by dividing the fluorescence intensity at 37°C minus the background fluorescence intensity at 4°C by the DAR value of the antibody-conjugated dye. A higher fluorescence value indicates a greater endocytic efficiency of the antibody into cells.

[0419] The results are shown in Figure 30. The data show that PR300159, PR300159-B71, PR300186, and PR300186-B71 exhibited good endocytic activity towards COV644 tumor cells.

[0420] Example 25. Experiments using antibody-drug conjugates PR300159 and PR300186 on cells expressing MSLN HT29-huMSLN cells were seeded at 2000 cells per well in 96-well plates and serially diluted PR300159, PR300159-B71, PR300186, PR300186-B71, or hIgG-B71 was added. After 6 days, cell viability was measured using the Promega CellTiter Glo assay (G7572).

[0421] The results are shown in Figure 31. The results demonstrate that PR300159-B71 and PR300186-B71 exhibit significant killing effects on CHOK1-huMSLN.

[0422] Example 26. Human ovarian cancer COV644 model COV644 cells (ECACC, catalog: 07071908) were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C under 5% CO2 conditions. On the day of inoculation, COV644 cells in the logarithmic growth phase were harvested and cultured at 5x10 6 COV644 tumor cells were subcutaneously inoculated into each BALB / c nude mouse. The tumor cells were first resuspended in a mixture of PBS and Matrigel (1:1) before subcutaneous inoculation. The average tumor volume in each group of mice was 150 mm 3 When the tumor volume reached 100 mm, the mice were divided into groups for treatment. 78 mice were divided into 13 groups, after which treatment began. The treatment schedule was twice a week for a total of five treatments via tail vein injection. After treatment began, tumor volume and body weight were measured twice a week, and tumor volume was calculated using the following formula: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 Observation was terminated 21 days after administration, and all mice were euthanized. Data analysis was performed using the t-test.

[0423] Figures 32A and 32B show the in vivo pharmacodynamic results for PR300159-B71, PR300159-B81, and PR300186-B81. At 21 days post-administration, the mean tumor volume in the control group of mice was 3154.82 mm. The group treated with the test drug IgG-B81 (1 mg / kg) had a mean tumor volume of 1508 mm. 3 The tumor growth inhibition rate (TGI, %) was 2.57%, which was not significantly different from the vehicle control group (p=0.82). The group treated with the test drug IgG-B81 (5 mg / kg) had a mean tumor volume of 1514.88 mm on the 21st day after administration. 3The group treated with the test drug PR300159-B71 (1 mg / kg) showed a mean tumor volume of 853.97 mm on the 21st day after administration. 3 The group treated with the test drug PR300159-B71 (5 mg / kg) achieved complete tumor eradication on day 21 after administration, showing a significant difference (p<0.001) compared with the vehicle control group and a tumor growth inhibition rate (TGI, %) of 72.93%. The group treated with the test drug PR300159-B71 (5 mg / kg) achieved complete tumor eradication on day 21 after administration, showing a significant difference (p<0.001) compared with the vehicle control group and a tumor growth inhibition rate (TGI, %) of 100%. The group treated with the test drug PR300159-B81 (1 mg / kg) had a mean tumor volume of 1356.25 mm on day 21 after administration. 3 The group treated with the test drug PR300159-B81 (5 mg / kg) achieved complete tumor eradication on day 21 after administration, showing a significant difference (p<0.001) compared with the vehicle control group and a tumor growth inhibition rate (TGI, %) of 100%. The group treated with the test drug PR300186-B81 (1 mg / kg) achieved complete tumor eradication on day 21 after administration, showing a significant difference (p<0.001) compared with the vehicle control group and a tumor growth inhibition rate (TGI, %) of 100%. The group treated with the test drug PR300186-B81 (1 mg / kg) achieved a mean tumor volume of 1544.39 mm on day 21 after administration. 3 The tumor growth inhibition rate (TGI, %) was 51.05%, which was not significantly different from the vehicle control group (p<0.001). The group treated with the test drug PR300186-B81 (5 mg / kg) had a mean tumor volume of 500.16 mm on the 21st day after administration. 3 This showed a significant difference (p<0.001) compared to the vehicle control group, with a tumor growth inhibition rate (TGI, %) of 84.15%. PR300159-B71, PR300159-B81, and PR300186-B81 demonstrated significant antitumor efficacy in the COV644 in vivo model.

[0424] All animals tolerated the drug well throughout the treatment period, and no severe weight loss or deaths were observed.

Claims

1. A compound of formula (I) or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof: Ab-[S-X-(Y) ｎ -Z-E-D] ｑ (I) an antibody-drug conjugate which is During the ceremony, Ab is an antibody that specifically binds to mesothelin (MSLN) or an antigen-binding fragment thereof; S is a sulfur bond connecting Ab and X; X is C ３～１０ cycloalkyl, 5- to 10-membered heterocyclic group, C ６～１０ aryl, or 5-10 membered heteroaryl group; X may optionally be R ＊ substituted with 1, 2, 3, 4, or 5 instances of R ＊ is H, deuterium, halogen, CN, NO ２ , O.H., C. １～６ Alkyl, C １～６ Haloalkyl, C ２～６ Alkenyl, or C ２～６ alkynyl; Y is -Y １ -Y ２ -Y ３ - and Y is Y １ to X via Y ３ is linked to Z via Y １ is -C(O)-, -C(O)O-, -OC(O)-, -C(O)NH-, C ２～６ Alkenyl, C ２～６ Alkynyl, C ３～１０ cycloalkyl, 5- to 10-membered heterocyclic group, C ６～１０ aryl, or 5-10 membered heteroaryl; Y ２ is a bond or C １～６ alkylene; Y ３ is selected from —C(O)—, —OC(O)—, or —NHC(O)—; Y is optionally substituted with 1, 2, 3, 4, or 5 instances of R#; R# is H, deuterium, halogen, CN, NO ２ , O.H., C. １～６ Alkyl, C １～６ Haloalkyl, C ２～６ Alkenyl, or C ２～６ alkynyl; Z is a peptide containing 2 to 8 amino acids, preferably a dipeptide, tripeptide, or tetrapeptide; E is absent or is a spacer unit connecting Z and D; D is a biologically active molecular drug or a derivative thereof; n is selected from 0, 1, 2, 3, 4, or 5, preferably 0, 1, 2, or 3; q is an integer selected from 1 to 10, preferably 8; Antibody-drug conjugates.

2. X is C ６～１０ selected from an aryl or a 5-10 membered heteroaryl group; X may optionally be R ＊ substituted with 1, 2, 3, 4, or 5 instances of R ＊ is H, deuterium, halogen, CN, NO ２ , OH, or C １～６ alkyl; Preferably, X is a 5-6 membered heteroaryl group; X may optionally be R ＊ substituted with one, two, or three instances of; R ＊ is H, deuterium, halogen, or C １～４ alkyl; More preferably, X is pyrimidinyl, e.g. 【Chemical 1】 That is, The antibody-drug conjugate of claim 1.

3. Y is -Y １ -Y ２ -Y ３ - and Y is Y １ to X via Y ３ is linked to Z via During the ceremony, Y １ is C ２～６ Alkenyl or C ２～６ alkynyl; Y ２ is a bond or C １～６ alkylene; Y ３ is selected from —C(O)— or —NHC(O)—; Y is optionally substituted with 1, 2, 3, 4, or 5 instances of R#; R# is H, deuterium, halogen, CN, NO ２ , OH, or C １～６ alkyl; Preferably, Y is -Y １ -Y ２ -Y ３ - and Y is Y １ to X via Y ３ is connected to Z via During the ceremony, Y １ is C ２～４ Alkenyl or C ２～４ alkynyl; Y ２ is C １～４ alkylene; Y ３ is —C(O)—; Y is optionally substituted with 1, 2, or 3 instances of R#; R# is H, deuterium, halogen, or C １～４ alkyl; More preferably, Y is 【Chemistry 2】 That is, The antibody-drug conjugate according to claim 1 or 2.

4. Z is val-cit, val-cit-gly, gly-gly, gly-gly-gly, gly-gly-gly-gly, val-gly-gly, val-g ln-gly, val-glu-gly, phe-lys-gly, leu-lys-gly, gly-val-lys-gly, val-lys-gly-gly, v selected from al-lys-gly, val-lys-ala, val-lys-leu, leu-leu-gly, gly-gly-phe-gly, gly-gly-phe-gly-gly, val-gly, val-lys-β-ala, or the above dipeptides, tripeptides, and tetrapeptides after modification by substitution; Preferably, Z is 【Chemistry 3】 and R １ and R ２ are independently H, deuterium, C １～６ Alkyl, C １～６ Haloalkyl, C ２～６ Alkenyl, or C ２～６ alkynyl; More preferably, Z is 【Chemistry 4】 and R １ and R ２ are independently H, deuterium, halogen, or C １～６ Alkyl, preferably H or C １～４ selected from alkyl, The antibody-drug conjugate according to any one of claims 1 to 3.

5. E is absent or -NH-C １～６ Alkylene- or -O-C １～６ alkylene-, wherein said —NH—C １～６ Alkylene- and —O—C １～６ Alkylene- is H, halogen, C １～６ Alkyl, and C １～６ optionally substituted with 1, 2, or 3 substituents selected from haloalkyl; Preferably, E is absent or -NH-C １～４ alkylene-, and the —NH—C １～４ Alkylene- is H, halogen, C １～４ Alkyl, and C １～４ optionally substituted with 1, 2, or 3 substituents selected from haloalkyl; More preferably, E is —NH—(CH ２ ) １～４ -, for example -NH-CH ２ -is, The antibody-drug conjugate according to any one of claims 1 to 4.

6. D is selected from metal complexes; glycopeptide antibiotics; DNA topoisomerase inhibitors; drugs that interfere with DNA synthesis; drugs that target structural proteins; tumor signaling pathway inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor angiogenesis inhibitors; cyclin inhibitors; serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors, or histidine kinase inhibitors; and other active substances that inhibit tumor cell proliferation or promote tumor cell apoptosis or necrosis; Preferably, D is oxaliplatin; bleomycin or pingjiangmycin; camptothecin, camptothecin derivatives (e.g., hydroxycamptothecin, 9-aminocamptothecin, etc.), SN-38, irinotecan, topotecan, belotecan, or rubitecan; actinomycin D, doxorubicin, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide; methotrexate, 5-fluorouracil, selected from lauracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, or nelarabine; vinca alkaloids, vincristine, vinblastine, paclitaxel, docetaxel, or cabazitaxel; maytansinoid derivatives; calicheamicin derivatives; auristatin derivatives; pyrrolobenzodiazepine dimer derivatives; melphalan; mitomycin C; or chlorambucil; More preferably, D is 【Chemistry 5】 and During the ceremony, R Ｄ is absent or selected from —NH— or —O—; L Ｄ is -C ０～６ Alkylene -O-C ０～６ Alkylene- or -C ０～６ Alkylene -O-C ０～６ Alkylene -C(O)NH-CHR Ｄ４ - selected from; R Ｄ１ and R Ｄ２ are independently H, halogen, C １～６ Alkyl, C １～６ Haloalkyl, or C １～６ alkoxy; or R Ｄ１ and R Ｄ２ together with the carbon atoms to which they are attached form a ring, e.g., C ６～１０ aryl, 6- to 10-membered heteroaryl group, C ３～７ forming a cycloalkyl or a 3- to 7-membered heterocyclic group, preferably a 5- or 6-membered heterocyclic group; R Ｄ３ and R Ｄ４ are independently H, halogen, C １～６ Alkyl, C １～６ Haloalkyl, or C １～６ alkoxy; or R Ｄ３ and R Ｄ４ together with the carbon atoms to which they are attached form a ring, e.g., C ６～１０ Aryl, 5-10 heteroaryl, C ５～１０ Cycloalkyl or 5- to 10-membered heterocyclic group, preferably C ５～６ Forming a cycloalkyl; More preferably, D is 【Chemistry 6】 Selected from: The antibody-drug conjugate according to any one of claims 1 to 5.

7. X is C ６～１０ selected from an aryl or a 5-10 membered heteroaryl group; X may optionally be R ＊ substituted with 1, 2, 3, 4, or 5 instances of R ＊ is H, deuterium, halogen, CN, NO ２ , OH, or C １～６ alkyl; Y is -Y １ -Y ２ -Y ３ - and Y is Y １ to X via Y ３ is linked to Z via During the ceremony, Y １ is C ２～６ Alkenyl or C ２～６ alkynyl; Y ２ is a bond or C １～６ alkylene; Y ３ is selected from —C(O)— or —NHC(O)—; Y is optionally substituted with 1, 2, 3, 4, or 5 instances of R#; R# is H, deuterium, halogen, CN, NO ２ , OH, or C １～６ alkyl; Z is val-cit, val-cit-gly, gly-gly, gly-gly-gly, gly-gly-gly-gly, val-gly-gly, val-g ln-gly, val-glu-gly, phe-lys-gly, leu-lys-gly, gly-val-lys-gly, val-lys-gly-gly, v selected from al-lys-gly, val-lys-ala, val-lys-leu, leu-leu-gly, gly-gly-phe-gly, gly-gly-phe-gly-gly, val-gly, val-lys-β-ala, or the above dipeptides, tripeptides, and tetrapeptides after modification by substitution; E is absent or -NH-C １～６ Alkylene- or -O-C １～６ alkylene-, wherein said —NH—C １～６ Alkylene- and -O-C １～６ Alkylene- is H, halogen, C １～６ Alkyl, and C １～６ optionally substituted with 1, 2, or 3 substituents selected from haloalkyl; D is selected from metal complexes; glycopeptide antibiotics; DNA topoisomerase inhibitors; drugs that interfere with DNA synthesis; drugs that target structural proteins; tumor signaling pathway inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor angiogenesis inhibitors; cyclin inhibitors; serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors, or histidine kinase inhibitors; and other active substances that inhibit tumor cell proliferation or promote tumor cell apoptosis or necrosis. The antibody-drug conjugate according to any one of claims 1 to 6.

8. X is a 5-6 membered heteroaryl group; X may optionally be R ＊ substituted with one, two, or three instances of; R ＊ is H, deuterium, halogen, or C １～４ alkyl; Y is -Y １ -Y ２ -Y ３ - and Y is Y １ to X via Y ３ is connected to Z via During the ceremony, Y １ is C ２～４ Alkenyl or C ２～４ alkynyl; Y ２ is C １～４ alkylene; Y ３ is —C(O)—; Y is optionally substituted with 1, 2, or 3 instances of R#; R# is H, deuterium, halogen, or C １～４ alkyl; Z is 【Chemistry 7】 and E is absent or -NH-C １～４ alkylene-, and the —NH—C １～４ Alkylene- is H, halogen, C １～４ Alkyl, or C １～４ optionally substituted with 1, 2, or 3 substituents selected from haloalkyl; R １ and R ２ are independently H, deuterium, halogen, C １～６ Alkyl, C １～６ Haloalkyl, C ２～６ Alkenyl, or C ２～６ alkynyl; D is oxaliplatin; bleomycin or pingjiangmycin; camptothecin, camptothecin derivatives (e.g., hydroxycamptothecin, 9-aminocamptothecin, etc.), SN-38, irinotecan, topotecan, belotecan, or rubitecan; actinomycin D, doxorubicin, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide; methotrexate, 5-fluorouracil, selected from lauracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, or nelarabine; vinca alkaloids, vincristine, vinblastine, paclitaxel, docetaxel, or cabazitaxel; maytansinoid derivatives; calicheamicin derivatives; auristatin derivatives; pyrrolobenzodiazepine dimer derivatives; melphalan; mitomycin C; or chlorambucil; Preferably, D is 【Chemistry 8】 and During the ceremony, R Ｄ is absent or selected from —NH— or —O—; L Ｄ is -C ０～６ Alkylene -O-C ０～６ Alkylene- or -C ０～６ Alkylene -O-C ０～６ Alkylene -C(O)NH-CHR Ｄ４ - selected from; R Ｄ１ and R Ｄ２ are independently H, halogen, C １～６ Alkyl, C １～６ Haloalkyl, or C １～６ alkoxy; or R Ｄ１ and R Ｄ２ together with the carbon atoms to which they are attached form a ring, e.g., C ６～１０ aryl, 5- to 10-membered heteroaryl group, C ３～７ forming a cycloalkyl or a 3- to 7-membered heterocyclic group, preferably a 5- or 6-membered heterocyclic group; R Ｄ３ and R Ｄ４ are independently H, halogen, C １～６ Alkyl, C １～６ Haloalkyl, or C １～６ alkoxy; or R Ｄ３ and R Ｄ４ together with the carbon atoms to which they are attached form a ring, e.g., C ６～１０ aryl, 5- to 10-membered heteroaryl group, C ５～１０ Cycloalkyl or 5- to 10-membered heterocyclic group, preferably C ５～６ forming a cycloalkyl, The antibody-drug conjugate according to any one of claims 1 to 7.

9. X is pyrimidinyl, preferably 【Chemistry 9】 and Y is 【Chemistry 10】 and Z is 【Chemistry 11】 and R １ and R ２ are independently H, deuterium, halogen, C １～６ Alkyl, preferably H or C １～４ alkyl; E is -NH-CH ２ - and; D is 【Chemistry 12】 Selected from: n is selected from 1, 2, or 3, preferably 1; q is selected from 4, 5, 6, 7, 8, 9, or 10, preferably 8; The antibody-drug conjugate according to any one of claims 1 to 8.

10. The antibody-drug conjugate comprises a compound of formula (II): 【Chemistry 13】 or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof; During the ceremony, Ab is anti-MSLN antibody; R １ and R ２ are independently １～６ Alkyl, preferably C １～４ alkyl; D is 【Chemistry 14】 and q is an integer selected from 1 to 10, for example, an integer selected from 4, 5, 6, 7, 8, 9, or 10, preferably 8; The antibody-drug conjugate according to any one of claims 1 to 9.

11. The antibody-drug conjugate may comprise the following compound: 【Chemistry 15-1】 【Chemistry 15-2】 or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof; wherein Ab is an anti-MSLN antibody; q is an integer selected from 1 to 10, for example, an integer selected from 4, 5, 6, 7, 8, 9, or 10, preferably 8; The antibody-drug conjugate according to any one of claims 1 to 10.

12. the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL); (1) the VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 27; (2) the VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 25, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 28; (3) the VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 26, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 27; (4) The VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 29; (5) The VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 30; (6) The VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 31; (7) The VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 32; (8) The VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 33; (9) The VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 25, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 29; (10) The VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 63, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 67; (11) The VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 64, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 67; (12) The VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 65, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (13) The VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 65, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 68; (14) The VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 63, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 68; (15) The VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 66, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 67; (16) The VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 64, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 69; (17) The VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 66, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 69; (18) The VH comprises HCDR1, HCDR2, and HCDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 63, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 69; or (19) The VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 65, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 69; The antibody-drug conjugate according to any one of claims 1 to 11.

13. (1) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13; (2) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 15, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 16, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18; (3) The VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 15, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13; (4) The VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 19; (5) The VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 20; (6) The VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 21; (7) The VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 22; or (8) The VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23; or (9) The VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 15, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 16, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 19; (10) The VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 45, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 52, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; or (11) The VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 52, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; or (12) The VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 59, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 52, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; or (13) The VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 59, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 61, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (14) The VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 45, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 61, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (15) The VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 60, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 52, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; or (16) The VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 62, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (17) The VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 60, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 62, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (18) The VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 45, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 62, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; (19) The VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 59, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49; the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 62, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56; The antibody-drug conjugate of claim 12.

14. (1) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:27; (2) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 25, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28; (3) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 27; (4) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29; (5) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30; (6) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31; (7) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32; (8) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33; (9) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 25, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29; (10) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (11) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (12) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 65, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (13) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 65, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68; (14) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68; (15) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 66, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (16) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; (17) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 66, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; (18) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; or (19) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 65, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

69. The antibody-drug conjugate according to claim 12 or 13.

15. The antibody comprises a heavy chain (HC) and a light chain (LC): (1) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37; (2) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38; (3) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 36, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37; (4) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39; (5) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 40; (6) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 41; (7) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42; (8) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43; (9) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39; (10) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (11) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (12) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (13) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 75; (14) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 75; (15) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (16) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76; (17) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76; (18) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76; or (19) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

76. The antibody-drug conjugate according to any one of claims 12 to 14.

16. The antibody-drug conjugate of any one of claims 12 to 15, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

17. The antigen-binding fragments include Fab, Fab', F(ab') ２ 17. The antibody-drug conjugate of claim 12, wherein the antibody-drug conjugate is selected from Fd, Fd', Fv, scFv, and ds-scFv.

18. The antibody-drug conjugate of any one of claims 12 to 17, wherein the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

19. The antibody-drug conjugate may comprise the following compound: 【Chemistry 16-1】 【Chemistry 16-2】 or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof; wherein Ab is an anti-MSLN antibody; q is an integer selected from 1 to 10, for example, an integer selected from 4, 5, 6, 7, 8, 9, or 10, preferably 8; The anti-MSLN antibody comprises a heavy chain (HC) and a light chain (LC): (1) the HC comprises the amino acid sequence set forth in SEQ ID NO: 34, and the LC comprises the amino acid sequence set forth in SEQ ID NO: 37; (2) the HC comprises the amino acid sequence set forth in SEQ ID NO: 34, and the LC comprises the amino acid sequence set forth in SEQ ID NO: 43; (3) the HC comprises the amino acid sequence set forth in SEQ ID NO: 70, and the LC comprises the amino acid sequence set forth in SEQ ID NO: 74; or (4) The HC comprises the amino acid sequence shown in SEQ ID NO: 72, and the LC comprises the amino acid sequence shown in SEQ ID NO: 76; The antibody-drug conjugate according to any one of claims 1 to 18.

20. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 19, and optionally a pharmaceutically acceptable carrier or excipient.

21. 21. The pharmaceutical composition of claim 20, further comprising a second therapeutic agent, wherein the second therapeutic agent is selected from a chemotherapeutic agent, a monoclonal antibody drug, a bispecific / multispecific antibody drug, a recombinant protein drug, a nucleotide drug (including siRNA and antisense oligonucleotide), a small molecule drug, an immunomodulatory drug, and a cell therapy drug.

22. A method for treating cancer in a subject, comprising administering to the subject an effective amount of the antibody-drug conjugate of any one of claims 1 to 19 or the pharmaceutical composition of claim 20 or 21.

23. 23. The method of claim 22, wherein the cancer is selected from colon cancer, mesothelioma, ovarian cancer, lung cancer, breast cancer, pancreatic cancer, gastric cancer, prostate cancer, squamous cell carcinoma, and cholangiocarcinoma.

24. 24. The method of claim 22 or 23, further comprising administering a second therapeutic agent to the subject.

25. 25. The method of claim 24, wherein the second therapeutic agent is selected from a chemotherapeutic agent, a monoclonal antibody drug, a bispecific / multispecific antibody drug, a recombinant protein drug, a nucleotide drug (including siRNA and antisense oligonucleotides), a small molecule drug, an immunomodulatory drug, and a cell therapy drug.

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