MSLN antibody drug conjugates

Abstract

An antibody drug conjugate is a compound of formula (I) or a tautomer or a pharmaceutically acceptable salt thereof: Ab-[S-X-(Y) n-Z-E-D] q (I) wherein Ab is an antibody that specifically binds mesothelin (MSLN) or an antigen-binding fragment thereof, and D is a bioactive molecular drug or a derivative thereof. A pharmaceutical composition comprising an antibody drug conjugate of formula (I). Antibody drug conjugates of formula (I) and their pharmaceutical compositions for the preparation of medicaments for the treatment of cancer.

Description

MSLN Antibody Drug Conjugate Technical Field

[0001] The present invention belongs to the field of targeted therapy, and in particular to the field of antibody-drug conjugates. Background Art

[0002] Mesothelin (MSLN) is a cell surface molecule expressed as a 71 kD precursor protein and further processed into a 40 kD glycoprotein that is anchored to the cell surface by glycosylphosphatidylinositol (GPI) on the cell surface. The expression of MSLN in normal tissues is very limited. It is expressed in mesothelial cells along the pleura, pericardium, and peritoneum, where it appears to play a role in cell adhesion (Chang et al. (1996) PNAS 93: 136-140). Soluble cleaved MSLN has also been proposed to play a role in megakaryocyte stimulation, but knockout in mice did not show any developmental defects, so its biological role is still unclear (Yamaguchi et al. (1994) J Biol Chem. 269 (2): 805-8; Bera et al. (2000) Mol Cell Biol. 20 (8): 2902-6).

[0003] In contrast, MSLN is highly expressed in some human cancers, including almost all mesotheliomas and pancreatic cancers, as well as approximately 70% of ovarian cancers and 50% of lung adenocarcinomas (Hassan and Ho (2008) Eur. J. Cancer 44:46-53; Miettinen and Sarlomo-Rikala (2003) Am. J. Surg. Pathol. 27:150-8; Ordonez (2003) Am. J. Surg. Pathol. 27:1418-1428; Ho et al. (2007) Clin. Cancer Res. 13:1571-5). The high expression level of MSLN makes it an attractive candidate for targeted therapy, as it plays an important role in promoting tumor proliferation and invasion (Servais et al. (2012) Clin. Cancer Res. 18(9):2478-2489).

[0004] MSLN interacts with MUC16, mediates cell adhesion, plays an important role in the peritoneal implantation of ovarian cancer cells, and increases the motility and invasiveness of pancreatic cancer cells (Rump et al. (2004) J Biol Chem. 279(10):9190-8; Gubbels et al. (2006) Mol Cancer 5(1):50; Coehlo et al. Expert Rev Anticancer Ther. 18(2):177-186; Chen et al. (2013) Sci Rep. 3:1870). Pancreatic tumors in particular often progress too rapidly before patients feel any symptoms of the disease, and the average survival time is very short. This prognosis is very poor because the tumors are often unable to be (completely) surgically removed and have already metastasized. Chemotherapy also fails to significantly improve survival time or cure rates. However, despite many attempts by scientists, antibodies that simply target MSLN still have some shortcomings in effectiveness and cannot become an excellent targeted therapy. Amatuximab, the fastest-developed and most famous anti-mesothelin monoclonal antibody, has performed poorly 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 dilemma, antibody-drug conjugates (ADCs) have been introduced for MSLN targeted therapy. Compared to traditional antibodies, ADCs targeting MSLN offer superior tumor-killing efficacy because they combine the targeting properties of antibodies with the high efficacy of small molecule drugs. Currently, the most rapidly developing ADC in this field is Anetumab ravtansine, developed by Bayer. Phase I clinical data have demonstrated good safety and efficacy, and the drug is currently being tested in multiple tumor types with high MSLN expression. Therefore, there is an urgent need for targeted therapies targeting MSLN. However, ADC drugs themselves have developed rapidly in recent years, and many superior linker-toxins have been continuously developed. However, the more traditional non-degradable linkers and DM4 toxin used in Anetumab ravtansine suffer from high toxicity, a narrow safety window, and low effective drug release rates in target organs (WO 2010 / 124797A1). At the same time, MSLN itself is cleaved by various proteases at near-membrane sites, generating free MSLN (soluble MSLN, sMSLN) (Mol Cancer Ther; 15(7)July 2016). The sequence of this free MSLN is highly similar to that of the MSLN on the membrane, and therefore it is recognized by most therapeutic antibodies targeting MSLN, including Anetumab and Amatuximab. Experiments have shown that free MSLN will competitively bind to MSLN antibodies, thereby affecting the efficacy of MSLN-targeted therapy.

[0006] Therefore, there is an urgent need in this field to develop a better antibody-drug conjugate targeting MSLN, which, on the one hand, can demonstrate better anti-tumor efficacy and better plasma stability; on the other hand, its tumor inhibition effect is less affected by free MSLN.

[0007] Summary of the Invention

[0008] The present invention provides an anti-MSLN antibody-drug conjugate and use thereof in treating cancer.

[0009] In a first aspect, the present invention provides an antibody drug conjugate, which is a compound of formula (I) or a tautomer, stereoisomer or pharmaceutically acceptable salt thereof: Ab-[SX-(Y) n -ZED] q (I)

[0010] in,

[0011] Ab is an antibody or antigen-binding fragment thereof that specifically binds to mesothelin (MSLN);

[0012] S is the sulfide bond connecting Ab and X;

[0013] X is selected from 3-10 membered cycloalkyl, 5-10 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl;

[0014] X is optionally substituted with 1, 2, 3, 4 or 5 R*;

[0015] R* is selected from H, deuterium, halogen, CN, NO2, OH, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl;

[0016] Y is -Y1-Y2-Y3-, where Y is connected to X through Y1 and to Z through Y3;

[0017] in,

[0018] Y1 is selected from -C(O)-, -C(O)O-, -OC(O)-, -C(O)NH-, C 2-6 Alkenyl, C 2-6 Alkynyl, 3-10 membered cycloalkyl, 5-10 membered heterocyclyl, C6-10 aryl or 5-10 membered heteroaryl;

[0019] Y2 is a bond, or C 1-6 alkylene;

[0020] Y3 is selected from -C(O)-, -OC(O)- or -NHC(O)-;

[0021] Y is optionally substituted with 1, 2, 3, 4 or 5 R#;

[0022] R# is selected from H, deuterium, halogen, CN, NO2, OH, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl;

[0023] Z is a peptide comprising 2 to 8 amino acids; preferably a dipeptide, tripeptide or tetrapeptide;

[0024] E is absent or is a spacer unit that optionally links Z and D;

[0025] D is a bioactive molecule drug or its derivative

[0026] n is selected from 0, 1, 2, 3, 4 or 5, preferably 0, 1, 2 or 3;

[0027] q is an integer selected from 1 to 10, preferably 8.

[0028] In some embodiments, the antibody drug conjugate is a compound of formula (II) or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof:

[0029] in,

[0030] Ab is anti-MSLN antibody;

[0031] R1 and R2 are independently selected from C 1-6 Alkyl, preferably C 1-4 alkyl;

[0032] D is

[0033] q is an integer selected from 1-10, for example, selected from 4, 5, 6, 7, 8, 9 or 10, preferably 8.

[0034] In some embodiments, the antibody drug conjugate is the following compound or its tautomer, stereoisomer or pharmaceutically acceptable salt:

[0035] Ab is anti-MSLN antibody;

[0036] q is an integer selected from 1-10, for example, selected from 4, 5, 6, 7, 8, 9 or 10, preferably 8.

[0037] In some embodiments, the antibody drug conjugate is the following compound or its tautomer, stereoisomer or pharmaceutically acceptable salt:

[0038] Ab is anti-MSLN antibody;

[0039] q is an integer selected from 1-10, for example, selected from 4, 5, 6, 7, 8, 9 or 10, preferably 8.

[0040] In some embodiments, the antibody drug conjugate is the following compound or its tautomer, stereoisomer or pharmaceutically acceptable salt:

[0041] Ab is anti-MSLN antibody;

[0042] q is an integer selected from 1-10, for example, selected from 4, 5, 6, 7, 8, 9 or 10, preferably 8.

[0043] In some embodiments, the antibody drug conjugate is the following compound or its tautomer, stereoisomer or pharmaceutically acceptable salt:

[0044] Ab is anti-MSLN antibody;

[0045] q is an integer selected from 1-10, for example, selected from 4, 5, 6, 7, 8, 9 or 10, preferably 8.

[0046] In some embodiments, the antibody drug conjugate is the following compound or its tautomer, stereoisomer or pharmaceutically acceptable salt:

[0047] Among them, Ab is anti-MSLN antibody;

[0048] q is an integer selected from 1-10, for example, selected from 4, 5, 6, 7, 8, 9 or 10, preferably 8.

[0049] In some embodiments, the Ab in the antibody drug conjugate of the present invention is an antibody or an antigen-binding fragment thereof that specifically binds to mesothelin (MSLN), wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein

[0050] (1) the VH comprises HCDR1, HCDR2, and HCDR3 of a VH having the amino acid sequence of SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of a VL having the amino acid sequence of SEQ ID NO: 27;

[0051] (2) the VH comprises HCDR1, HCDR2, and HCDR3 of a VH having the amino acid sequence of SEQ ID NO: 25, and the VL comprises LCDR1, LCDR2, and LCDR3 of a VL having the amino acid sequence of SEQ ID NO: 28;

[0052] (3) the VH comprises HCDR1, HCDR2, and HCDR3 of a VH having the amino acid sequence of SEQ ID NO: 26, and the VL comprises LCDR1, LCDR2, and LCDR3 of a VL having the amino acid sequence of SEQ ID NO: 27;

[0053] (4) the VH comprises HCDR1, HCDR2, and HCDR3 of a VH having the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of a VL having the amino acid sequence shown in SEQ ID NO: 29;

[0054] (5) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 30;

[0055] (6) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 31;

[0056] (7) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 32;

[0057] (8) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 33;

[0058] (9) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 25, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 29;

[0059] (10) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 63, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 67;

[0060] (11) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 64, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 67;

[0061] (12) the VH comprises HCDR1, HCDR2, and HCDR3 of a VH having the amino acid sequence of SEQ ID NO: 65, and the VL comprises LCDR1, LCDR2, and LCDR3 of a VL having the amino acid sequence of SEQ ID NO: 67;

[0062] (13) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 65, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 68;

[0063] (14) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 63, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 68;

[0064] (15) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 66, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 67;

[0065] (16) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 64, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 69;

[0066] (17) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 66, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 69;

[0067] (18) the VH comprises HCDR1, HCDR2, and HCDR3 of a VH having the amino acid sequence of SEQ ID NO: 63, and the VL comprises LCDR1, LCDR2, and LCDR3 of a VL having the amino acid sequence of SEQ ID NO: 69; or

[0068] (19) The VH comprises HCDR1, HCDR2 and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 65, and the VL comprises LCDR1, LCDR2 and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 69.

[0069] In some embodiments, the Ab in the antibody-drug conjugate of the present invention is an antibody or an antigen-binding fragment thereof that specifically binds to mesothelin (MSLN), wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0070] (1) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 13;

[0071] (2) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 15, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 16, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 18;

[0072] (3) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 15, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 13;

[0073] (4) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 19;

[0074] (5) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 20;

[0075] (6) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 21;

[0076] (7) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 22;

[0077] (8) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 23;

[0078] (9) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 15, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 16, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 19;

[0079] (10) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 45, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 47, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 52, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56;

[0080] (11) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 47, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 52, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56;

[0081] (12) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 59, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 52, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56;

[0082] (13) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 59, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 61, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56;

[0083] (14) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 45, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 47, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 61, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56;

[0084] (15) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 60, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 52, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56;

[0085] (16) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 47, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 62, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56;

[0086] (17) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 60, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 62, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56;

[0087] (18) the VH comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 45, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 47, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 62, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56; or

[0088] (19) The VH comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 59, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 62, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 56.

[0089] In some embodiments, the Ab in the antibody drug conjugate of the present invention is an antibody or antigen-binding fragment thereof that specifically binds to mesothelin (MSLN), wherein the antibody comprises VH and VL, wherein

[0090] (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;

[0091] (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;

[0092] (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;

[0093] (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;

[0094] (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;

[0095] (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;

[0096] (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;

[0097] (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;

[0098] (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;

[0099] (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;

[0100] (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;

[0101] (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;

[0102] (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;

[0103] (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;

[0104] (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;

[0105] (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;

[0106] (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;

[0107] (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

[0108] (19) The VH comprises an amino acid sequence that has 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 that has 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.

[0109] In some embodiments, the Ab in the antibody drug conjugate of the present invention is an antibody or an antigen-binding fragment thereof that specifically binds to mesothelin (MSLN), wherein the antibody comprises a heavy chain (HC) and a light chain (LC), and wherein:

[0110] (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;

[0111] (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;

[0112] (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;

[0113] (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;

[0114] (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;

[0115] (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;

[0116] (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;

[0117] (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;

[0118] (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;

[0119] (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;

[0120] (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;

[0121] (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;

[0122] (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;

[0123] (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;

[0124] (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;

[0125] (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;

[0126] (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;

[0127] (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

[0128] (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.

[0129] In some embodiments, the antibody drug conjugate of the present invention comprises an antibody that specifically binds to mesothelin (MSLN), and the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0130] In some embodiments, the antibody drug conjugate of the present 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.

[0131] In some embodiments, the antibody drug conjugate of the present 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.

[0132] In a second aspect, the present invention provides a pharmaceutical composition comprising the antibody drug conjugate of the present invention, and optionally a pharmaceutically acceptable carrier or excipient.

[0133] In some embodiments, the composition of the present invention further comprises a second therapeutic agent selected from chemotherapeutic agents, monoclonal antibody drugs, bi / multispecific antibody drugs, recombinant protein drugs, nucleotide drugs (siRNA, antisense oligonucleotides), small molecule drugs, immunomodulatory drugs and cell therapy drugs.

[0134] In a third aspect, the present invention provides a method for treating cancer in a subject, comprising administering an effective amount of an antibody drug conjugate of the present invention or a pharmaceutical composition of the present invention to the subject. 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 bile duct cancer. In some embodiments, the method of the present invention further comprises administering a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent is selected from chemotherapeutics, monoclonal antibody drugs, bi / multi-specific antibody drugs, recombinant protein drugs, nucleotide drugs (siRNA, antisense oligonucleotides), small molecule drugs, immunomodulatory drugs and cell therapy drugs.

[0135] In a fourth aspect, the present invention provides an antibody drug conjugate of the present invention or a 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 bile duct cancer. In some embodiments, the antibody of the present invention or the 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 chemotherapeutics, monoclonal antibody drugs, dual / multi-specific antibody drugs, recombinant protein drugs, nucleotide drugs (siRNA, antisense oligonucleotides), small molecule drugs, immunomodulatory drugs and cell therapy drugs.

[0136] In a fifth aspect, the present invention provides an antibody drug conjugate of the present invention or a pharmaceutical composition of the present invention for use in the preparation of a drug for the treatment of 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 bile duct cancer. In some embodiments, the drug is used in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from chemotherapeutics, monoclonal antibody drugs, bi / multi-specific antibody drugs, recombinant protein drugs, nucleotide drugs (siRNA, antisense oligonucleotides), small molecule drugs, immunomodulatory drugs and cell therapy drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] 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, PR300186-10 to human MSLN protein.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] Figure 6. Internalization of PR300186 and its PTM variants PR300186-3, PR300186-9, and PR300186-10 in COV644 cells.

[0143] Figure 7. Internalization of PR300159-8 and PR300186-10 in COV644 cells.

[0144] Figure 8. Internalization of PR300159-8 and PR300186-10 in NCI-H226 cells.

[0145] Figure 9. Internalization of PR300159-8 and PR300186-10 in HPAC cells.

[0146] Figure 10. MSLN shedding in the HT29-huMSLN wild-type 2C3 cell line and the HT29-huMSLN mutant 1A11 cell line.

[0147] Figure 11. Cytotoxicity of PR300159-8-B81 antibody drug conjugate against huMSLN-expressing cells HT29-huMSLN.

[0148] Figure 12. Stability results of PR300159-8-B81 in human, cynomolgus monkey, SD rat, ICR mouse plasma and phosphate buffered saline (PBS).

[0149] Figure 13. Stability results of PR300186-10-B81 in human, cynomolgus monkey, SD rat, ICR mouse plasma and phosphate buffered saline (PBS).

[0150] Figure 14. Tumor volume in mice treated with different doses of PR300159-8-B81 and PR300186-10-B81 in the HT29-huMSLN wild-type 2C3 model.

[0151] Figure 15. Body weight of mice treated with different doses of PR300159-8-B81 and PR300186-10-B81 in the HT29-huMSLN wild-type 2C3 model.

[0152] Figure 16. Tumor volume in mice treated with different doses of PR300159-8-B81 and PR300186-10-B81 in the HT29-huMSLN mutant 1A11 model.

[0153] Figure 17. Body weight of mice after treatment with different doses of PR300159-8-B81 and PR300186-10-B81 in the HT29-huMSLN mutant 1A11 model.

[0154] Figure 18. Tumor volume in mice treated with different doses of PR300159-8-B81 and PR300186-10-B81 in the COV644 model.

[0155] Figure 19. Body weight of mice after treatment with different doses of PR300159-8-B81 and PR300186-10-B81 in the COV644 model.

[0156] Figure 20. Tumor volume in mice treated with different doses of PR300159-8-DXD, PR300159-8-B81 and PR300186-10-B81 in the OVCAR3 model.

[0157] Figure 21. Body weight of mice after treatment with different doses of PR300159-8-DXD, PR300159-8-B81 and PR300186-10-B81 in the OVCAR3 model.

[0158] Figure 22. Tumor volume in mice treated with different doses of PR300159-8-B81 and PR300186-10-B81 in the SKOV3 model.

[0159] Figure 23. Body weight of mice treated with different doses of PR300159-8-B81 and PR300186-10-B81 in the SKOV3 model.

[0160] Figure 24. Tumor volume in mice treated with 1 mg / kg of PR300159-8-DXD, BAY-949343, PR300159-8-B81, and PR300186-10-B81 in the NCI-H226 model.

[0161] Figure 25. Tumor volume in mice treated with 5 mg / kg of PR300159-8-DXD, BAY-949343, PR300159-8-B81, and PR300186-10-B81 in the NCI-H226 model.

[0162] Figure 26. Body weight changes of mice after treatment with 1 mg / kg of PR300159-8-DXD, BAY-949343, PR300159-8-B81 and PR300186-10-B81 in the NCI-H226 model.

[0163] Figure 27. Body weight changes of mice after treatment with 5 mg / kg of PR300159-8-DXD, BAY-949343, PR300159-8-B81 and PR300186-10-B81 in the NCI-H226 model.

[0164] Figure 28. Binding of PR300159-B71, PR300159, PR300186-B71 and PR300186 to CHOK-huMSLN cells.

[0165] Figure 29. Binding of PR300159-B81, PR300159, PR300186-B81 and PR300186 to COV644 cells.

[0166] Figure 30. Internalization of PR300159, PR300159-B71, PR300186, and PR300186-B71 in COV644 cells.

[0167] Figure 31. Cytotoxicity of PR300159 and PR300159-B71 antibody drug conjugates on CHOK1-huMSLN cells expressing huMSLN (Figure 31A); and cytotoxicity of PR300186 and PR300186-B71 antibody drug conjugates on CHOK1-huMSLN cells expressing huMSLN (Figure 31B).

[0168] Figure 32. Tumor volume (Figure 32A) and body weight (Figure 32B) of mice after treatment with different doses of PR300159-B71, PR300159-B81 and PR300186-B81 in the COV644 mouse tumor model.

[0169] Detailed Description of the Invention

[0170] The above features and advantages of the present invention as well as other features and advantages thereof will be more clearly understood from the following detailed description of embodiments with reference to the accompanying drawings.

[0171] The embodiments described herein with reference to the accompanying drawings are illustrative, explanatory, and for general understanding of the present invention. The examples should not be construed as limiting the scope of the present invention.

[0172] definition

[0173] Chemical definition

[0174] Definitions of specific functional groups and chemical terms are described in more detail below.

[0175] When a numerical range is listed, it is intended to include every value and sub-range within the stated range. For example, "C 1-6 "Alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 、C 1-5 、C 1-4 、C 1-3 、C 1-2 、C 2-6 、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 alkyl.

[0176] “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 groups are preferred. 1-6 Examples of alkyl groups 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). The term “C 1-6 "Alkyl" also includes heteroalkyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkyl group may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Conventional alkyl abbreviations include: Me(-CH3), Et(-CH2CH3), iPr(-CH(CH3)2), nPr(-CH2CH2CH3), n-Bu(-CH2CH2CH2CH3), or i-Bu(-CH2CH(CH3)2).

[0177] “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. 2-6 Examples of alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. The term "C 2-6 "Alkenyl" also includes heteroalkenyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). An alkenyl group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0178] “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 groups 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. The term "C2-6 "Alkynyl" also includes heteroalkynyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkynyl groups can be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0179] “C 0-6 "Alkylene" refers to a chemical bond or "C 1-6 "Alkylene".

[0180] “C 1-6 "Alkylene" refers to the removal of C 1-6 In some embodiments, C 1-4 Alkylene, C 2-4 Alkylene and C 1-3 Alkylene is preferred. Unsubstituted alkylene includes, but is not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexylene (-CH2CH2CH2CH2CH2CH2-), and the like. Exemplary substituted alkylenes, for example, alkylenes substituted with one or more alkyl (methyl) groups, 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- ), substituted propylene (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), and the like.

[0181] "Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0182] Therefore, “C 1-6 "Haloalkyl" refers to the above-mentioned "C 1-6 Alkyl", which is substituted by one or more halogen groups. In some embodiments, C 1-4 Halogenated alkyl is particularly preferred, more preferably C 1-2Haloalkyl. Exemplary haloalkyl groups include, but are not limited to, -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, and the like. The haloalkyl group can be substituted at any available point of attachment, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0183] “C 3-10 "Cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms. In some embodiments, "C 3-10 "Cycloalkyl" is a saturated ring. In other embodiments, "C 3-10 The cycloalkyl group may be an unsaturated ring, for example, comprising 1, 2, 3 or more unsaturated bonds, such as olefinic and acetylenic bonds. In some embodiments, C 3-7 Cycloalkyl and C 3-6 Cycloalkyl is particularly preferred, more preferably C 5- 6 cycloalkyl. Cycloalkyl also includes a ring system in which the above-mentioned cycloalkyl ring is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the cycloalkyl ring, and in such a case, the number of carbons continues to represent the number of carbons in the cycloalkyl system. Exemplary cycloalkyls include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), and the like. The cycloalkyl group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0184] "3-10 membered heterocyclyl" refers to a group of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as valence permits. In some embodiments, a 5-10 membered heterocyclyl is preferred, which is a 3 to 10 membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms; in some embodiments, a 3-7 membered heterocyclyl is preferred, which is a 3 to 7 membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms; a 3-6 membered heterocyclyl is preferred, which is a 3 to 6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; a 4-8 membered heterocyclyl is preferred, which is a 4 to 8 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; and a 5-6 membered heterocyclyl is more preferred, which is a 5 to 6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms. Heterocyclyl also includes a ring system in which the above-mentioned heterocyclyl ring is fused to one or more cycloalkyl groups, wherein the point of attachment is on the cycloalkyl ring, or a ring system in which the above-mentioned heterocyclyl ring is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring; and in such a case, the number of ring members continues to represent the number of ring members in the heterocyclyl ring system. Exemplary 3-membered heterocyclyls containing one heteroatom include, but are not limited to, aziridine, oxirane, and thiorenyl. Exemplary 4-membered heterocyclyls containing one heteroatom include, but are not limited to, azetidinyl, oxetane, and thiidine. Exemplary 5-membered heterocyclyls containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxaslfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to, hexahydrotriazinyl. Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thienyl. Exemplary 5-membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclyl) include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, benzoxazolinonyl, and the like.Exemplary 6-membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as 6,6-bicyclic heterocyclyl groups) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like. The heterocyclyl group may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0185] “C 6-10 "Aryl" refers to a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic arrangement) having 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, an aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms ("C 10 Aryl also includes ring systems in which an aryl ring as described above is fused to one or more cycloalkyl or heterocyclyl groups, and the point of attachment is on the aryl ring, in which case the number of carbon atoms continues to refer to the number of carbon atoms in the aryl ring system. Aryl groups can be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0186] "5-10 membered heteroaryl" refers to a group of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic arrangement) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom as long as valence permits. Heteroaryl bicyclic ring systems may include one or more heteroatoms in one or both rings. Heteroaryl also includes ring systems in which the above-mentioned heteroaryl ring is fused to one or more cycloalkyl or heterocyclyl groups, and the point of attachment is on the heteroaryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the heteroaryl ring system. In some embodiments, "5-10 membered heteroaryl" is a saturated ring. In other embodiments, "5-10 membered heteroaryl" may be an unsaturated ring, for example, comprising 1, 2, 3 or more unsaturated bonds, such as olefinic and acetylenic bonds. In some embodiments, 5-6 membered heteroaryls are particularly preferred, which are 5-6 membered monocyclic or bicyclic 4n+2 aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms. Exemplary 5-membered heteroaryls containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thienyl. Exemplary 5-membered heteroaryls containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryls containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl), and thiadiazolyl. Exemplary 5-membered heteroaryls containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryls containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryls containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azacycloheptatrienyl, oxepinyl, and thieptatrienyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indanyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. A heteroaryl group can be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0187] The divalent groups formed by removing another hydrogen from the above-defined alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl groups are collectively referred to as "subunits". The ring-forming groups such as cycloalkyl, heterocyclyl, aryl and heteroaryl groups are collectively referred to as "cyclyls".

[0188] Alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and the like are defined herein as optionally substituted groups.

[0189] Exemplary substituents on carbon atoms include, but are not limited to, halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa 、-ON(R bb )2、-N(R bb )2、-N(R bb )3 + X - 、-N(OR cc )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 、-NRbb 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, heterocyclyl, aryl and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0190] Or the two geminal hydrogen atoms on the carbon atom are replaced by groups =O, =S, =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 replace;

[0191] R aa Each of R is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R aa The groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0192] R bb Each of the following is independently selected from: 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 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, heterocyclyl, aryl and heteroaryl, or two R bb The groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0193] R ccEach of R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R cc The groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0194] R dd Each of the is independently selected from: 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、-NR ff 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, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R gg Group substitution, or two geminal R dd Substituents may combine to form =O or =S;

[0195] R ee Each of R is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently replaced by 0, 1, 2, 3, 4 or 5 R gg group substitution;

[0196] R ff Each of R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R ff The groups are combined to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R gg group substitution;

[0197] R gg Each of the 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 1-6 alkyl), -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 1-6alkyl), 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 Halogenated alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C6-C 10 Aryl, C3-C7 heterocyclic, C5-C 10 heteroaryl; or two geminal R gg Substituents may combine to form =O or =S; wherein X - For the counter ion.

[0198] Exemplary substituents on nitrogen atoms include, but are not limited to, 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, heterocyclyl, aryl and heteroaryl, or two R attached to the nitrogen atom ccThe groups are combined to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd Group substituted, and wherein R aa 、R bb 、R cc and R dd As mentioned above.

[0199] Other definitions

[0200] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0201] Unless otherwise specified or clearly contradicted by context, the articles "a," "an," and "the" as used herein are intended to include "at least one" or "one or more." Thus, as used herein, these articles refer to one or more than one (i.e., at least one) of the objects. For example, "a component" refers to one or more components, i.e., more than one component may be contemplated for use or use in implementing the described embodiments.

[0202] Unless otherwise indicated or defined, the term "comprise" and variations thereof such as "comprises" and "comprising" should be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps. The term "comprise" encompasses the meanings of "including" as well as "consisting of," for example, a composition "comprising" X may consist exclusively of X or may include additional ingredients, for example, the composition may consist of X + Y.

[0203] The term "about" with respect to a value x is optional and means, for example, x+10% or x±5%.

[0204] "Stereoisomers" are compounds that have identical chemical constitutions but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric (cis / trans) isomers, atropisomers, and the like.

[0205] "Chiral" refers to a molecule that is non-superimposable on its mirror image; "achiral" refers to a molecule that is superimposable on its mirror image.

[0206] "Enantiomers" refer to two non-superimposable isomers of a compound that are mirror images of each other.

[0207] "Diastereoisomers" refers to stereoisomers that have two or more chiral neutral groups whose molecules are not mirror images of each other. Diastereoisomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities. Diastereomeric mixtures can be separated by high-resolution analytical procedures such as electrophoresis and chromatography, for example, HPLC.

[0208] Stereochemical definitions and conventions used herein generally follow SP 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.

[0209] Many organic compounds exist in optically active forms, meaning they have the ability to rotate the plane of plane-polarized light. When describing an optically active compound, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule about its chiral center(s). The prefixes d and l or (+) and (-) are the symbols used to designate the rotation of plane-polarized light caused by the compound, where (-) or l indicates that the compound is left-handed. A compound prefixed with (+) or d is right-handed. A specific stereoisomer is an enantiomer, and a mixture of such isomers 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 stereoselectivity or stereospecificity in a chemical reaction or process.

[0210] Any asymmetric atom (e.g., carbon, etc.) of the compounds disclosed herein can exist in a racemic or enantiomerically enriched form, such as in the (R)-, (S)-, or (R,S)-configuration. In certain embodiments, each asymmetric atom has 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 in terms of the (R)- or (S)-configuration.

[0211] Depending on the choice of starting materials and process, the compounds of the present invention may exist as one of the possible isomers or a mixture thereof, such as a racemate or a mixture of diastereoisomers (depending on the number of asymmetric carbon atoms). Optically active (R)- or (S)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If the compound contains a double bond, the substituents may be in the E or Z configuration; if the compound contains a disubstituted cycloalkyl group, the cycloalkyl substituents may be in the cis or trans configuration.

[0212] Any resulting mixture of stereoisomers can be separated into the pure or substantially pure geometric isomers, enantiomers, and diastereomers on the basis of the differences in the constituent physicochemical properties, for example, by chromatography and / or fractional crystallization.

[0213] Any racemate of the resulting final product or intermediate can be separated into its optical antipodes by methods familiar to those skilled in the art using known methods, such as by separating the diastereomeric salts obtained. The racemic products can also be separated by chiral chromatography, such as high performance liquid chromatography (HPLC) using a chiral adsorbent. In particular, enantiomers can be prepared by asymmetric synthesis, for example, see Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Principles of Asymmetric Synthesis (2 nd Ed.Robert E.Gawley, Jeffrey Aubé, Elsevier, Oxford, UK, 2012); Eliel, ELStereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, SHTables of Resolving Agents and Optical Resolutions p.268 (ELEliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972); Chiral Separation Techniques: A Practical Approach (Subramanian, G.Ed., Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2007).

[0214] The term "tautomer" or "tautomeric form" refers to structural isomers with different energies that can be converted into each other through a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of the tautomers can be reached. For example, proton tautomers (also known as prototropic tautomers) include interconversions performed by proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions performed by the reorganization of some bonding electrons. A specific example of keto-enol tautomerism is the interconversion of pentane-2,4-dione and 4-hydroxypent-3-ene-2-one tautomers. Another example of tautomerism is phenol-keto tautomerism. A specific example of phenol-keto tautomerism is the interconversion of pyridine-4-ol and pyridine-4(1H)-one tautomers. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

[0215] As used herein, "pharmaceutically acceptable salts" refer to organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art, as described in S.M. Berge et al., "Describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19." Pharmaceutically acceptable salts formed from non-toxic acids include, but are not limited to, inorganic acid salts formed by reaction with amino groups, such as hydrochlorides, hydrobromides, phosphates, sulfates, and perchlorates, and organic acid salts such as acetates, oxalates, maleates, tartrates, citrates, succinates, and malonates, or salts obtained by other methods described in the literature, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C1-C4 alkyl) 4 salts. The present invention also contemplates quaternary ammonium salts formed by any compound containing a N group. Water-soluble 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 appropriate, non-toxic ammonium, quaternary ammonium salts and amine cations formed by counter ions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C 1-8 Sulfonates and aromatic sulfonates.

[0216] Unless otherwise indicated or defined, all terms used have their ordinary meanings in the art, which are clear to a skilled person. For example, refer to standard manuals such as Leuenberger, HGW, Nagel, B. and Klbl, 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., "Immunobiology" (6th Ed.) Ed.), Garland Science Publishing / Churchill Livingstone, New York (2005).

[0217] As used herein, the term "subject" refers to an animal. Typically, the animal is a mammal. Subject also refers, for example, to primates (e.g., humans, male or female), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, and the like. In certain embodiments, the subject is a primate. In other embodiments, the subject is a human.

[0218] The term "patient" used in the present invention refers to humans (including adults and children) or other animals. In some embodiments, "patient" refers to humans.

[0219] The terms "antibody drug conjugate," "antibody-drug conjugate," "antibody conjugate," "conjugate," "immunoconjugate," and "ADC" are used interchangeably to refer to an antibody or antigen-binding fragment thereof covalently linked to a drug. The drug is typically, for example, a small molecule drug, a toxin, a cytotoxic agent, a therapeutic agent, or the like.

[0220] As used herein, the term "antibody" refers to an immunoglobulin molecule that can specifically bind to a specific antigen. Antibodies generally include heavy chains and light chains, the heavy chain comprising a heavy chain variable region and a heavy chain constant region, and the light chain comprising a light chain variable region and a light chain constant region. The variable regions of the antibody heavy and light chains comprise binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, which include various cells of the immune system (such as effector cells) and components of the complement system (such as C1q). Most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL), which together constitute the part of the antibody that binds to the antigen.

[0221] The "light chain variable region" (VL) or "heavy chain variable region" (VH) both contain four "framework" regions separated by three "complementarity determining regions" or "CDRs." The framework regions serve to align the CDRs so that the CDRs specifically bind to an antigenic epitope. The CDRs include the amino acid residues primarily responsible for antigen binding. From amino-terminus 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. The CDR1, CDR2, and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; the CDR1, CDR2, and CDR3 of the VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0222] The amino acid assignments for each VL and VH domain conform to any conventional definition of a CDR. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), the Chothia definition (Chothia & Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); a composite of the Chothia-Kabat CDR (also known as the Chothia and Kabat combined CDRs), in which each CDR is a composite of a Chothia CDR and a Kabat CDR; the AbM definition, which is used by the Oxford Molecular Antibody Modeling software; and the contact definition of Martin et al. (See the world wide web bioinfo.org.uk / abs for the above conventional CDR definition systems). Kabat provides a widely used numbering system (the Kabat numbering system) in which corresponding residues between different heavy chains or corresponding residues between different light chains are assigned the same number.

[0223] The present invention contemplates CDRs defined according to any one of these numbering systems, although preferred embodiments contemplate CDRs defined by a combination of Chothia and Kabat.

[0224] Table 1. CDR definition method for the antibodies of the present invention (see http: / / bioinf.org.uk / abs / )

[0225] In Table 1, Laa-Lbb may 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-Hbb may 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-L34 may refer to the amino acid sequence from position 24 to position 34 starting from the N-terminus of the antibody light chain (according to the Chothia numbering system); H26-H32 may refer to the amino acid sequence from position 26 to position 32 starting from the N-terminus of the antibody heavy chain (according to the Chothia numbering system).

[0226] The term "antibody" as used herein should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) comprising at least two different antigen-binding regions. Antibodies may include additional modifications, such as unnatural amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies may also include post-translationally modified antibodies, fusion proteins comprising antigenic determinants of antibodies, and immunoglobulin molecules comprising any other modifications to the antigen recognition sites, as long as these antibodies exhibit the desired biological activity.

[0227] 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 (eg, MSLN). It has been shown that fragments of a full-length antibody can perform the antigen-binding function of an antibody.

[0228] Examples of antigen-binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment consisting of two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fab' fragment, which is essentially a Fab with a portion of the hinge region (see FUNDAMENTAL IMMUNOLOGY (Paul ed., 3.sup.rd ed. 1993)); (iv) an Fd fragment consisting of the VH and CH1 domains; (v) an Fd' fragment having the VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (vi) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (vii) a dAb fragment (Ward et al., (1989) Nature 58:105-110; 341:544-546), which consists of a VH domain; (viii) an isolated complementarity determining region (CDR); and (ix) a nanobody, which comprises a heavy chain variable region of one variable domain and two constant domains. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be connected using recombinant methods by a synthetic linker that makes them a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called single-chain Fv (scFv)); for example, see Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also included in the term "antigen-binding fragment" of an antibody. Additionally, the term includes "linear antibodies" comprising a pair of tandem Fd fragments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form an antigen binding region, as well as modifications of any of the foregoing fragments that retain antigen binding activity.

[0229] These antigen-binding fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments screened for utility in the same manner as intact antibodies.

[0230] As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as 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) between the antigen and the antibody. D ) represents the strength of the binding between the antigenic determinant (epitope) and the antigen binding site on the antibody: K DThe smaller the value, the stronger the binding strength between the antigenic determinant (epitope) and the antibody. Alternatively, affinity can also be expressed as the affinity constant (KA), which is 1 / K D .

[0231] Avidity is a measure of the strength of binding between an antibody and its associated antigen. Avidity is related to the affinity between an antigenic determinant (epitope) and its antigen binding site on the antibody, as well as the number of relevant binding sites present on the antibody. Typically, antibodies will be expressed as 10 -5 to 10 -12 The dissociation constant (KD) of the molecule is 10 M or less, preferably 10 -7 to 10 -12 M or less, more preferably 10 -8 to 10 -12 M, and / or at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , for example at least 10 12 M -1 Any binding affinity greater than 10 - 4 A KD value of M is generally considered to be nonspecific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined by any suitable detection format known in the art, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA), biolayer interferometry (BLI), and sandwich competition assays, and various variations thereof known in the art.

[0232] The term "k on ” or “k a ” refers to the association rate constant for the association of an antibody with an antigen to form an antibody / antigen complex. This rate can be determined using standard assays, such as Biacore or ELISA assays.

[0233] The term "k off ” or “k d ” refers to the dissociation rate constant for the dissociation of the antibody from the antibody / antigen complex. This rate can be determined using standard assays, such as Biacore or ELISA assays.

[0234] The term "K D ” refers to the equilibrium dissociation constant of a specific antibody-antigen interaction. K D By k a / k dThe rate can be determined using standard assays, such as Biacore or ELISA assays. The antibody or antigen-binding fragment thereof in the conjugate of the present application can be determined at the equilibrium dissociation constant (K D ≤1 mM, ≤100 nM or ≤10 nM or any amount therebetween binds to the target antigen.

[0235] As used herein, the term "sequence identity" refers to the degree to which two sequences (amino acids) have identical residues at the same position in the alignment. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" means that the amino acid sequence has X% identity to SEQ ID NO: Y, and is specifically expressed as X% of the residues in the amino acid sequence being identical to the residues in the sequence disclosed in SEQ ID NO: Y. Typically, computer programs are 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), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0236] Furthermore, when determining the degree of sequence identity between two amino acid sequences, one skilled in the art may consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which one amino acid residue is replaced by another amino acid residue of similar chemical structure and has 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.

[0237] Particularly preferred conservative substitutions are as follows: Ala for Gly or Ser; Arg for Lys; Asn for Gln or for His; Asp for Glu; Cys for Ser; Gln for Asn; Glu for Asp; Gly for Ala or for Pro; His for Asn or for Gln; Ile for Leu or for Val; Leu for Ile or for Val; Lys for Arg, for Gln or for Glu; Met for Leu, for Tyr or for Ile; Phe for Met, for Leu or for Tyr; Ser for Thr; Thr for Ser; Trp for Tyr; Tyr for Trp; and / or Phe for Val, for Ile or for Leu.

[0238] 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 encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, as long as the fragment specifically binds to MSLN.

[0239] As used herein with respect to an antibody or antigen-binding fragment, "internalization" refers to an antibody or antigen-binding fragment that, upon binding to a cell, is able to pass through the cellular lipid bilayer membrane to an internal compartment (i.e., "internalized"), preferably to a degradation compartment in the cell.

[0240] The terms "therapeutic agent," "drug," or "drug moiety" refer to an agent that modulates a biological process and / or possesses biological activity.

[0241] The term "cytotoxic agent" refers to a substance that causes cell death primarily by interfering with the expression activity and / or function of the cell. Examples of cytotoxic agents include, but are not limited to, antimitotic agents such as eribulin, auristatins (e.g., monomethyl auristatin E (MMAE)).

[0242] The term "antibody:drug ratio" or "drug-to-antibody ratio" or "DAR" refers to the number of drug moieties attached per antibody, ie, the drug loading.

[0243] As used herein, the term "treat" refers to administering an agent or performing a procedure in order to obtain an effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of achieving a partial or complete cure of a disease and / or disease symptoms. As used herein, "treatment" may include the treatment of a disease or disorder (e.g., cancer) in a mammal, particularly a human, and includes: (a) preventing the disease or disease symptoms from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease (e.g., including a disease that may be associated with or caused by the primary disease); (b) inhibiting the disease, i.e., preventing its development; and (c) alleviating the disease, i.e., causing the disease to regress. Treatment may refer to any indicator of successful cancer treatment, improvement, or prevention, including any objective or subjective parameter, such as remission; remission reduces symptoms or makes the disease condition more tolerable for the patient. Thus, the term "treatment" includes administering an ADC or composition disclosed herein to prevent or delay, alleviate, or prevent or inhibit the development of symptoms or conditions associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination or prevention of a disease, disease symptoms or disease side effects in a subject.

[0244] The term "cancer" refers to the physiological condition in mammals that is characterized by unregulated cell growth in a population of cells.

[0245] "Tumor-associated antigens" refer to antigens that are differentially expressed in cancer cells compared to normal cells. Tumor-associated antigens can therefore be used to target cancer cells.

[0246] "Pharmaceutical composition" refers to a preparation that is in a form that allows administration and subsequent provision of the desired biological activity and / or therapeutic effect of the active ingredient and does not contain other components that are toxic and unacceptable to the subject to which the preparation is administered. Pharmaceutical compositions can be sterile.

[0247] "Pharmaceutical excipients" include substances such as adjuvants, carriers, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like.

[0248] "Pharmaceutically acceptable" means suitable, within the scope of sound medical judgment, for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, etc., and commensurate with a reasonable benefit / risk ratio.

[0249] As used herein, an "effective amount" of an ADC is an amount sufficient to carry out a particular stated purpose, such as an amount that produces a therapeutic effect upon administration, such as a reduction in tumor growth rate or tumor volume, a reduction in cancer symptoms, or some other sign of therapeutic efficacy. An effective amount can be determined in a conventional manner for the stated purpose. The term "therapeutically effective amount" refers to an amount of an ADC that effectively treats a disease or condition in a subject. In the case of cancer, a therapeutically effective amount of an ADC can 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 an amount effective to achieve a prophylactic result at the necessary dosage and time period. Typically, because a prophylactic dose is used in a subject before or in the early stages of the disease, the prophylactic effective amount will be less than the therapeutically effective amount.

[0250] Certain embodiments of the present invention will now be described in more detail, examples of which are illustrated by the accompanying structural formulas and chemical formulae. The present invention is intended to encompass all substitutions, modifications, and equivalent technical solutions, which are all included within the scope of the invention as defined in the claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used to practice the present invention. The present invention is in no way limited to the methods and materials described herein. In the event that one or more of the combined documents, patents, and similar materials differ from or contradict the present application (including but not limited to defined terms, term applications, described technologies, etc.), the present application shall prevail.

[0251] It will be further appreciated that certain features of the invention, which, for clarity, are described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which, for brevity, are described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0252] Unless otherwise specified, all technical terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. All patents and publications related to the present invention are incorporated herein by reference in their entirety.

[0253] Unless otherwise indicated, the following definitions shall apply as used herein. For purposes of the present invention, the chemical elements are as per the Periodic Table of the Elements, CAS version, and Handbook of Chemistry and Physics, 75th edition, 1994. In addition, general principles of organic chemistry may be found in "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 entire contents of which are incorporated herein by reference. DETAILED DESCRIPTION

[0254] The following embodiments are provided to further illustrate the present invention. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0255] In one embodiment, the present invention relates to a MSLN antibody drug conjugate, which is a compound of formula (I) or a tautomer, stereoisomer or pharmaceutically acceptable salt thereof: Ab-[SX-(Y) n -ZED] q (I)

[0256] in,

[0257] Ab is an antibody or antigen-binding fragment thereof that specifically binds to mesothelin (MSLN);

[0258] S is the sulfide bond connecting Ab and X;

[0259] X is selected from C 3-10 Cycloalkyl, 5-10 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl;

[0260] X is optionally substituted with 1, 2, 3, 4 or 5 R*;

[0261] R* is selected from H, deuterium, halogen, CN, NO2, OH, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl;

[0262] Y is -Y1-Y2-Y3-, where Y is connected to X through Y1 and to Z through Y3;

[0263] in,

[0264] Y1 is selected from -C(O)-, -C(O)O-, -OC(O)-, -C(O)NH-, C 2-6 Alkenyl, C 2-6 Alkynyl, 3-10 membered cycloalkyl, 5-10 membered heterocyclyl, C6-10 aryl or 5-10 membered heteroaryl;

[0265] Y2 is a bond, or C 1-6 alkylene;

[0266] Y3 is selected from -C(O)-, -OC(O)- or -NHC(O)-;

[0267] Y is optionally substituted with 1, 2, 3, 4 or 5 R#;

[0268] R# is selected from H, deuterium, halogen, CN, NO2, OH, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl;

[0269] Z is a peptide comprising 2 to 8 amino acids; preferably a dipeptide, tripeptide or tetrapeptide;

[0270] E is absent or is a spacer unit that optionally links Z and D;

[0271] D is a bioactive molecule drug or its derivative

[0272] n is selected from 0, 1, 2, 3, 4 or 5, preferably 0, 1, 2 or 3;

[0273] q is an integer selected from 1 to 10, preferably 8.

[0274] Ab

[0275] In one embodiment, Ab is an MSLN antibody; in another embodiment, Ab is an MSLN antigen-binding fragment.

[0276] X

[0277] In one embodiment, X is C 3-10 In another embodiment, X is a 5-10 membered heterocyclic group; In another embodiment, X is C 6-10 In another embodiment, X is a 5-10 membered heteroaryl; in another embodiment, X is a 5-6 membered heteroaryl.

[0278] In one embodiment, X is a pyrimidinyl group, for example

[0279] In one embodiment, X is unsubstituted; in another embodiment, X is substituted with 1 R*; in another embodiment, X is substituted with 2 R*; in another embodiment, X is substituted with 3 R*; in another embodiment, X is substituted with 4 R*; in another embodiment, X is substituted with 5 R*.

[0280] Y

[0281] In one embodiment, Y is -Y1-Y2-Y3-, where Y is linked to X through Y1 and to Z through Y3.

[0282] In one embodiment, Y is

[0283] In one embodiment, Y is unsubstituted; in another embodiment, Y is substituted with 1 R#; in another embodiment, Y is substituted with 2 R#; in another embodiment, Y is substituted with 3 R#; in another embodiment, Y is substituted with 4 R#; in another embodiment, Y is substituted with 5 R#.

[0284] Y1

[0285] In one embodiment, Y1 is -C(O)-; in another embodiment, Y1 is -C(O)O-; in another embodiment, Y1 is -OC(O)-; in another embodiment, Y1 is -C(O)NH-; in another embodiment, Y1 is C 2-6 In another embodiment, Y1 is C 2-4 In another embodiment, Y1 is C 2-6 Alkynyl; In another embodiment, Y1 is C 2-4 Alkynyl; In another embodiment, Y1 is C 3-10 Cycloalkyl; in another embodiment, Y1 is a 5-10 membered heterocyclic group; in another embodiment, Y1 is C 6-10Aryl; In another embodiment, Y1 is a 5-10 membered heteroaryl.

[0286] Y2

[0287] In one embodiment, Y2 is a bond; in another embodiment, Y2 is C 1-6 Alkylene; In another embodiment, Y2 is C 1-4 Alkylene.

[0288] Y3

[0289] In one embodiment, Y3 is -C(O)-; in another embodiment, Y3 is -OC(O)-; in another embodiment, Y3 is -NHC(O)-.

[0290] Z

[0291] In one embodiment, Z is a linker peptide comprising a peptide of 2 to 8 amino acids; preferably a dipeptide, tripeptide or tetrapeptide.

[0292] 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 that can be cleaved by cathepsin B is valine-citrulline (Val-Cit) (Dubowchik et al. (2002) Bioconjugate Chem. 13:855-69).

[0293] In a specific 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-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 modified by substitution.

[0294] In one embodiment, Z is Wherein, R1 and R2 are independently selected from H, deuterium, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl.

[0295] In one embodiment, Z is Wherein, R1 and R2 are independently selected from H, deuterium, halogen or C 1-6 Alkyl, preferably H or C 1-4 alkyl.

[0296] E

[0297] E is absent or is a spacer unit that optionally connects Z and D; E is absent or is selected from -NH-C 1-6 Alkylene- or -OC 1-6 Alkylene-, the -NH-C 1-6 Alkylene and -OC 1-6 Alkylene- may optionally be selected from H, halogen, C 1-6 Alkyl and C 1-6 substituted by 1, 2 or 3 substituents in a haloalkyl group;

[0298] Preferably, E is absent or is -NH-C 1-4 Alkylene-, the -NH-C 1-4 Alkylene- may optionally be selected from H, halogen, C 1-4 Alkyl and C 1-4 substituted by 1, 2 or 3 substituents in a haloalkyl group;

[0299] More preferably, E is -NH-(CH2) 1-4 -, for example -NH-CH2-.

[0300] In some embodiments, E can be a spacer unit linking Z and D. The spacer can be a functional group that facilitates the attachment of XYZ to the camptothecin derivative, or it can provide an additional structural component to further facilitate the release of the camptothecin derivative from the conjugate.

[0301] D

[0302] In one embodiment, D is a biologically active molecule drug or a derivative thereof.

[0303] In one embodiment, D is selected from the group consisting of metal complexes, glycopeptide antibiotics, DNA topoisomerase inhibitors, drugs that interfere with DNA synthesis, drugs that act on structural proteins, tumor signaling pathway inhibitors, proteasome inhibitors, histone deacetylase inhibitors, tumor angiogenesis inhibitors, cell cycle protein inhibitors, serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors, or histidine kinase inhibitors, and other active substances that inhibit tumor cell growth and promote tumor cell apoptosis or necrosis.

[0304] In one embodiment, D is selected from: oxaliplatin; bleomycin or bleomycin; camptothecin, camptothecin derivatives (such as 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; maytansine derivatives; calicheamicin derivatives; auristatin derivatives; pyrrolobenzodiazepine dimer derivatives; melphalan; mitomycin C; or chlorambucil.

[0305] In one embodiment, D is

[0306] in,

[0307] R D absent, or selected from -NH- or -O-;

[0308] L D Selected from -C 0-6 Alkylene-OC 0-6 Alkylene- or -C 0-6 Alkylene-OC 0-6 Alkylene-C(O)NH-CHR D4 -;

[0309] R D1 and R D2 independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy; or, R D1 、R D2 Together with the carbon atoms to which they are attached, they form C 3-7 Cycloalkyl or 3-7 membered heterocyclic group, preferably 5-6 membered heterocyclic group;

[0310] R D3 and RD4 independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy; or, R D3 、R D4 Together with the carbon atom to which they are attached, they form C 5-10 Cycloalkyl or 5-10 membered heterocyclic group, preferably C 5-6 Cycloalkyl.

[0311] In one embodiment, D is selected from:

[0312] R*

[0313] In one embodiment, R* is H; in another embodiment, R* is deuterium; in another embodiment, R* is halogen; in another embodiment, R* is CN; in another embodiment, R* is NO2; in another embodiment, R* is OH; in another embodiment, R* is C 1-6 Alkyl; in another embodiment, R* is C 1-4 Alkyl; in another embodiment, R* is C 1-6 In another embodiment, R* is C 1-4 In another embodiment, R* is C 2-6 In another embodiment, R* is C 2-4 In another embodiment, R* is C 2-6 Alkynyl; In another embodiment, R* is C 2-4 Alkynyl.

[0314] R#

[0315] In one embodiment, R# is H; in another embodiment, R# is deuterium; in another embodiment, R# is halogen; in another embodiment, R# is CN; in another embodiment, R# is NO2; in another embodiment, R# is OH; in another embodiment, R# is C 1-6 Alkyl; In another embodiment, R# is C 1-4 Alkyl; In another embodiment, R# is C 1-6 In another embodiment, R# is C 1-4 In another embodiment, R# is C 2-6 In another embodiment, R# is C 2-4In another embodiment, R# is C 2-6 Alkynyl; In another embodiment, R# is C 2-4 Alkynyl.

[0316] n

[0317] In one embodiment, n is 0; in another embodiment, n is 1; in another embodiment, n is 2; in another embodiment, n is 3; in another embodiment, n is 4; and in another embodiment, n is 5.

[0318] q

[0319] In one embodiment, q is an integer selected from 1 to 10; in another embodiment, q is 1; in another embodiment, q is 2; in another embodiment, q is 3; in another embodiment, q is 4; in another embodiment, q is 5; in another embodiment, q is 6; in another embodiment, q is 7; in another embodiment, q is 8; in another embodiment, q is 9; in another embodiment, q is 10.

[0320] Any technical solution or any combination thereof in any of the above specific embodiments may be combined with any technical solution or any combination thereof in any other specific embodiment. For example, any technical solution of Ab or any combination thereof may be combined with any technical solution of 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 combinations of these technical solutions, which are not listed one by one due to space limitations.

[0321] In a more specific embodiment, the present invention provides an MSLN antibody drug conjugate of the above formula (I), wherein,

[0322] X is selected from C 6-10 Aryl or 5-10 membered heteroaryl;

[0323] X is optionally substituted with 1, 2, 3, 4 or 5 R*;

[0324] R* is selected from H, deuterium, halogen, CN, NO2, OH, C 1-6 alkyl;

[0325] Preferably,

[0326] X is a 5-6 membered heteroaryl group;

[0327] X is optionally substituted with 1, 2 or 3 R*;

[0328] R* is selected from H, deuterium, halogen or C 1-4 alkyl;

[0329] More preferably,

[0330] X is a pyrimidinyl group, for example

[0331] In a more specific embodiment, the present invention provides an MSLN antibody drug conjugate of the above formula (I), wherein,

[0332] Y is -Y1-Y2-Y3-, where Y is connected to X through Y1 and to Z through Y3;

[0333] in,

[0334] Y1 is selected from C 2-6 Alkenyl or C 2-6 Alkynyl;

[0335] Y2 is a bond, or C 1-6 alkylene;

[0336] Y3 is selected from -C(O)- or -NHC(O)-;

[0337] Y is optionally substituted with 1, 2, 3, 4 or 5 R#;

[0338] R# is selected from H, deuterium, halogen, CN, NO2, OH, C 1-6 alkyl;

[0339] Preferably,

[0340] Y is -Y1-Y2-Y3-, where Y is connected to X through Y1 and to Z through Y3;

[0341] in,

[0342] Y1 is selected from C 2-4 Alkenyl or C 2-4 Alkynyl;

[0343] Y2 is C 1-4 alkylene;

[0344] Y3 is -C(O)-;

[0345] Y is optionally substituted with 1, 2 or 3 R#;

[0346] R# is selected from H, deuterium, halogen or C 1-4 alkyl;

[0347] More preferably,

[0348] Y is

[0349] In a more specific embodiment, the present invention provides an MSLN antibody drug conjugate of the above formula (I), wherein,

[0350] 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, va l-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 above dipeptide, tripeptide, tetrapeptide modified by substitution;

[0351] Preferably,

[0352] Z is

[0353] R1 and R2 are independently selected from H, deuterium, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl;

[0354] More preferably,

[0355] Z is

[0356] R1 and R2 are independently selected from H, deuterium, halogen or C 1-6 Alkyl, preferably H or C 1-4 alkyl.

[0357] In a more specific embodiment, the present invention provides an MSLN antibody drug conjugate of the above formula (I), wherein,

[0358] E is absent or selected from -NH-C 1-6 Alkylene- or -OC 1-6 Alkylene-, the -NH-C 1-6 Alkylene and -OC 1-6 Alkylene- may optionally be selected from H, halogen, C 1-6 Alkyl and C 1-6 substituted by 1, 2 or 3 substituents in a haloalkyl group;

[0359] Preferably, E is absent or is -NH-C 1-4 Alkylene-, the -NH-C1-4 Alkylene- may optionally be selected from H, halogen, C 1-4 Alkyl and C 1-4 substituted by 1, 2 or 3 substituents in a haloalkyl group;

[0360] More preferably, E is -NH-(CH2) 1-4 -, for example -NH-CH2-.

[0361] In a more specific embodiment, the present invention provides an MSLN antibody drug conjugate of the above formula (I), wherein,

[0362] D is selected from metal complexes; glycopeptide antibiotics; DNA topoisomerase inhibitors; drugs that interfere with DNA synthesis; drugs that act on structural proteins; tumor signaling pathway inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor angiogenesis inhibitors; cell cycle protein inhibitors; serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors or histidine kinase inhibitors; and other active substances that inhibit tumor cell growth and promote tumor cell apoptosis or necrosis;

[0363] Preferably,

[0364] D is selected from oxaliplatin; bleomycin or bleomycin; camptothecin, camptothecin derivatives (such as 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; maytansine derivatives; calicheamicin derivatives; auristatin derivatives; pyrrolobenzodiazepine dimer derivatives; melphalan; mitomycin C; or chlorambucil;

[0365] More preferably,

[0366] D is

[0367] in,

[0368] R D absent, or selected from -NH- or -O-;

[0369] L D Selected from -C 0-6 Alkylene-OC 0-6 Alkylene- or -C 0-6 Alkylene-OC 0-6 Alkylene-C(O)NH-CHRD4 -;

[0370] R D1 and R D2 independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy; or, R D1 、R D2 Together with the carbon atoms to which they are attached, they form C 3-7 Cycloalkyl or 3-7 membered heterocyclic group, preferably 5-6 membered heterocyclic group;

[0371] R D3 and R D4 independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy; or, R D3 、R D4 Together with the carbon atom to which they are attached, they form C 5-10 Cycloalkyl or 5-10 membered heterocyclic group, preferably C 5-6 Cycloalkyl;

[0372] More preferably,

[0373] D is selected from:

[0374] In a more specific embodiment, the present invention provides an MSLN antibody drug conjugate of the above formula (I), wherein,

[0375] X is selected from C 6-10 Aryl or 5-10 membered heteroaryl;

[0376] X is optionally substituted with 1, 2, 3, 4 or 5 R*;

[0377] R* is selected from H, deuterium, halogen, CN, NO2, OH, C 1-6 alkyl;

[0378] Y is -Y1-Y2-Y3-, where Y is connected to X through Y1 and to Z through Y3;

[0379] in,

[0380] Y1 is selected from C 2-6 Alkenyl or C 2-6 Alkynyl;

[0381] Y2 is a bond, or C 1-6 alkylene;

[0382] Y3 is selected from -C(O)- or -NHC(O)-;

[0383] Y is optionally substituted with 1, 2, 3, 4 or 5 R#;

[0384] R# is selected from H, deuterium, halogen, CN, NO2, OH or C 1-6 alkyl;

[0385] 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, va l-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 above dipeptide, tripeptide, tetrapeptide modified by substitution;

[0386] E is absent or selected from -NH-C 1-6 Alkylene- or -OC 1-6 Alkylene-, the -NH-C 1-6 Alkylene and -OC 1-6 Alkylene- may optionally be selected from H, halogen, C 1-6 Alkyl and C 1-6 substituted by 1, 2 or 3 substituents in a haloalkyl group;

[0387] D is selected from metal complexes; glycopeptide antibiotics; DNA topoisomerase inhibitors; drugs that interfere with DNA synthesis; drugs that act on structural proteins; tumor signaling pathway inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor angiogenesis inhibitors; cell cycle protein inhibitors; serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors or histidine kinase inhibitors; and other active substances that inhibit tumor cell growth and promote tumor cell apoptosis or necrosis.

[0388] In a more specific embodiment, the present invention provides an MSLN antibody drug conjugate of the above formula (I), wherein,

[0389] X is a 5-6 membered heteroaryl group;

[0390] X is optionally substituted with 1, 2 or 3 R*;

[0391] R* is selected from H, deuterium, halogen or C 1-4 alkyl;

[0392] Y is -Y1-Y2-Y3-, where Y is connected to X through Y1 and to Z through Y3;

[0393] in,

[0394] Y1 is selected from C 2-4 Alkenyl or C 2-4 Alkynyl;

[0395] Y2 is C 1-4 alkylene;

[0396] Y3 is -C(O)-;

[0397] Y is optionally substituted with 1, 2 or 3 R#;

[0398] R# is selected from H, deuterium, halogen or C 1-4 alkyl;

[0399] Z is

[0400] E does not exist or is -NH-C 1-4 Alkylene-, the -NH-C 1-4 Alkylene- may optionally be selected from H, halogen, C 1-4 Alkyl and C 1-4 substituted by 1, 2 or 3 substituents in a haloalkyl group;

[0401] R1 and R2 are independently selected from H, deuterium, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl;

[0402] D is selected from oxaliplatin; bleomycin or bleomycin; camptothecin, camptothecin derivatives (such as 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; maytansine derivatives; calicheamicin derivatives; auristatin derivatives; pyrrolobenzodiazepine dimer derivatives; melphalan; mitomycin C; or chlorambucil;

[0403] Preferably,

[0404] D is

[0405] in,

[0406] R D absent, or selected from -NH- or -O-;

[0407] L D Selected from -C 0-6 Alkylene-OC 0-6 Alkylene- or -C 0-6 Alkylene-OC 0-6 Alkylene-C(O)NH-CHR D4 -;

[0408] R D1 and R D2 independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy; or, R D1 、R D2 Together with the carbon atoms to which they are attached, they form C 3-7 Cycloalkyl or 3-7 membered heterocyclic group, preferably 5-6 membered heterocyclic group;

[0409] R D3 and R D4 independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy; or, R D3 、R D4 Together with the carbon atom to which they are attached, they form C 5-10 Cycloalkyl or 5-10 membered heterocyclic group, preferably C 5-6 Cycloalkyl.

[0410] In a more specific embodiment, the present invention provides an MSLN antibody drug conjugate of the above formula (I), wherein,

[0411] X is a pyrimidinyl group, preferably

[0412] Y is

[0413] Z is

[0414] R1 and R2 are independently selected from H, deuterium, halogen, C 1-6 Alkyl, preferably H or C 1-4 alkyl;

[0415] E is -NH-CH2-;

[0416] D is selected from:

[0417] n is selected from 1, 2 or 3, preferably 1;

[0418] q is selected from 4, 5, 6, 7, 8, 9 or 10, preferably 8.

[0419] In a more specific embodiment, the present invention provides an MSLN antibody drug conjugate, wherein the MSLN antibody drug conjugate is a compound of formula (II) or a tautomer, stereoisomer or pharmaceutically acceptable salt thereof:

[0420] in,

[0421] Ab is anti-MSLN antibody;

[0422] R1 and R2 are independently selected from C 1-6 Alkyl, preferably C 1-4 alkyl;

[0423] D is selected from:

[0424] q is selected from integers of 1-10, preferably selected from 4, 5, 6, 7, 8, 9 or 10, and most preferably 8.

[0425] Anti-MSLN antibody

[0426] 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), and wherein

[0427] (1) the VH comprises HCDR1, HCDR2, and HCDR3 of a VH having the amino acid sequence of SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of a VL having the amino acid sequence of SEQ ID NO: 27;

[0428] (2) the VH comprises HCDR1, HCDR2, and HCDR3 of a VH having the amino acid sequence of SEQ ID NO: 25, and the VL comprises LCDR1, LCDR2, and LCDR3 of a VL having the amino acid sequence of SEQ ID NO: 28;

[0429] (3) the VH comprises HCDR1, HCDR2, and HCDR3 of a VH having the amino acid sequence of SEQ ID NO: 26, and the VL comprises LCDR1, LCDR2, and LCDR3 of a VL having the amino acid sequence of SEQ ID NO: 27;

[0430] (4) the VH comprises HCDR1, HCDR2, and HCDR3 of a VH having the amino acid sequence of SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of a VL having the amino acid sequence of SEQ ID NO: 29;

[0431] (5) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 30;

[0432] (6) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 31;

[0433] (7) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 32;

[0434] (8) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 33;

[0435] (9) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 25, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 29;

[0436] (10) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 63, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 67;

[0437] (11) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 64, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 67;

[0438] (12) the VH comprises HCDR1, HCDR2, and HCDR3 of a VH having the amino acid sequence of SEQ ID NO: 65, and the VL comprises LCDR1, LCDR2, and LCDR3 of a VL having the amino acid sequence of SEQ ID NO: 67;

[0439] (13) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 65, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 68;

[0440] (14) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 63, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 68;

[0441] (15) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 66, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 67;

[0442] (16) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 64, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 69;

[0443] (17) the VH comprises HCDR1, HCDR2, and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 66, and the VL comprises LCDR1, LCDR2, and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 69;

[0444] (18) the VH comprises HCDR1, HCDR2, and HCDR3 of a VH having the amino acid sequence of SEQ ID NO: 63, and the VL comprises LCDR1, LCDR2, and LCDR3 of a VL having the amino acid sequence of SEQ ID NO: 69; or

[0445] (19) The VH comprises HCDR1, HCDR2 and HCDR3 of VH having the amino acid sequence shown in SEQ ID NO: 65, and the VL comprises LCDR1, LCDR2 and LCDR3 of VL having the amino acid sequence shown in SEQ ID NO: 69.

[0446] In some embodiments, LCDRs 1-3 and HCDRs 1-3 are defined by the EU Kabat definition / numbering system. In some embodiments, LCDRs 1-3 and HCDRs 1-3 are defined by the Chothia definition / numbering system. In some embodiments, LCDRs 1-3 and HCDRs 1-3 are defined by the AbM definition / numbering system. In some preferred embodiments, LCDRs 1-3 and HCDRs 1-3 are defined by the combined Kabat and Chothia numbering systems.

[0447] In some embodiments, the Ab in the antibody-drug conjugate of the present invention is an antibody or an antigen-binding fragment thereof that specifically binds to mesothelin (MSLN), wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0448] (1) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 13;

[0449] (2) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 15, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 16, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 18;

[0450] (3) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 15, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 13;

[0451] (4) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 19;

[0452] (5) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 20;

[0453] (6) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 21;

[0454] (7) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 22;

[0455] (8) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 23;

[0456] (9) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 15, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 16, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 19;

[0457] (10) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 45, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 47, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 52, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56;

[0458] (11) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 47, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 52, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56;

[0459] (12) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 59, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 52, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56;

[0460] (13) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 59, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 61, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56;

[0461] (14) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 45, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 47, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 61, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56;

[0462] (15) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 60, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 52, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56;

[0463] (16) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 47, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 62, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56;

[0464] (17) the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 60, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 62, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56;

[0465] (18) the VH comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 45, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 47, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 62, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56; or

[0466] (19) The VH comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 59, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 62, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 56.

[0467] In some embodiments, CDRs are determined by the Kabat Chothia combinatorial system.

[0468] In some embodiments, the Ab in the antibody drug conjugate of the present invention is an antibody or antigen-binding fragment thereof that specifically binds to mesothelin (MSLN), wherein the antibody comprises VL and VH, wherein

[0469] (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;

[0470] (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;

[0471] (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;

[0472] (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;

[0473] (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;

[0474] (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;

[0475] (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;

[0476] (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;

[0477] (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;

[0478] (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;

[0479] (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;

[0480] (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;

[0481] (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;

[0482] (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;

[0483] (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;

[0484] (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;

[0485] (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;

[0486] (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

[0487] (19) The VH comprises an amino acid sequence that has 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 that has 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.

[0488] In some embodiments, the VL comprises a functional variant of the amino acid sequence shown in any one of SEQ ID NOs: 27-33 and 67-69 formed by inserting, deleting and / or replacing one or more amino acids, provided that the antibody comprising the VL of the functional variant retains the ability to bind to MSLN. In some embodiments, the VH comprises a functional variant of the amino acid sequence shown in any one of SEQ ID NOs: 24-26 and 63-66 formed by inserting, deleting and / or replacing one or more amino acids, provided that the antibody comprising the VH of the functional variant retains the ability to bind to MSLN.

[0489] Functional variants comprise or consist 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: 27-33 and 67-69 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: 27-33 and 67-69, respectively. A functional variant of any one of SEQ ID NOs: 24-26 and 63-66 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: 24-26 and 63-66, respectively.

[0490] In the context of functional variants, the number of inserted, deleted and / or substituted amino acids preferably does not exceed 40% of the total number of amino acids in the parent amino acid sequence, more preferably does not exceed 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, more preferably 15-20%. 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 preferred embodiments, the number of inserted, deleted and / or substituted amino acids is 1, 2, 3, 4, 5, 6 or 7.

[0491] In some embodiments, insertions, deletions and / or substitutions can be performed in framework (FR) regions, such as FR1, FR2, FR3 and / or FR4.

[0492] In a preferred embodiment, VL comprises the amino acid sequence as shown in SEQ ID NO: 27, and VH comprises the amino acid sequence as shown in SEQ ID NO: 24; or VL comprises the amino acid sequence as shown in SEQ ID NO: 28, and VH comprises the amino acid sequence as shown in SEQ ID NO: 25; or VL comprises the amino acid sequence as shown in SEQ ID NO: 27, and VH comprises the amino acid sequence as shown in SEQ ID NO: 26; or VL comprises the amino acid sequence as shown in SEQ ID NO: 29, and VH comprises the amino acid sequence as shown in SEQ ID NO: 24; or VL comprises the amino acid sequence as shown in SEQ ID NO: 30, and VH comprises the amino acid sequence as shown in SEQ ID NO: 24; or VL comprises the amino acid sequence as shown in SEQ ID NO: 31, and VH comprises the amino acid sequence as shown in SEQ ID NO: 24; or VL comprises the amino acid sequence as shown in SEQ ID NO: 32, and VH comprises the amino acid sequence as shown in SEQ ID NO: 24; or VL comprises the amino acid sequence as shown in SEQ ID NO: 33, and VH comprises the amino acid sequence as shown in SEQ ID NO: 24; or VL comprises the amino acid sequence as shown in SEQ ID NO: 34. NO: 29, VH comprises the amino acid sequence shown in SEQ ID NO: 25; or VL comprises the amino acid sequence shown in SEQ ID NO: 67, and VH comprises the amino acid sequence shown in SEQ ID NO: 63; or VL comprises the amino acid sequence shown in SEQ ID NO: 67, and VH comprises the amino acid sequence shown in SEQ ID NO: 64; or VL comprises the amino acid sequence shown in SEQ ID NO: 67, and VH comprises the amino acid sequence shown in SEQ ID NO: 65; or VL comprises the amino acid sequence shown in SEQ ID NO: 68, and VH comprises the amino acid sequence shown in SEQ ID NO: 65; or VL comprises the amino acid sequence shown in SEQ ID NO: 68, and VH comprises the amino acid sequence shown in SEQ ID NO: 63; or VL comprises the amino acid sequence shown in SEQ ID NO: 67, and VH comprises the amino acid sequence shown in SEQ ID NO: 66; or VL comprises the amino acid sequence shown in SEQ ID NO: 69, and VH comprises the amino acid sequence shown in SEQ ID NO: 64; or VL comprises the amino acid sequence shown in SEQ ID NO: NO:69, VH comprises the amino acid sequence shown in SEQ ID NO:66; or VL comprises the amino acid sequence shown in SEQ ID NO:69, VH comprises the amino acid sequence shown in SEQ ID NO:63;Or VL comprises the amino acid sequence shown in SEQ ID NO: 69, and VH comprises the amino acid sequence shown in SEQ ID NO: 65. ;

[0493] In some embodiments, the Ab in the antibody drug conjugate of the present invention is an antibody or an antigen-binding fragment thereof that specifically binds to mesothelin (MSLN), wherein the antibody comprises a heavy chain (HC) and a light chain (LC), and wherein:

[0494] (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;

[0495] (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;

[0496] (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;

[0497] (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;

[0498] (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;

[0499] (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;

[0500] (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;

[0501] (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;

[0502] (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;

[0503] (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;

[0504] (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;

[0505] (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;

[0506] (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;

[0507] (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;

[0508] (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;

[0509] (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;

[0510] (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;

[0511] (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

[0512] (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.

[0513] In some embodiments, the light chain comprises a functional variant of the amino acid sequence shown in any one of SEQ ID NOs: 37-43 and 74-76 formed by insertion, deletion and / or substitution of one or more amino acids, provided that the antibody comprising the light chain of the functional variant retains the ability to bind to MSLN. In some embodiments, the heavy chain comprises a functional variant of the amino acid sequence shown in any one of SEQ ID NOs: 34-36 and 70-73 formed by insertion, deletion and / or substitution of one or more amino acids, provided that the antibody comprising the VH of the functional variant retains the ability to bind to MSLN.

[0514] For example, a functional variant of any one of SEQ ID NOs: 37-43 and 74-76 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: 37-43 and 74-76. For example, a functional variant of any one of SEQ ID NOs: 34-36 and 70-73 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: 34-36 and 70-73.

[0515] In some embodiments, the number of amino acids inserted, deleted and / or substituted preferably does not exceed 40% of the total number of amino acids in the parent amino acid sequence, more preferably does not exceed 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, more preferably 15-20%. For example, the number of amino acids inserted, deleted and / or substituted can be 1-50, preferably 1-20, more preferably 1-10, still more preferably 1-5. In preferred embodiments, the number of amino acids inserted, deleted and / or substituted is 1, 2, 3, 4, 5, 6 or 7.

[0516] In some embodiments, insertions, deletions and / or substitutions can be performed in the framework (FR) regions, e.g., FR1, FR2, FR3 and / or FR4; and / or constant regions, e.g., CL, CH1, CH2 and / or CH3.

[0517] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Examples of conservative substitutions are described above.

[0518] In a preferred embodiment, the light chain comprises the amino acid sequence as shown in SEQ ID NO:37, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:34; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:38, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:35; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:37, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:36; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:39, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:34; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:40, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:34; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:41, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:34; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:42, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:34; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:43, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:44. 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, 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, 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, and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:73; or 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 the light chain comprises the amino acid sequence set forth in SEQ ID NO:77 NO:76, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO:73; or the light chain comprises the amino acid sequence shown in SEQ ID NO:76, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO:70; or the light chain comprises the amino acid sequence shown in SEQ ID NO:76, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO:72.

[0519] In some embodiments, the antibody comprises an Fc region. In some embodiments, the Fc region can be of any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and can comprise one or more mutations or modifications. In one embodiment, the Fc region is of the IgG1 isotype or is derived therefrom, optionally with one or more mutations or modifications. In one embodiment, the Fc region is a human IgG1 Fc.

[0520] In one embodiment, the Fc region lacks effector function. For example, the Fc region can be an IgG1 isotype, or a non-IgG1 type, such as IgG2, IgG3, or IgG4, which has been mutated so that the ability to mediate effector function (e.g., ADCC) has been reduced or even eliminated. For example, Dall'Acqua WF et al., J Immunol. 177(2):1129-1138 (2006) and Hezareh M, J Virol.; 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.

[0521] In some embodiments, the antibody is mutated at one or more post-translational modification sites. In one embodiment, the Fc region includes a mutation that removes an acceptor site for Asn-linked glycosylation, or is manipulated to eliminate effector function of the antibody.

[0522] Post-translational modifications (PTMs) are widely present in proteins expressed in mammalian cells. In addition to conserved PTM sites in antibodies, such as the conserved N-glycosylation site on the CH2 domain of IgG1 antibodies, other PTM sites that appear within the antigen binding site (i.e., CDR region) of an antibody may reduce antigen binding activity or reduce chemical stability. For example, deamidation or isomerization may make the molecule unstable and uneven. In order to reduce sequence susceptibility, PTM motifs can be removed by mutation. The VH or VL sequence is scanned for the presence of PTM motifs, such as isomerization motifs (e.g., DG). The "hotspot" residues (e.g., D or g in the DG motif) are then mutated to corresponding residues in the germline sequence or other residues with similar biophysical properties.

[0523] In some embodiments, the antibody drug conjugate of the present invention comprises an antibody that specifically binds to mesothelin (MSLN), and 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), and the antibody is 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), and the antibody is a subtype selected from IgG1, IgG2, IgG3, and IgG4.

[0524] In some embodiments, the antibody drug conjugate of the present 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.

[0525] In some embodiments, the antibody drug conjugate of the present 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.

[0526] In some embodiments, the antibody drug conjugate of the present invention comprises an antibody or antigen-binding fragment thereof that specifically binds to mesothelin, wherein the antibody is monovalent, bivalent, or multivalent.

[0527] In some embodiments, the antibody drug conjugate of the present invention comprises an antibody or antigen-binding fragment thereof that specifically binds to mesothelin, wherein the antibody or antigen-binding fragment is attached to a fluorescent label or a radioactive label or a cytotoxic agent.

[0528] In some embodiments, the antibody-drug conjugate of the present invention comprises an antibody or an antigen-binding fragment thereof that specifically binds to mesothelin, wherein the antibody is a bispecific antibody that further comprises a second antigen-binding region that specifically binds to a tumor-associated antigen, an immune cell antigen, or an immune checkpoint molecule.

[0529] In some embodiments, the antibody drug conjugate of the present invention is the following compound or its tautomer, stereoisomer or pharmaceutically acceptable salt:

[0530] Ab is anti-MSLN antibody;

[0531] q is an integer selected from 1-10, for example, selected from 4, 5, 6, 7, 8, 9 or 10, preferably 8.

[0532] In some embodiments, the antibody drug conjugate of the present invention is the following compound or its tautomer, stereoisomer or pharmaceutically acceptable salt:

[0533] Ab is anti-MSLN antibody;

[0534] q is an integer selected from 1-10, for example, selected from 4, 5, 6, 7, 8, 9 or 10, preferably 8.

[0535] In some embodiments, the antibody drug conjugate of the present invention is the following compound or its tautomer, stereoisomer or pharmaceutically acceptable salt:

[0536] Ab is anti-MSLN antibody;

[0537] q is an integer selected from 1-10, for example, selected from 4, 5, 6, 7, 8, 9 or 10, preferably 8.

[0538] In some embodiments, the antibody drug conjugate of the present invention is the following compound or its tautomer, stereoisomer or pharmaceutically acceptable salt:

[0539] Ab is anti-MSLN antibody;

[0540] q is an integer selected from 1-10, for example, selected from 4, 5, 6, 7, 8, 9 or 10, preferably 8.

[0541] In some embodiments, the antibody drug conjugate of the present invention is the following compound or its tautomer, stereoisomer or pharmaceutically acceptable salt:

[0542] Among them, Ab is anti-MSLN antibody;

[0543] q is an integer selected from 1-10, for example, selected from 4, 5, 6, 7, 8, 9 or 10, preferably 8.

[0544] In some preferred embodiments, the MSLN antibody comprises a heavy chain (HC) and a light chain (LC), and wherein:

[0545] (1) the HC comprises the amino acid sequence shown in SEQ ID NO: 34, and the LC comprises the amino acid sequence shown in SEQ ID NO: 37;

[0546] (2) the HC comprises the amino acid sequence shown in SEQ ID NO: 34, and the LC comprises the amino acid sequence shown in SEQ ID NO: 43;

[0547] (3) the HC comprises the amino acid sequence shown in SEQ ID NO: 70, and the LC comprises the amino acid sequence shown in SEQ ID NO: 74; or

[0548] (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.

[0549] Bispecific antibodies

[0550] In some embodiments, the antibody in the antibody-drug conjugate of the present invention can 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, which comprises an antibody or antigen-binding fragment thereof that specifically binds to MSLN and further comprises a second antigen-binding region that binds a second antigen. In some embodiments, the second antigen can be a tumor-associated antigen, an immune checkpoint molecule, or an immune cell antigen.

[0551] Many tumor-associated antigens relevant to specific cancers have been identified in the art. In some embodiments, tumor-associated antigens are antigens that can stimulate a significant tumor-specific immune response. Some of these antigens are encoded by normal cells, but are not necessarily expressed by normal cells. These antigens can be described as antigens that are usually silent (i.e., not expressed) in normal cells, antigens that are only expressed at certain stages of differentiation, and antigens that are expressed over time, such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cell genes, such as oncogenes (e.g., activated ras oncogenes), suppressor genes (e.g., mutated p53), and fusion proteins produced by internal deletions or chromosomal translocations. Other cancer antigens can be encoded by viral genes, such as tumor viruses carrying RNA and DNA. Many other tumor-associated antigens and antibodies against them are known and / or commercially available, and can also be produced by those skilled in the art.

[0552] In some embodiments, a bispecific antibody comprises a single polypeptide chain comprising a first and a second antigen binding region and, optionally, an Fc region.

[0553] Pharmaceutical composition

[0554] In a second aspect, the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate according to the first aspect of the present invention, and optionally a pharmaceutically acceptable carrier or excipient.

[0555] The present invention provides pharmaceutical compositions comprising the antibody drug conjugates of the present invention. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). For example, in some embodiments, the composition for intravenous administration is typically a solution in a sterile isotonic aqueous buffer.

[0556] The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous injection, intradermal administration, subcutaneous administration, oral administration (e.g., inhalation), transdermal administration (i.e., topical administration), transmucosal administration, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following ingredients: a sterile diluent, such as water for injection, saline, fixed oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methyl paraben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid (EDTA); a buffer, such as acetate, citrate, or phosphate, and an agent for adjusting tension, such as sodium chloride or glucose. The pH value can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be packaged in ampoules, disposable syringes, or multiple-dose bottles made of glass or plastic.

[0557] Treatment

[0558] In a third aspect, the present invention provides a method for treating cancer in a subject, comprising administering to the subject an effective amount of the antibody drug conjugate of the first aspect of the invention or the pharmaceutical composition of the second aspect. The antibody drug conjugates provided herein can be used to slow or inhibit the progression of mesothelin-positive cancers, or to inhibit the 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 the growth, replication or metastasis of cancer cells, or to inhibit signs or symptoms of cancer. Suitable subjects may include subjects 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 bile duct cancer.

[0559] The administration of the antibody drug conjugates disclosed herein can also be administered in combination with other anticancer agents or therapeutic treatments (e.g., surgical resection of a tumor). Any suitable anticancer agent can be administered in combination with the antibody drug conjugates disclosed herein. Exemplary anticancer agents include, but are not limited to, chemotherapeutics, such as mitotic inhibitors, alkylating agents, antimetabolites, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, antisurvival agents, biological response modifiers, antihormones (e.g., antiandrogens), and anti-angiogenic agents. Other anticancer treatments include radiotherapy and other antibodies directed against cancer cells.

[0560] Another common treatment method used to treat certain types of cancer is surgery, such as surgical removal of metastatic tumors. Another example of treatment is radiation therapy, such as the administration of radioactive material or energy (e.g., external beam therapy) to a tumor site to help eradicate the tumor or shrink it prior to surgical removal.

[0561] In a fourth aspect, the present invention provides an antibody drug conjugate of the present invention or a 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 bile duct cancer. In some embodiments, the antibody of the present invention or the 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, siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

[0562] In a fifth aspect, the present invention provides the use of an antibody-drug conjugate of the present invention or a pharmaceutical composition of the present invention in the preparation of 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 bile duct cancer. 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, siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

[0563] Example

[0564] Example 1. Preparation of initial antibodies and variants

[0565] To obtain MSLN-specific antibodies, Harbour H2L2 transgenic mice (https: / / harbourantibodies.com / ) were immunized using various methods. Immunization yielded a number of antibodies that bind to the MSLN extracellular domain (ECD) protein and MSLN-expressing cells, such as COV644 cells (an ovarian epithelial-mucinous carcinoma cell line expressing MSLN).

[0566] Specifically, recombinant human MSLN ECD His-tag protein (Acro Biosystem, Catalog #MSN-H5223) or recombinant cynomolgus monkey MSLN ECD His-tag protein (having the amino acid sequence shown in SEQ ID NO: 77) was used as an immunogen to immunize Harbor H2L2 transgenic mice.

[0567] use The Optofluidic system was used for single B cell screening to obtain antibody heavy and light chain sequences from single plasma cells. The general procedure involved extraction and purification of total RNA from single plasma cell lysates, reverse transcription to cDNA synthesis, amplification and purification of the cDNA, amplification of DNA sequences encoding the antibody heavy and light chains, cloning and transfection, and Sanger sequencing. The obtained sequences were analyzed for uniqueness and clustering, and then DNA sequences encoding the paired heavy and light chains were synthesized. Initial antibodies PR300159 and PR300186 were obtained.

[0568] The VH and VL sequences of the anti-MSLN antibodies PR300159 and PR300186 were further optimized by a PTM (post-translational modification) removal program. The design variants obtained by removing PTMs 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 antibodies derived from PR300159 with PTMs removed. PR300186-2, PR300186-3, PR300186-4, PR300186-5, PR300186-6, PR300186-7, PR300186-8, PR300186-9, and PR300186-10 are PTM-removing antibodies derived from PR300186.

[0569] Table 2. PTM variants derived from PR300159 and PR300186

[0570] Table 3. Amino acid sequences of heavy and light chains of antibodies

[0571] Table 4. Amino acid sequences of the heavy chain variable region and light chain variable region of the antibodies of the present invention

[0572] Table 5. Amino acid sequences of HCDR1 to HCDR3 of antibodies (according to Combined encoding rules)

[0573] Table 6. Amino acid sequences of LCDR1 to LCDR3 of antibodies (according to Combined coding rules)

[0574] Example 2. Binding activity of antibodies to human and cynomolgus monkey MSLN proteins

[0575] Human MSLN (Acro Biosystems, catalog number: MSN-H5223) or cynomolgus monkey MSLN protein (Harbourbiomed, lot number: 2019072202) was diluted to a concentration of 1 μg / mL in PBS. 100 μl of diluted human MSLN or cynomolgus monkey MSLN was added to each well of the ELISA microplate and incubated overnight at 4°C. The plates were blocked with ELISA blocking solution (containing 2% w / v BSA, 0.05% (v / v) Tween-20, pH 7.4 PBS buffer) at 37° C. for 1 hour, then washed and incubated with diluted anti-MSLN antibodies (PR300186, PR300186-2, PR300186-3, PR300186-4, PR300186-5, PR300186-6, PR300186-7, PR300186-8, PR300186-9 and PR300186-10) (at 15 μg / mL) (100 nM, 10-fold dilution, 8 points) at 37° C. for 1 hour. The plates were then washed and incubated with HRP-conjugated goat anti-human IgG (H+L) antibody (Jackson, Cat: 109-035-088) at 37° C. for 1 hour. 100 μL 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. 100 μL ELISA stop solution (Solarbio, Cat#: C1058) was added to terminate the reaction and the optical density at 450 nm (OD450 nm) was measured by an ELISA plate reader (Molecular Devices, Spectra max 384 plus).

[0576] 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 to human MSLN protein are shown in Figure 1 and Table 7, and the binding activity results to cynomolgus macaque MSLN protein are shown in Figure 2 and Table 7. The results showed that all of these antibodies had good binding activity to both human and cynomolgus macaque MSLN proteins.

[0577] Table 7. Binding activity of the antibodies of the present invention to human MSLN protein and cynomolgus monkey MSLN protein

[0578] Example 3. Binding activity of MSLN antibody to cells expressing MSLN

[0579] The 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 COV644 cell line (ECACC, catalog number: 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 a solution containing 1-2 × 10 5 50 μL cell suspension of 10 cells was added and incubated at 4°C for 1 hour. The cells were washed twice with staining buffer (PBS containing 2% FBS) and 100 μL of 1:1000 diluted fluorescently labeled anti-human IgG antibody (Alexa Fluor 488 Affinity Pure Goat Anti-Human IgG (H+L), Jackson ImmunoResearch, catalog number: 109-545-088) was added to each well. After incubation at 4°C for 1 hour, the cells were washed twice with staining buffer and subjected to flow cytometry.

[0580] As shown in FIG3 and Table 8, PR300159, PR300159-1, PR300159-3, PR300159-5, PR300159-6, PR300159-8 and PR300159-9 all have high affinity for COV644 cells.

[0581] Table 8. Antibody binding activity to cell surface MSLN detected by FACS

[0582] 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 had high affinity for COV644 cells.

[0583] Example 4. Determination of Antibody Internalization by MSLN-Expressing Cells

[0584] In this example, pHAb Amine Reactive Dye (Promega, Cat# G9841) was used to determine the internalization efficiency of anti-MSLN antibodies on different tumor cells. pHAb dye is a pH sensor dye that has very low fluorescence when the pH value is greater than 7. When the pH value of the solution decreases, the fluorescence increases significantly. When the antibody labeled with pHAb dye binds to the cell outer membrane under neutral pH conditions, no fluorescence or very low fluorescence is detected. After the pHAb dye-labeled antibody is internalized by the cell, strong fluorescence is detected in the low pH environment of the cell's endosomes and lysosomes.

[0585] The antibody was labeled with pHAb dye, and the DAR value was calculated according to the kit instructions. The pHAb dye-labeled antibody was incubated with COV644 at 4°C (the antibody internalization activity was very low at this temperature and was used as a background control) or 37°C for 24 hours. The fluorescence at the excitation light value (Ex) was then detected at 532nm and the emission light value (Em) was detected at 560nm. The antibody internalization result was calculated as follows: the fluorescence intensity at 37°C minus the fluorescence intensity at the background at 4°C, divided by the DAR value of the antibody-coupled dye. The higher the fluorescence value, the higher the internalization efficiency of the antibody on the cell.

[0586] As shown in Figures 5 to 6 and the results in Table 9, PR300159, PR300159-1, PR300159-3, PR300159-4, PR300159-5, PR300159-6, PR300159-7, PR300159-8, PR300159-9 and PR300186, PR300186-3, PR300186-9, and PR300186-10 all showed good internalization activity.

[0587] Table 9. Results of antibody internalization by MSLN-expressing cells

[0588] Example 5. Binding activity experiments of PR300159-8 and PR300186-10 on different tumor cells

[0589] The binding activity of PR300159-8 and PR300186-10 antibodies on the following tumor cells was detected by flow cytometry: breast cancer cell lines COV644 (ECACC, 07071908), OVCAR3 (ATCC, HTB-161), and SKOV3 (ATCC, HTB77); lung cancer cell lines NCl-H226 (ATCC, CRL5826), HCl-H1568 (ATCC, CRL5876); pancreatic cancer cell lines HPAC (ATCC, CRL2199) and CAPAN2 (ATCC, HTB80); and gastric cancer cell line MKN45 (CCTCC; GDC0220).

[0590] PR300159-8 and PR300186-10 antibodies were serially diluted in staining buffer (PBS containing 2% FBS). 50 μL of the diluted antibody solution was added to a 1-2×10 5 The cells were suspended in 50 μL of the cell suspension and incubated at 4°C for 1 hour. The cells were washed twice with a buffer solution (PBS containing 2% fetal bovine serum) and 100 μL of fluorescently labeled anti-human IgG antibody (Alexa Fluor 488 affinity pure goat anti-human IgG (H+L), Jackson ImmunoResearch, Catalog 109-545-088). After incubation at 4°C for 1 hour, the cells were washed twice with buffer and analyzed by flow cytometry.

[0591] The experimental results are shown in Table 10. The experimental results show that the PR300159-8 antibody has good binding activity on different tumor cells. The binding activity of the antibody PR300159-8 is higher than that of PR300186-10.

[0592] Table 10. Binding activity of PR300159-8 and PR300186-10 on various tumor cells

[0593] Example 6. Internalization activity experiments of PR300159-8 and PR300186-10 antibodies on various tumor cells

[0594] Flow cytometry was used to detect the internalization efficiency of PR300159-8 and PR300186-10 antibodies in the following tumor cells: breast cancer cell line COV644 (ECACC, 07071908); lung cancer cell line NCl-H226 (ATCC, CRL5826); and pancreatic cancer cell line HPAC (ATCC, CRL2199).

[0595] In this example, pHAb Amine Reactive Dye (Promega, Cat# G9841) was used to determine the internalization efficiency of anti-MSLN antibodies on different tumor cells. pHAb dye is a pH sensor dye that has very low fluorescence when the pH value is greater than 7. When the pH value of the solution decreases, the fluorescence increases significantly. When the antibody labeled with pHAb dye binds to the cell outer membrane under neutral pH conditions, no fluorescence or very low fluorescence is detected. After the pHAb dye-labeled antibody is internalized by the cell, strong fluorescence is detected in the low pH environment of the cell's endosomes and lysosomes.

[0596] The antibody was labeled with pHAb dye, and the DAR value was calculated according to the kit instructions. The pHAb dye-labeled antibody was incubated with COV644 at 4°C (the antibody internalization activity was very low at this temperature and was used as a background control) or 37°C for 24 hours. The fluorescence at the excitation light value (Ex) was then detected at 532nm and the emission light value (Em) was detected at 560nm. The antibody internalization result was calculated as follows: the fluorescence intensity at 37°C minus the fluorescence intensity at the background at 4°C, divided by the DAR value of the antibody-coupled dye. The higher the fluorescence value, the higher the internalization efficiency of the antibody on the cell.

[0597] As shown in Figures 7, 8 and 9, PR300159-8 and PR300186-10 had good internalization activity in COV644, NCl-H226 and HPAC cells, and the cell internalization activity of antibody PR300159-8 was higher than that of PR300186-10.

[0598] Example 7. Synthesis of Bioactive Molecules and Intermediates Used in the Synthesis of Drug-Linker Compounds

[0599] 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)

[0600] 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)

[0601] To a 75% sulfuric acid solution (5 mL) of (S)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolane[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1A, 500 mg) was added ferrous sulfate heptahydrate (570 mg dissolved in 1 mL of water) and 4,4-dimethoxychlorobutane (3.89 g) under ice-cooling conditions. The reaction mixture was stirred for three minutes, followed by the dropwise addition of hydrogen peroxide (29%, 2.5 mL). The reaction mixture was stirred at 0°C for 5 minutes, then warmed to room temperature and stirred for 3 hours. The reaction solution 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 a crude product. The crude product was further purified using a C18 column (acetonitrile / 0.05% formic acid in water: 5%-60%) to give the target compound A1B (yellow solid, 400 mg, yield: 67%).

[0602] LCMS (ESI) [M+H] + :468.9;

[0603] 1 H NMR(400MHz,DMSO-d6)δ7.65(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).

[0604] 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)

[0605] 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 10% sulfuric acid (5 mL) and reacted at 110°C for 48 hours. Saturated sodium bicarbonate (30 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (10 mL x 5), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The product was purified by HPLC (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).

[0606] LCMS (ESI) [M+H] + :451.0;

[0607] 1 H NMR(400MHz,DMSO-d6)δ7.63(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).

[0608] 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)

[0609] Step 1:

[0610] To a solution of compound A2A (10 g) in 1,2-dichloroethane (200 mL) at 0°C, 1 mol / L boron trichloride (96 mL) was added dropwise, followed by 4-chlorobutyronitrile (9.9 g). The mixture was stirred at 80°C for 2 hours. The reaction mixture was cooled to room temperature, 2 mol / L hydrochloric acid (90 mL) was added, and the mixture was refluxed at 80°C with stirring 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 a crude product. The crude product was then separated and purified by column chromatography (petroleum ether:ethyl acetate = 10 / 1) to obtain the target compound A2B (4 g).

[0611] LCMS (ESI) [M+H] + :230.0.

[0612] Step 2:

[0613] To compound A2B (50 mg) were added (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (A2C, 35 mg), p-toluenesulfonic acid monohydrate (41.4 mg), and dichloromethane (30 mL). The solution was clarified and mixed, then concentrated under reduced pressure and evacuated to a vacuum using an oil pump. The reaction was incubated at 120°C under vacuum for 3 hours. LCMS indicated completion of the reaction. The reaction solution was cooled to room temperature, water (20 mL) was added, and the mixture was extracted with dichloromethane (20 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure to yield the crude product. The crude product was purified by column chromatography (dichloromethane to methanol = 20:1) to yield the target compound A2D (80 mg) as a white solid.

[0614] LCMS (ESI) [M+H] + :457.0.

[0615] Step 3:

[0616] Compound A2D (75 mg) was dissolved in hexamethylphosphoric triamide, and pure water (0.8 mL) was added. The reaction mixture was stirred at 100°C for 72 hours. LCMS confirmed the reaction was complete. The target compound A2 (10 mg) was purified by preparative chromatography (0.01% TFA in water, MeCN).

[0617] LCMS (ESI) [M+H] + :439.2;

[0618] 1H NMR (400MHz, DMSO-d6) δ8.22(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,2 H),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).

[0619] Example 7.3: (S)-2-amino-N-((3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-

[0620] Synthesis of 10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)propoxy)methyl)acetamide (B1)

[0621] 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, then washed with aqueous sodium bicarbonate solution and aqueous hydrochloric acid solution, respectively. The organic solvent was removed under reduced pressure to obtain a crude product. The crude product was separated and purified by column chromatography (dichloromethane:methanol = 10 / 1) to obtain the target compound Compound B1B (240 mg).

[0622] LCMS (ESI) [M+H] + :759.5.

[0623] Step 2: Dissolve compound B1B (240 mg) in DMF (5 mL) and add piperidine (1 mL). Stir the mixture for 20 minutes, remove low-boiling components under reduced pressure, and use the residue directly in the next step. A small amount of the crude product was purified by reverse-phase chromatography (acetonitrile / 0.05% FA in water: 5% to 50%) to obtain the target compound B1.

[0624] ESI-MS (m / z): 537.4 [M+H] + ;

[0625] 1H NMR (400MHz, DMSO-d6) δ9.13(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).

[0626] 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)

[0627] Step 1:

[0628] Compound A2 (160 mg, 0.365 mmol) was dissolved in N,N-dimethylformamide (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 then added to the reaction mixture, and the mixture was stirred at room temperature overnight. LCMS confirmed the reaction was complete. The reaction mixture was directly purified by reverse-phase chromatography (acetonitrile / 0.05% FA in water: 5% to 50%) to obtain the target compound B2B (80 mg, 29.0% yield) as a white solid.

[0629] LCMS (ESI) [M+H] + =747.4;

[0630] 1H NMR(400MHz, DMSO-d6)δ8.74(t,J=6.4Hz,1H),8.28–8.17(m,1H),7.99–7.88(m,3H),7.73(d,J=7.3H z,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).

[0631] Step 2: B2B (240 mg) was dissolved in DMF (5 mL), piperidine (1 mL) was added, the mixture was stirred for 20 minutes, and low-boiling components were removed under reduced pressure. The residue was directly used for the next synthesis.

[0632] ESI-MS (m / z): 525.2 [M+H] + .

[0633] 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.

[0634] ESI-MS (m / z): 525.1 [M+H] + ;

[0635] 1 H NMR (400MHz, DMSO) δ9.13(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).

[0636] Example 7.5: N 6 ,N 6 -dimethyl-N 2Synthesis of -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (C1)

[0637] 6-(2-(Methylsulfonyl)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 by C18 reverse phase column (acetonitrile and 0.05% formic acid aqueous solution) to obtain the target compound N 6 ,N 6 -dimethyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (C1, white solid, 327 mg).

[0638] LCMS (ESI) [M+H] + :524.4.

[0639] 1 H NMR(400MHz,)δ9.13(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).

[0640] Example 7.6: N 6 ,N 6 -Diethyl-N 2 Synthesis of -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (C2)

[0641] Step 1:

[0642] 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 stirred at room temperature for 10 minutes. Sodium triacetoxyborohydride (12.8 g, 60.25 mmol) was then added to the reaction mixture and stirred at room temperature for 1 hour. LCMS indicated the reaction was complete. A saturated aqueous solution of ammonium chloride was added to the reaction mixture and stirred for one hour. The mixture was then dried by evaporation. The filtrate was filtered and purified using a C18 reverse phase column (acetonitrile to 0.05% formic acid in water: 5% to 55%) to obtain the target compound C2B (4.57 g, 82.0% yield) as a white solid.

[0643] LCMS (ESI) [M+H] + =436.4;

[0644] 1 H NMR(400MHz,DMSO-d6)δ7.70(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,2 H),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).

[0645] Step 2:

[0646] Compound C2B (1.6 g, 3.68 mmol) was dissolved in methanol (80 mL) at room temperature. Pd / C (0.16 g) was then added to the reaction mixture, and the mixture was stirred at room temperature under hydrogen for 12 hours. LCMS indicated the reaction was complete. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to afford the target compound C2C (900 mg, 82% yield) as an off-white solid.

[0647] 1H NMR(400MHz, DMSO-d6)δ8.04(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).

[0648] Step 3:

[0649] Compound 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (268 mg, 1 mmol) was dissolved in DMF (8 mL). HATU (380 mg, 1 mmol) and triethylamine (322 mg, 2.5 mmol) were added sequentially. After stirring at room temperature for 20 minutes, compound C2C (301 mg, 1 mmol) was added and stirring continued at room temperature for 30 minutes. After completion of the reaction, the reaction solution was directly purified on a C18 reverse phase column (acetonitrile and 0.05% formic acid in water) to obtain the target compound C2 (280 mg, 51% yield) as a white solid.

[0650] LCMS (ESI) [M+H] + =552.3;

[0651] 1 H NMR(400MHz,DMSO-d6)δ9.13(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).

[0652] Example 7.7: N 6 ,N 6 -Dipropyl-N 2Synthesis of -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (C3)

[0653] Step 1:

[0654] Compound C2A (5.0 g, 12 mmol) was dissolved in dichloromethane (100 mL). Propionaldehyde (4.2 g, 72.3 mmol) was added to the reaction mixture and stirred at room temperature for 10 minutes. Sodium triacetoxyborohydride (12.8 g, 60.25 mmol) was then added to the reaction mixture and stirred at room temperature for 1 hour. LCMS indicated the reaction was complete. A saturated aqueous solution of ammonium chloride was added to the reaction mixture and stirred for one hour. The mixture was then dried by evaporation. The filtrate was filtered and purified using a C18 column with reversed phase separation (acetonitrile to 0.05% formic acid in water: 5% to 55%) to obtain the target compound C3A (4.57 g, 82.0% yield) as a white solid.

[0655] LCMS (ESI) [M+H] + =464.0;

[0656] 1 H NMR(400MHz, DMSO-d6)δ7.81(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),2.6 5–2.52(m,6H),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).

[0657] Step 2:

[0658] Compound C3A (2.0 g, 4.32 mmol) was dissolved in methanol (80 mL) at room temperature. Pd / C (0.16 g) was then added to the reaction mixture, and the mixture was stirred under hydrogen at room temperature for 12 hours. LCMS indicated the reaction was complete. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to afford the target compound C3B (1.2 g, 85.5% yield) as a white solid.

[0659] Step 3:

[0660] Compound 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (100 mg, 0.373 mmol) was dissolved in N,N-dimethylformamide (1 mL), and then HATU (142 mg, 0.373 mmol) and N,N-diisopropylethylamine (120 mg, 0.93 mmol) were added. The system was stirred for 30 minutes, and then compound C3B (122 mg, 0.371 mmol) was added. The reaction solution was stirred at room temperature for 1 hour. After the reaction was completed by LCMS detection, the reaction solution was directly purified by C18 reverse phase column (acetonitrile and 0.05% formic acid aqueous solution system) to obtain the target compound N 6 ,N 6 -Dipropyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (C3, 50 mg, yield 28%) was a light yellow solid.

[0661] LCMS (ESI) [M+H] + =580.0;

[0662] 1 H NMR(400MHz,DMSO-d6)δ8.24(s,2H),7.98–7.93(m,2H),4.24–4.16(m,1H),4 .10(d,J=5.2Hz,1H),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).

[0663] Example 7.8: N 6 ,N 6 -Dibutyl-N 2 Synthesis of -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (C4)

[0664] Step 1:

[0665] Compound C2A (10.0 g) was dissolved in dichloromethane (200 mL), and n-butyraldehyde (10.4 g) was added to the reaction mixture. The mixture was stirred at room temperature for 10 minutes. Sodium triacetoxyborohydride (25.6 g) was then added portionwise to the reaction mixture, and the mixture was stirred at room temperature for 1 hour. LCMS indicated the reaction was complete. A saturated aqueous solution of ammonium chloride was added to the reaction mixture, and the mixture was stirred for one hour. The mixture was then dried by evaporation. The filtrate was filtered and purified using a C18 column with reversed phase separation (acetonitrile to 0.05% formic acid in water: 5% to 55%) to afford the target compound C4A (4.9 g) as a white solid.

[0666] LCMS (ESI) [M+H] + =492.7;

[0667] 1 H NMR (400MHz, CDCl3) δ7.35–7.30(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).

[0668] Step 2:

[0669] Compound C4A (5.0 g) was dissolved in methanol (100 mL) at room temperature. Pd / C (10%, 1 g) was then added to the reaction mixture, and the mixture was stirred under hydrogen for 12 hours. LCMS indicated the reaction was complete. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to afford the target compound C4B (3.68 g) as a white solid.

[0670] LCMS (ESI) [M+H] + =358.3.

[0671] Step 3:

[0672] The compound 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (1.0 g) was dissolved in N,N-dimethylformamide (15 mL), and then N,N-diisopropylethylamine (1.2 g) and HATU (1.4 g) were added in sequence. The reaction was stirred for 30 minutes, and then compound C4B (1.3 g) was added. The reaction solution was stirred at room temperature for 1 hour. After the reaction was completed by LCMS detection, the reaction solution was directly purified by preparative chromatography (0.01% trifluoroacetic acid aqueous solution, acetonitrile) to obtain the target compound N 6 ,N 6 -Dibutyl-N 2-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (C4, 500 mg) was obtained as a yellow solid.

[0673] LCMS (ESI) [M+H] + =608.7;

[0674] 1H NMR (400MHz, DMSO) δ9.37 (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 ),1.78–1.71(m,1H),1.62–1.56(m,4H),1.34–1.32(m,8H),0.94–0.85(m,12H).

[0675] Example 8. Synthesis of drug-linker compounds

[0676] 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]dioxolano[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-triazahexadec-14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (DL-01)

[0677] Compound C3 (43 mg, 0.074 mmol) and compound B1 (40 mg, 0.075 mmol) were dissolved in N,N-dimethylformamide (1 mL), and then HBTU (28 mg, 0.075 mmol) and N,N-diisopropylethylamine (24 mg, 0.187 mmol) were added sequentially. The reaction solution was stirred at room temperature for 1 hour. After the reaction was completed by LCMS detection, the reaction solution was directly purified by preparative chromatography (0.01% trifluoroacetic acid aqueous solution, acetonitrile) to obtain the target compound (DL-01, 8.5 mg, yield 10%) as a yellow solid.

[0678] LCMS (ESI) [M+H] + =1098.6;

[0679] 1 H NMR (400MHz, DMSO-d6) δ9.10(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).

[0680] 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]dioxolano[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-triazahexadec-14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (DL-02)

[0681] Compound C2 (40 mg, 0.075 mmol) and compound B1 (41 mg, 0.075 mmol) were dissolved in DMF (1 mL), and then HOBt (15.2 mg, 0.113 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (21.5 mg, 0.113 mmol) were added, and then triethylamine (23 mg, 0.225 mmol) was added. After the addition, the reaction solution was stirred at room temperature for 16 hours. After the reaction was completed by LCMS, the reaction solution was concentrated to obtain a crude product. The crude product was purified by preparative chromatography (0.01% TFA in 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]dioxolano[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-triazahexadec-14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (16 mg, yield 20%) was obtained as a yellow solid.

[0682] LCMS (ESI) [M+H] + =1070.6;

[0683] 1 H NMR (400MHz, DMSO-d6) δ9.13–9.08(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=6.5 Hz,1H),4.20–4.10(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).

[0684] 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]dioxolano[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-triazahexadec-14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (DL-03)

[0685] Compound B1 (100 mg, 0.186 mmol), compound N 6 ,N 6 -dimethyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-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 the reaction was complete, the reaction mixture was analyzed by LCMS and purified by preparative chromatography (0.01% TFA in 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]dioxolano[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-triazahexadec-14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (DL-03, 20 mg, yield 10%) was obtained as a yellow solid.

[0686] LCMS (ESI) [M+H] + =1042.5;

[0687] 1H NMR(400MHz,DMSO-d6)δ9.28(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).

[0688] 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-triazahexadec-14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (DL-04)

[0689] Compound C4 (50 mg, 0.08 mmol) and compound B2 (43 mg, 0.08 mmol) were dissolved in N,N-dimethylformamide (1.5 mL). DIPEA (26 mg, 0.21 mmol) and HBTU (31 mg, 0.08 mmol) were then added sequentially. The mixture was stirred at room temperature for 1 hour. LCMS confirmed the reaction was complete, and the reaction solution was directly purified by high-efficiency preparative chromatography (0.01% trifluoroacetic acid in water, acetonitrile) to afford the target compound DL-04 (13.57 mg) as a yellow solid.

[0690] LCMS (ESI) [M+H] + =1114.6;

[0691] 1H NMR (400MHz, DMSO) δ9.09 (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,2 H),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).

[0692] 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-triazapentadecan-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (DL-05)

[0693] Compound N 6 ,N 6 -dimethyl-N 2-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-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 in sequence. After the addition, the reaction solution was stirred at room temperature for 30 minutes, and then 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, 1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-[1,2-b]-1,2-dimethylamino)-1,2-dimethylamino)-1,2-dimethylamino)-1,2-dimethylamino)-2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,2-dimethylamino)-1,2-dimethylamino)-1,2-dimethylamino)-1,2-dimethylamino)-2 ... 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-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide DL-05 (8.1 mg, 7% yield).

[0694] LCMS (ESI) [M+H] + =1085.5;

[0695] 1H NMR (400MHz, DMSO-d6) δ9.31(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,2 H),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).

[0696] Example 9. Preparation of anti-MSLN antibody-drug conjugates

[0697] Antibody Drug Conjugate PR300159-B81, PR300159-8-B81, PR300186-B81, or PR300186-10-B81 Sample Preparation

[0698] Ab was the anti-MSLN antibody PR300159, PR300159-8, PR300186, or PR300186-10.

[0699] 30 mg of anti-MSLN antibody PR300159-8 or PR300186-10 was diluted with diluent (20 mM The solution was diluted with 1% PB (pH 7.5), and a sodium edetate solution was added to a final concentration of 5 mM, mixed. A TCEP solution (5.5 times the equivalent of the antibody) was added, mixed, and allowed to stand at room temperature for 60 minutes. DL-01 dissolved in dimethyl sulfoxide (DMSO) (12 times the equivalent of the antibody) was added to the above solution, mixed, and allowed to stand at room temperature for 2 hours to obtain a coupled sample. After the reaction, the sample was replaced with a 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 containing the ADC conjugates of the anti-MSLN antibody PR300159-8 or PR300186-10, designated PR300159-B81, PR300159-8-B81, PR300186-B81, and PR300186-10-B81, respectively. The DAR value was determined by mass spectrometry as described in Example 10 to be 8.0.

[0700] Antibody Drug Conjugate PR300159-B71 or PR300186-B71 Sample Preparation

[0701] Ab was the anti-MSLN antibody PR300159 or PR300186.

[0702] 30 mg of the anti-MSLN antibody PR300159 or PR300186 was diluted with a diluent (20 mM PB, pH 7.5), and a sodium edetate solution was added to a final concentration of 5 mM, mixed. A TCEP solution (5.5 times the equivalent of the antibody) was added, mixed, and allowed to stand at room temperature for 60 minutes. To this solution, DL-03 (12 times the equivalent of the antibody) dissolved in dimethyl sulfoxide was added, mixed, and allowed to stand at room temperature for 2 hours to obtain a conjugated sample. After the reaction, the sample was replaced with a 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 containing the ADC conjugate of the anti-MSLN antibody PR300159 or PR300186, designated PR300159-B71 and PR300186-B71, respectively. The DAR value was determined by mass spectrometry as described in Example 10 to be 8.0.

[0703] Example 10. Determination of the DAR value of the coupled sample by mass spectrometry

[0704] Detection: PR300159-8-B81 was analyzed by LC-MS molecular weight and DAR value using the following conditions:

[0705] Chromatographic conditions:

[0706] Column: Xbridge Protein BEH SEC (2.5 μm, 4.6*150 mm)

[0707] Mobile phase A: 0.1% FA / H2O; Mobile phase B: 0.1% FA / ACN

[0708] Column temperature: 30°C

[0709] Sample chamber temperature: 8°C

[0710] Flow rate: 0.3 mL / min

[0711] Injection volume: 1 μL

[0712] Table 11. Chromatographic conditions

[0713] Sample treatment: Take 50 μg of sample, add 2 μL of 1 M DTT, add ultrapure water to 50 μL and dilute to a concentration of about 1.0 mg / mL, mix well, and reduce at room temperature for 30 min.

[0714] LC / MS model: UPLC (AB SCIEX), high-resolution mass spectrometer (AB SCIEX)

[0715] Mass spectrometry conditions: Gas1:45; Gas2:45; CUR:30; TEM:450; ISVF:5000; DP:120; CE:12; mass range: 600-4000

[0716] The results are shown in Table 12 below.

[0717] Table 12. Theoretical molecular weight and measured molecular weight

[0718] In Table 12, mAb represents an unconjugated monoclonal antibody; LC represents the antibody light chain; HC represents the antibody heavy chain; DAR1 represents a conjugate comprising a light chain or heavy chain conjugated to one toxin molecule; DAR2 represents a conjugate comprising a light chain or heavy chain conjugated to two toxin molecules; and DAR3 represents a conjugate comprising a light chain or heavy chain conjugated to three toxin molecules. The theoretical molecular weight of the monoclonal antibody is calculated based on the G0F glycoform. Hereinafter, mAb, LC, HC, DAR1, DAR2, and DAR3 are as described above.

[0719] The test results showed that the proportions of antibody light chains coupled to 0 to 1 toxin molecules (LC, DAR1) on PR300159-8-B81 were 0% and 100%, respectively, and the proportions of heavy chains coupled to 0 to 3 toxin molecules (mAb, DAR1, DAR2, DAR3) were 0%, 0%, 0%, and 100.0%, respectively. Based on this, the drug-antibody ratio (DAR value) of PR300159-8-B81 and PR300186-10-B81 was calculated to be 8.0.

[0720] Example 11. Construction of HT29-huMSLN wild-type cell line and HT29-huMSLN mutant cell line

[0721] The HT29-huMSLN wild-type cell line (stable wild-type human MSLN) was prepared in the laboratory, and the specific procedures are as follows.

[0722] HT-29 cell suspensions were plated at a density of 5E5 cells / well in a 6-well plate and incubated overnight at 37°C. A plasmid encoding the human MSLN gene was added to a tube and diluted with Opti-MEM (Gibco, Cat. No. 31985070). The transfection reagent, Lipofectamine 3K, was added to a new tube and diluted in Opti-MEM at a 3:1 ratio of Lipofectamine 3K to DNA. The diluted Lipofectamine 3K was added to the diluted plasmid, mixed, and incubated at room temperature for 15 minutes. The mixture was added to the cells, gently shaken to mix, and incubated at 37°C overnight. The supernatant was then discarded and replaced with fresh culture medium supplemented with 0.3 μg / mL G418. The culture medium was incubated at 37°C, with fresh G418-containing culture medium replaced every three days. After the cells have grown confluently, perform FACS verification. Dilute the verified cell pool to 5 cells / mL and inoculate five 96-well plates at 100 μL / well. Culture at 37°C for 14 days. Pick a single clone for FACS verification. After verification, continue to expand the culture and freeze.

[0723] The HT29-huMSLN mutant cell line (stable mutant human MSLN) was generated in the laboratory. Construction methods were similar to those for the HT29-huMSLN wild-type cell line, with the human mutant MSLN gene being transfected, in which the YLVLDLSV in the wild-type human MSLN gene was mutated to GGGGS.

[0724] Example 12. Detection of MSLN Shedding by HT29-huMSLN Wild-Type 2C3 Cells and HT29-huMSLN Mutant 1A11 Cells

[0725] 5×10 5Seed HT29-huMSLN wild-type 2C3 cells and HT29-huMSLN mutant 1A11 cells into 6-well plates at 1 mL / well. The next day, remove the culture medium and replace it with 1 mL of fresh culture medium. Collect 100 mL of culture medium every hour and store at -80°C for subsequent detection of cell shedding of MSLN.

[0726] PR300159 was diluted to a concentration of 5 μg / mL and added to a 96-well plate, 100 μL per well, and incubated at 4°C overnight. The 96-well plate was washed three times with PBST solution, and then a 2% BSA solution in PBS was added and incubated at 37°C for 1 hour. Human MSLN protein (Acro Biosystems, Catalog #MSN-H522) was diluted two-fold starting from 0.2 μg / mL, with a total of 11 dilution points used as a standard curve. The collected cell supernatant was added to a 96-well plate and incubated at 37°C for 1 hour. After washing three times with PBST solution, the biotinylated anti-MSLN positive control antibody PR300175 (which has a different binding epitope than PR300159, with the heavy and light chain sequences shown as SEQ ID NOs: 78 and 79, respectively) was added and incubated at 37°C for 1 hour. After washing three times with PBST solution, add SA-HRP secondary antibody (5000× dilution) and incubate at 37°C for 30-60 minutes. After washing three times with PBST solution, add TMB color development solution for 5-15 minutes, and then add stop solution to stop color development.

[0727] As shown in FIG10 , compared with the HT29-huMSLN wild-type 2C3 cell line, the HT29-huMSLN mutant 1A11 cell line only exhibited a small amount of MSLN shedding.

[0728] Example 13. Cytotoxicity of PR300159-8-B81 Antibody Drug Conjugate to MSLN-Expressing Cells

[0729] HT29-huMSLN wild-type 2C3 cells were added to a 96-well plate at 2000 cells / well, and then serially diluted PR300159-8, hIgG-B81 (human IgG control linked to DL-01), and PR300159-8-B81 were added. After 6 days, cell viability was detected using Promega (G7572) CellTiter Glo.

[0730] As shown in the experimental results of Figure 11, PR300159-8-B81 showed a significant killing effect on HT-29-huMSLN.

[0731] Example 14. Pharmacokinetic Study of PR300159-8-B81 and PR300186-10-B81 Antibody Drug Conjugates

[0732] Six male SD rats were selected and the antibody drug conjugate was administered intravenously at a dose of 5 mg / kg. Plasma and serum were collected before administration and 10 minutes, 2 hours, 8 hours, 24 hours (day 1), day 3, day 7, day 10, day 14, day 21, and day 28 after administration.

[0733] Collection of plasma samples: Place whole blood in a tube containing K2-EDTA, centrifuge at 2-8°C for 6 minutes, and freeze at around -80°C until analysis.

[0734] Serum samples were collected by allowing whole blood to clot for 30 minutes, then centrifuged at 2200 g for 10 minutes at 4°C and the separated serum samples were frozen at -80°C until analysis.

[0735] Detection of Antibody-Drug Conjugates (Bound Antibodies): This example uses an ELISA method to quantitatively determine the concentration of antibody-drug conjugates in mouse serum, as follows. The antibody-drug conjugates in mouse serum are captured using a mouse anti-toxin A1 Fab monoclonal antibody coated in a 96-well plate. Detection is then performed using an HRP-labeled goat anti-human Fc secondary antibody.

[0736] Total Antibody Detection: This example uses an ELISA method to quantitatively determine the total antibody concentration in mouse serum. The ELISA method uses a sheep anti-human Fc monoclonal antibody coated on a 96-well plate to capture the antibodies in mouse serum, and then adds an HRP-labeled goat anti-human IgG secondary antibody for detection.

[0737] Detection of toxin molecule A1: In this example, the LC-MS / MS method was used to detect the concentration of small molecules in serum. 100 μL of acetonitrile containing 0.5 ng / mL internal standard (YL0010098) was added to 30 μL of plasma sample to precipitate the protein. The mixture was vortexed for 1 minute and centrifuged. The supernatant was analyzed by LC-MS / MS (Waters XEVO TQ-XS).

[0738] Phoenix WinNonlin software version 7.0 was used to analyze the plasma drug concentration data using the non-compartmental model (NCA) to calculate its pharmacokinetic parameters.

[0739] Table 13 shows the pharmacokinetic parameters of PR300159-8-B81 and PR300186-10-B81. The results showed that after administration of PR300159-8-B81 and PR300186-10-B81, the half-lives of the total antibody of PR300159-8-B81 and PR300186-10-B81 in rats were 203 and 232 hours, respectively, while the half-lives of the antibody-drug conjugates (ADCs) of PR300159-8-B81 and PR300186-10-B81 in rats were 193 and 250 hours, respectively.

[0740] Table 13. Pharmacokinetic parameters of PR300159-8-B81 and PR300186-10-B81

[0741] Example 15. Stability Study of PR300159-8-B81 and PR300186-10-B81 Antibody Drug Conjugates in Human, Cynomolgus Monkey, SD Rat, and ICR Mouse Plasma

[0742] The antibacterial agent Proclin was added to plasma from various species (human, cynomolgus macaque, SD rat, ICR mouse) and PBS containing 1% BSA to a final concentration of 0.1%. Aseptic operation was performed at 37°C. The stock solution of the antibody-drug conjugate was diluted to a working solution of 20 mg / mL using PBS or 1 mM EDTA buffer, pH 7. 4 μL of the working solution of PR300159-8-B81 or PR300186-10-B81 was mixed with 396 μL of plasma from various species (human, cynomolgus macaque, SD rat, or ICR mouse) or PBS containing 1% BSA to prepare plasma samples of 200 μg / mL PR300159-8-B81 or PR300186-10-B81. The amount of organic solvent did not exceed 1%. The samples were incubated at 37°C in a 5% CO2 incubator, sealed with film, and shaken at 80 times / minute. Incubate for 0 hours, 4 hours, 24 hours, 2 days, 3 days, 7 days, 10 days, 14 days, 17 days, 21 days, and 28 days. Transfer 20 μL of plasma sample, add 200 μL of precipitant (including internal standard), and mix thoroughly to precipitate protein. After protein precipitation, all samples were centrifuged, the supernatant was collected, diluted with water, and analyzed by LC-MS / MS.

[0743] FIG12 and FIG13 show the stability results of PR300159-8-B81 and PR300186-10-B81 in human, cynomolgus monkey, SD rat, ICR mouse plasma and phosphate buffered saline (PBS).

[0744] The results in FIG12 show that within 28 days, the release percentage of the cytotoxic drug in PR300159-8-B81 in the plasma of humans and cynomolgus monkeys was less than 0.5%, the release percentage in the plasma of SD rats was less than 1.5%, and the release percentage in the plasma of ICR mice was less than 2.5%.

[0745] The results in FIG13 show that within 28 days, the percentage of release of the cytotoxic drug in PR300186-10-B81 in the plasma of humans and cynomolgus monkeys was less than 1.0%, the percentage of release in the plasma of SD rats was less than 2.5%, and the percentage of release in the plasma of ICR mice was less than 4.0%.

[0746] Example 16. Colon cancer HT29-huMSLN wild-type cell line model

[0747] 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 and 5% CO2. HT29-huMSLN wild-type 2C3 cells were harvested during the exponential growth phase on the day of inoculation. Tumor cells were resuspended in a 1:1 mixture of PBS and Matrigel and then inoculated subcutaneously. 1×10 tumor cells were inoculated subcutaneously per BALB / c nude mouse. 6 HT29-huMSLN wild-type 2C3 cells. When the average tumor volume of each group of mice was 100 mm 3 30 mice were randomly divided into 6 groups and the drug was administered after grouping. The administration cycle was twice a week for a total of 6 doses. The administration method was tail vein injection. The dosage was 5 mg / kg body weight or 1 mg / kg body weight. After the start of the drug administration, the body weight and tumor volume were measured twice a week. The tumor volume was calculated as follows: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 The experimental observation was terminated on day 38 after administration, and all mice were euthanized. Data were analyzed using the t-test.

[0748] Figure 14 shows the results of in vivo pharmacodynamic studies of PR300159-8-B81 and PR300186-10-B81. On day 38 after administration, the average tumor volume of mice in the vehicle control group was 1131 mm 3 The average tumor volume of mice in the hIgG1-B81 (5 mg / kg) control group was 934 mm on day 38 after administration. 3 The average tumor volume of the test drug PR300159-8-B81 (1 mg / kg) treatment group was 34 mm on day 38 after administration. 3, compared with the vehicle control group, there was a significant difference (p value <0.001), and the tumor inhibition rate TGI was 97 (%). In the test drug PR300159-8-B81 (5 mg / kg) treatment group, the mouse tumors were completely eliminated on the 38th day after administration, which was significantly different from the vehicle control group (p value <0.001), and the tumor inhibition rate TGI (%) was 100%. In the test drug PR300186-10-B81 (1 mg / kg) treatment group, the average tumor volume was 168 mm on the 38th day after administration. 3 , showing a significant difference compared to the vehicle control group (p value < 0.001), with a tumor inhibition rate (TGI) of 85%. The test drug PR300186-10-B81 (5 mg / kg) treatment group completely eliminated tumors in mice on day 38 after administration, demonstrating a significant tumor inhibition effect compared to the vehicle control group (p value < 0.001), with a tumor inhibition rate (TGI) of 100%.

[0749] Figure 15 shows the body weight results of mice. Throughout the treatment period, the animals showed good drug tolerance, and no severe weight loss or animal death occurred.

[0750] Example 17. Colon cancer HT29-huMSLN mutant cell line model

[0751] 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 and 5% CO2. HT29-huMSLN mutant 1A11 cells were collected in the exponential growth phase on the day of inoculation and 1×10 cells were subcutaneously inoculated into each BALB / c nude mouse. 6 HT29-huMSLN mutant 1A11 cells. Tumor cells were first resuspended in a mixture of PBS and Matrigel (1:1) and then inoculated subcutaneously. When the average tumor volume of each group of mice was 100 mm 3 The mice were divided into 5 groups and the administration was started after the grouping. The administration cycle was twice a week for a total of 6 times. The administration method was tail vein injection. The dosage was 1 mg / kg body weight or 5 mg / kg body weight. After the start of the administration, the body weight and tumor volume were measured twice a week. The tumor volume was calculated as follows: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 The experimental observation was terminated on day 32 after administration, and all mice were euthanized. Data were analyzed using the t-test.

[0752] Figure 16 shows the results of the study on the anti-tumor effects of PR300159-8-B81 and PR300186-10-B81. Figure 17 shows the body weight of mice.

[0753] As shown in Figure 16, on day 32 after administration, the average tumor volume of mice in the control group was 1109 mm 3 The average tumor volume of the test drug PR300159-8-B81 (1 mg / kg) treatment group was 60.4 mm on day 32 after administration. 3 , compared with the vehicle control group, there was a significant difference (p value <0.001), and the tumor inhibition rate TGI was 95%. The test drug PR300159-8-B81 (5 mg / kg) treatment group completely eliminated the tumor of mice on the 32nd day after administration, which was significantly different from the vehicle control group (p value <0.001), and the tumor inhibition rate TGI (%) was 100%. The test drug PR300186-10-B81 (1 mg / kg) treatment group had an average tumor volume of 59.5 mm on the 32nd day after administration. 3 , with a significant difference compared to the vehicle control group (p value < 0.001), and a tumor inhibition rate (TGI) of 95%. The test drug PR300186-10-B81 (5 mg / kg) treatment group completely eliminated the tumors in mice on day 32 after administration, showing a significant tumor inhibition effect compared to the vehicle control group (p value < 0.001), with a tumor inhibition rate (TGI) of 100%. This shows that PR300159-8-B81 and PR300186-10-B81 have equivalent anti-tumor effects in the HT29-huMSLN mutant cell model in vivo.

[0754] As shown in FIG17 , during the entire treatment period, the animals showed good drug tolerance, and no severe weight loss or animal death occurred.

[0755] Example 18. Ovarian Cancer COV644 Model

[0756] COV644 (ECACC, catalog: 07071908) cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C under 5% CO2 conditions. COV644 cells were harvested during the exponential growth phase on the day of inoculation. Tumor cells were resuspended in a mixture of PBS and Matrigel (1:1) and then inoculated subcutaneously. 5×10 cells were inoculated subcutaneously per BALB / c nude mouse. 6 COV644 tumor cells. When the average tumor volume of each group of mice was 150mm 3 The mice were divided into 5 groups at 37 ℃ and 1 hour after treatment. The mice were divided into 5 groups at 37 ℃ and 1 hour after treatment. The treatment period was twice a week for 5 times. The treatment was done by tail vein injection at a dose of 1 mg / kg body weight or 5 mg / kg body weight. After the start of treatment, the body weight and tumor volume were measured twice a week. The tumor volume was calculated as follows: tumor volume (mm 3) = 0.5 × tumor long diameter × tumor short diameter 2 The experimental observation was terminated on day 38 after administration, and all mice were euthanized. Data were analyzed using the t-test.

[0757] Figure 18 shows the results of in vivo pharmacodynamic studies of PR300159-8-B81 and PR300186-10-B81. On day 38 after administration, the average tumor volume of mice in the vehicle control group was 1429 mm 3 The average tumor volume of the test drug PR300159-8-B81 (1 mg / kg) treatment group was 993 mm on day 38 after administration. 3 , the tumor inhibition rate TGI (%) was 31%. The test drug PR300159-8-B81 (5 mg / kg) treatment group completely eliminated the tumor on the 38th day after administration, which was significantly different from the solvent control group (p value <0.001), and the tumor inhibition rate TGI (%) was 100%. The test drug PR300186-10-B81 (1 mg / kg) treatment group had an average tumor volume of 995 mm on the 38th day after administration. 3 The tumor inhibition rate (TGI) was 30%. In the test drug PR300186-10-B81 (5 mg / kg) treatment group, the tumors of 4 mice in the group were completely eliminated on the 38th day after administration, and the tumor volume of 1 mouse was 14 mm 3 The average tumor volume of this group of mice was 8 mm 3 Compared with the vehicle control group, PR300159-8-B81 showed significant tumor inhibition (p < 0.001), with a tumor inhibition rate (TGI) of 99%. PR300159-8-B81 and PR300186-10-B81 had equivalent anti-tumor effects in the COV644 in vivo model.

[0758] Figure 19 shows the results of mouse body weight. Throughout the treatment period, the animals showed good drug tolerance, and no severe weight loss or animal death occurred.

[0759] Example 19. Ovarian cancer OVCAR3 model

[0760] OVCAR3 cells were cultured in RPMI-1640 medium (containing 20% ​​fetal bovine serum and 0.01 mg / mL bovine insulin) at 37°C and 5% CO2. OVCAR3 cells were harvested during the exponential growth phase on the day of inoculation. Tumor cells were resuspended in a mixture of PBS and Matrigel (1:1) and then inoculated subcutaneously. 1×10 tumor cells were inoculated subcutaneously per BALB / c nude mouse. 7 CVCAR3 tumor cells, when the average tumor volume of each group of mice was 200 mm 3The mice were divided into 7 groups at 35 pm. The drug was administered twice a week for 5 times in total. The dosage was 1 mg / kg or 5 mg / kg body weight by tail vein injection. After the start of drug administration, the body weight and tumor volume were measured twice a week. The tumor volume was calculated as follows: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 The experimental observation was terminated on the 23rd day after administration, and all mice were euthanized. Data were analyzed using the t-test.

[0761] Among them, PR300159-8-DXD was synthesized by Shanghai Ruizhi Chemical Research Co., Ltd. with reference to the synthesis method of trastuzumab deruxtecan (DS-8201, Enhertu) (WO2015115091A1), replacing trastuzumab in DS-8201 with PR300159-8.

[0762] Figure 20 shows the results of in vivo pharmacodynamic studies of PR300159-8-DXD, PR300159-8-B81, and PR300186-10-B81. On day 23 after administration, the average tumor volume of mice in the control group was 1811 mm 3 The average tumor volume of the PR300159-8-DXD (1 mg / kg) treatment group was 51 mm on day 23 after administration. 3 , compared with the vehicle control group, there was a significant difference (p value was 0.001), and the tumor inhibition rate TGI (%) was 97%. The average tumor volume of the test drug PR300159-8-DXD (5mg / kg) treatment group on the 23rd day after administration was 47mm 3 Compared with the vehicle control group, there was a significant difference (p value was 0.001), and the tumor inhibition rate TGI (%) was 97%. The average tumor volume of the test drug PR300159-8-B81 (1 mg / kg) treatment group on the 23rd day after administration was 35 mm 3 , compared with the vehicle control group, there was a significant difference (p value was 0.001), and the tumor inhibition rate TGI (%) was 98%. The average tumor volume of the test drug PR300159-8-B81 (5 mg / kg) treatment group on the 23rd day after administration was 54 mm 3 Compared with the vehicle control group, there was a significant difference (p value was 0.001), and the tumor inhibition rate TGI (%) was 97%. The average tumor volume of the test drug PR300186-10-B81 (1 mg / kg) treatment group on the 23rd day after administration was 429 mm 3Compared with the vehicle control group, the PR300186-10-B81 (1 mg / kg) treatment group had a significant tumor inhibition effect (p value was 0.002), with a tumor inhibition rate (TGI) of 76%. The average tumor volume of the test drug PR300186-10-B81 (1 mg / kg) treatment group on day 23 after administration was 67 mm 3 Compared with the vehicle control group, PR300159-8-DXD, PR300159-8-B81, and PR300186-10-B81 exhibited significant antitumor effects at both 1 mg / kg and 5 mg / kg, with PR300186-10-B81 exhibiting a clear dose-dependent effect.

[0763] Figure 21 shows the results of mouse body weight. Throughout the treatment period, the animals showed good drug tolerance, and no severe weight loss or animal death occurred.

[0764] Example 20. Ovarian cancer SKOV3 model

[0765] SKOV3 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C under 5% CO2 conditions. SKOV3 cells in the exponential growth phase were harvested on the day of inoculation. Tumor cells were resuspended in a mixture of PBS and Matrigel (1:1) and then inoculated subcutaneously. 1×10 cells were inoculated subcutaneously per BALB / c nude mouse. 7 SKOV3 tumor cells, when the average tumor volume of each group of mice was 200 mm 3 The mice were divided into 7 groups and the administration was started after the grouping. The administration cycle was twice a week for a total of 4 times. The administration method was tail vein injection. After the start of the administration, the body weight and tumor volume were measured twice a week. The tumor volume was calculated as follows: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 The experimental observation was terminated on day 21 after administration, and all mice were euthanized. Data were analyzed using the t-test.

[0766] Figure 22 shows the results of in vivo pharmacodynamic studies of PR300159-8-B81 and PR300186-10-B81. On day 21 after administration, the average tumor volume of mice in the control group was 2011 mm 3 The average tumor volume of the test drug PR300159-8-B81 (1.28 mg / kg) treatment group was 657 mm on day 21 after administration. 3Compared with the vehicle control group, there was a significant difference (p value < 0.001), and the tumor inhibition rate TGI (%) was 67%. The average tumor volume of the test drug PR300159-8-B81 (6.4 mg / kg) treatment group on the 21st day after administration was 261 mm 3 Compared with the vehicle control group, there was a significant difference (p value < 0.001), and the tumor inhibition rate TGI (%) was 87%. The average tumor volume of the test drug PR300159-8-B81 (12.8 mg / kg) treatment group on the 21st day after administration was 122 mm 3 Compared with the vehicle control group, there was a significant difference (p value < 0.001), and the tumor inhibition rate TGI (%) was 94%. The average tumor volume of the test drug PR300186-10-B81 (0.78 mg / kg) treatment group on the 21st day after administration was 1064 mm 3 Compared with the vehicle control group, the PR300186-10-B81 (3.9 mg / kg) treatment group had a significant tumor inhibition effect (p value < 0.001), with a tumor inhibition rate (TGI) of 47%. The average tumor volume of the PR300186-10-B81 (3.9 mg / kg) treatment group on day 21 after administration was 478 mm. 3 Compared with the vehicle control group, the PR300186-10-B81 (7.8 mg / kg) treatment group had a significant tumor inhibition effect (p value < 0.001), with a tumor inhibition rate (TGI) of 76%. The average tumor volume of the PR300186-10-B81 (7.8 mg / kg) treatment group on day 21 after administration was 265 mm 3 Compared with the vehicle control group, PR300159-8-B81 and PR300186-10-B81 showed significant tumor inhibition (p<0.001), with a tumor inhibition rate (TGI) of 87%. PR300159-8-B81 and PR300186-10-B81 had significant anti-tumor effects in a dose-dependent manner.

[0767] Figure 23 shows the results of mouse body weight. Throughout the treatment period, the animals showed good drug tolerance, and no severe weight loss or animal death occurred.

[0768] Example 21. Lung cancer NCI-H226 model

[0769] NCI-H226 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C under 5% CO2 conditions. NCI-H226 cells were harvested during the exponential growth phase on the day of inoculation. Tumor cells were first resuspended in a 1:1 mixture of PBS and Matrigel and then inoculated subcutaneously. 5×10 cells were inoculated subcutaneously per BALB / c nude mouse. 6 NCI-H226 tumor cells. When the average tumor volume of each group of mice was 150mm 3The mice were divided into 11 groups and the drug was administered twice a week for 5 times. The dosage was 1 mg / kg or 5 mg / kg body weight by tail vein injection. After the start of the drug administration, the body weight and tumor volume were measured twice a week. The tumor volume was calculated as follows: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 The experimental observation was terminated on day 28 after administration, and all mice were euthanized. Data were analyzed using the t-test.

[0770] BAY-949343 is Anetumab ravtansine, an ADC developed by Bayer. It consists of the fully human anti-MSLN monoclonal antibody MF-T (anetumab) linked to the microtubule inhibitor maytansine DM4 via amino conjugation. The linker is N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), which has a pericytic effect (WO2010124797A1). BAY-949343 was synthesized by Shanghai Ruizhi Chemical Research Co., Ltd.

[0771] Figures 24 and 25 show the results of in vivo pharmacodynamic studies of PR300159-8-DXD, BAY-949343, PR300159-8-B81, and PR300186-10-B81. On day 28 after administration, the average tumor volume of mice in the vehicle control group was 1535 mm 3 The average tumor volume of the PR300159-8-DXD (1 mg / kg) treatment group was 743 mm on day 28 after administration. 3 Compared with the vehicle control group, there was a significant difference (p value was 0.05), and the tumor inhibition rate TGI (%) was 52%. The average tumor volume of the test drug PR300159-8-DXD (5mg / kg) treatment group on the 28th day after administration was 460mm 3 , there was a significant difference compared with the vehicle control group (p value was 0.01), and the tumor inhibition rate TGI (%) was 70%. The average tumor volume of the test drug BAY-949343 (1 mg / kg) treatment group on day 28 after administration was 497 mm 3 , there was a significant difference compared with the vehicle control group (p value was 0.01), and the tumor inhibition rate TGI (%) was 68%. The average tumor volume of the test drug BAY-949343 (5mg / kg) treatment group on day 28 after administration was 253mm 3Compared with the vehicle control group, there was a significant difference (p value was 0.004), and the tumor inhibition rate TGI (%) was 84%. The average tumor volume of the test drug PR300159-8-B81 (1 mg / kg) treatment group on day 28 after administration was 556 mm 3 Compared with the vehicle control group, there was a significant difference (p value was 0.02), and the tumor inhibition rate TGI (%) was 64%. The average tumor volume of the test drug PR300159-8-B81 (5 mg / kg) treatment group on the 28th day after administration was 164 mm 3 Compared with the vehicle control group, there was a significant difference (p value was 0.003), and the tumor inhibition rate TGI (%) was 89%. The average tumor volume of the test drug PR300186-10-B81 (1 mg / kg) treatment group on day 28 after administration was 526 mm 3 Compared with the vehicle control group, the PR300186-10-B81 (5 mg / kg) treatment group had a significant tumor inhibition effect (p value was 0.01), with a tumor inhibition rate (TGI) of 66%. The average tumor volume of the test drug PR300186-10-B81 (5 mg / kg) treatment group on day 28 after administration was 211 mm 3 Compared with the vehicle control group, the drug showed a significant tumor inhibition effect (p = 0.003), with a tumor inhibition rate (TGI) of 86%. PR300159-8-B81, PR300186-10-B81, and BAY-949343 had equivalent anti-tumor effects, and all showed superior anti-tumor effects to PR300159-8-DXD.

[0772] Figures 26 and 27 show the results of mouse body weight. Throughout the treatment period, the animals showed good drug tolerance, and no severe weight loss or animal death occurred.

[0773] Example 22. Binding activity assay of PR300159, PR300159-B71, PR300186 and PR300186-B71 on CHOK1 cells overexpressing human mesothelin (CHOK1-huMSLN)

[0774] Flow cytometry was used to detect the binding activity of PR300159, PR300186 antibodies and PR300159-B71, PR300186-B71 antibody drug conjugates (ADCs) on CHOK1-huMSLN cells.

[0775] PR300159, PR300186 antibodies and PR300159-B71, PR300186-B71 ADC were serially diluted in a buffer solution (PBS containing 2% FBS). 50 μL of the diluted antibody (30 μg / ml) was added to a 1-2×10 5The cells were suspended in 50 μL of the cell suspension and incubated at 4°C for 1 hour. The cells were washed twice with a buffer solution (PBS containing 2% serum) and 100 μL of fluorescently labeled anti-human IgG antibody (Alexa Fluor 488 affinity pure goat anti-human IgG (H+L), Jackson ImmunoResearch, Catalog 109-545-088). After incubation at 4°C for 1 hour, the cells were washed twice with buffer and analyzed by flow cytometry.

[0776] The experimental results are shown in Table 14 and Figure 28. The experimental results show that antibodies PR300159 and PR300186 and their corresponding ADCs PR300159-B71 and PR300186-B71 have comparable binding activity on cells.

[0777] Table 14. Binding activity of antibodies or ADCs to cell surface MSLN detected by FACS

[0778] Example 23. Binding activity experiments of PR300159, PR300159-B81, PR300186 and PR300186-B81 on tumor cells

[0779] Flow cytometry was used to detect the binding activity of PR300159, PR300186 antibodies and PR300159-B81, PR300186-B81 antibody drug conjugates (ADCs) on tumor cells. The tumor cells used in this example were breast cancer cell line COV644.

[0780] PR300159, PR300186 antibodies and PR300159-B81, PR300186-B81 ADC were serially diluted in a buffer solution (PBS containing 2% FBS). 50 μL of the diluted antibody solution was added to a solution containing 1-2×10 5 The cells were incubated in 50 μL of a cell suspension of COV644 cells at 4°C for 1 hour. The cells were washed twice with a buffer solution (PBS containing 2% serum) and 100 μL of fluorescently labeled anti-human IgG antibody (Alexa Fluor 488 affinity pure goat anti-human IgG (H+L), Jackson ImmunoResearch, Catalog 109-545-088). After incubation at 4°C for 1 hour, the cells were washed twice with buffer and analyzed by flow cytometry.

[0781] The experimental results are shown in Table 15 and Figure 29. The experimental results show that the antibody and its corresponding ADC have considerable binding activity on COV644 cells.

[0782] Table 15. Binding activity of antibodies and their ADCs to COV644 cells

[0783] Example 24. Endocytic activity of PR300159, PR300159-B71, PR300186 and PR300186-B71 on tumor cells

[0784] Flow cytometry was used to detect the endocytosis efficiency of PR300159 and PR300186 antibodies as well as PR300159-B71 and PR300186-B71 in tumor cells COV644. The tumor cells used in this example were breast cancer cell line COV644.

[0785] In this example, pHAb Amine Reactive Dye (Promega, Cat# G9841) was used to determine the endocytosis efficiency of anti-MSLN antibodies on tumor cells COV644. pHAb dye is a pH sensor dye that has very low fluorescence when the pH value is greater than 7. When the pH value of the solution becomes lower, the fluorescence increases significantly. When the antibody labeled with pHAb dye binds to the cell outer membrane under neutral pH conditions, no fluorescence or very low fluorescence is detected. After the pHAb dye-labeled antibody is internalized by the cell, strong fluorescence is detected in the low pH environment of the cell's endosomes and lysosomes.

[0786] The antibody was labeled with pHAb dye, and the DAR value was calculated according to the kit instructions. The pHAb dye-labeled antibody was incubated with COV644 at 4°C (the antibody internalization activity was very low at this temperature and was used as a background control) or 37°C for 24 hours. Fluorescence was then detected at an excitation light value (Ex) of 532nm and an emission light value (Em) of 560nm. The antibody internalization results were calculated as follows: the fluorescence intensity at 37°C minus the fluorescence intensity at the background at 4°C, divided by the DAR value of the antibody-conjugated dye. Higher fluorescence values ​​indicate higher internalization efficiency of the antibody on the cell.

[0787] The experimental results are shown in Figure 30. The experimental results show that PR300159, PR300159-B71, PR300186 and PR300186-B71 all have good endocytic activity on tumor cells COV644.

[0788] Example 25. Experimental study of PR300159 and PR300186 antibody drug conjugates on cells expressing MSLN

[0789] HT29-huMSLN were seeded at 2000 cells / well in a 96-well plate, and serially diluted PR300159, PR300159-B71, PR300186, PR300186-B71, or hIgG-B71 were added. Cell viability was measured 6 days later using Promega (G7572) CellTiter Glo.

[0790] The experimental results are shown in Figure 31. The experimental results show that PR300159-B71 and PR300186-B71 have a significant killing effect on CHOK1-huMSLN.

[0791] Example 26. Human ovarian cancer COV644 model

[0792] COV644 (ECACC, catalog: 07071908) cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. COV644 cells in the exponential growth phase were collected on the day of inoculation and 5×10 cells were subcutaneously inoculated into each BALB / c nude mouse. 6 COV644 tumor cells. Tumor cells were first resuspended in a mixture of PBS and Matrigel (1:1) and then inoculated subcutaneously. When the average tumor volume of each group of mice was 150 mm 3 The mice were divided into 13 groups and the administration was started after the grouping. The administration cycle was twice a week for a total of 5 times. The administration method was tail vein injection. After the start of the administration, the body weight and tumor volume were measured twice a week. The tumor volume was calculated as follows: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 The experimental observation was terminated on day 21 after administration, and all mice were subsequently euthanized. Data were analyzed using the t-test.

[0793] Figures 32A and 32B show the results of in vivo pharmacodynamic studies of PR300159-B71, PR300159-B81, and PR300186-B81. On day 21 after administration, the average tumor volume of the control group mice was 3154.82 mm 3 The average tumor volume of the test drug IgG-B81 (1 mg / kg) treatment group was 1508 mm on day 21 after administration. 3 There was no significant difference compared with the vehicle control group (p value was 0.82), and the tumor inhibition rate TGI (%) was 2.57%. The average tumor volume of the test drug IgG-B81 (5 mg / kg) treatment group on day 21 after administration was 1514.88 mm 3Compared with the vehicle control group, there was a significant difference (p value < 0.001), and the tumor inhibition rate TGI (%) was 51.98%. The average tumor volume of the test drug PR300159-B71 (1 mg / kg) treatment group on the 21st day after administration was 853.97 mm 3 , compared with the vehicle control group, there was a significant difference (p value <0.001), and the tumor inhibition rate TGI (%) was 72.93%. The test drug PR300159-B71 (5 mg / kg) treatment group completely eliminated the tumor on the 21st day after administration, which was significantly different from the vehicle control group (p value <0.001), and the tumor inhibition rate TGI (%) was 100%. The test drug PR300159-B81 (1 mg / kg) treatment group had an average tumor volume of 1356.25 mm on the 21st day after administration. 3 , compared with the vehicle control group, there was a significant difference (p value <0.001), and the tumor inhibition rate TGI (%) was 57.01%. The test drug PR300159-B81 (5 mg / kg) treatment group completely eliminated the tumor on the 21st day after administration, which was significantly different from the vehicle control group (p value <0.001), and the tumor inhibition rate TGI (%) was 100%. The test drug PR300186-B81 (1 mg / kg) treatment group had an average tumor volume of 1544.39 mm on the 21st day after administration. 3 There was no significant difference compared with the vehicle control group (p value < 0.001), and the tumor inhibition rate TGI (%) was 51.05%. The average tumor volume of the test drug PR300186-B81 (5 mg / kg) treatment group on day 21 after administration was 500.16 mm 3 Compared with the vehicle control group, there was a significant difference (p value < 0.001), and the tumor inhibition rate (TGI) was 84.15%. PR300159-B71, PR300159-B81, and PR300186-B81 had a good anti-tumor effect in the COV644 in vivo model.

[0794] Throughout the treatment period, the animals showed good drug tolerance and no severe weight loss or animal death occurred.

Claims

1. An antibody drug conjugate, which is a compound of formula (I) or its tautomers, stereoisomers or pharmaceutically acceptable salts: Ab-[SX-(Y) n -ZED] q (I) in, Ab is an antibody or antigen-binding fragment thereof that specifically binds to mesothelin (MSLN); S is the sulfide bond connecting Ab and X; X is selected from C 3-10 Cycloalkyl, 5-10 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl; X is optionally substituted with 1, 2, 3, 4 or 5 R*; R* is selected from H, deuterium, halogen, CN, NO2, OH, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl; Y is -Y1-Y2-Y3-, where Y is connected to X through Y1 and to Z through Y3; in, Y1 is selected from -C(O)-, -C(O)O-, -OC(O)-, -C(O)NH-, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 5-10 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl; Y2 is a bond, or C 1-6 alkylene; Y3 is selected from -C(O)-, -OC(O)- or -NHC(O)-; Y is optionally substituted with 1, 2, 3, 4 or 5 R#; R# is selected from H, deuterium, halogen, CN, NO2, OH, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl; Z is a peptide comprising 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 bioactive molecular drug or its derivative; 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.

2. The antibody drug conjugate according to claim 1, wherein X is selected from C 6-10 Aryl or 5-10 membered heteroaryl; X is optionally substituted with 1, 2, 3, 4 or 5 R*; R* is selected from H, deuterium, halogen, CN, NO2, OH, C 1-6 alkyl; Preferably, X is a 5-6 membered heteroaryl group; X is optionally substituted with 1, 2 or 3 R*; R* is selected from H, deuterium, halogen or C 1-4 alkyl; More preferably, X is a pyrimidinyl group, for example 3. The antibody drug conjugate according to claim 1 or 2, wherein Y is -Y1-Y2-Y3-, where Y is connected to X through Y1 and to Z through Y3; in, Y1 is selected from C 2-6 Alkenyl or C 2-6 Alkynyl; Y2 is a bond, or C 1-6 alkylene; Y3 is selected from -C(O)- or -NHC(O)-; Y is optionally substituted with 1, 2, 3, 4 or 5 R#; R# is selected from H, deuterium, halogen, CN, NO2, OH, C 1-6 alkyl; Preferably, Y is -Y1-Y2-Y3-, where Y is connected to X through Y1 and to Z through Y3; in, Y1 is selected from C 2-4 Alkenyl or C 2-4 Alkynyl; Y2 is C 1-4 alkylene; Y3 is -C(O)-; Y is optionally substituted with 1, 2 or 3 R#; R# is selected from H, deuterium, halogen or C 1-4 alkyl; More preferably, Y is 4. The antibody drug conjugate according to any one of claims 1 to 3, wherein 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, va l-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 above dipeptide, tripeptide, tetrapeptide modified by substitution; Preferably, Z is R1 and R2 are independently selected from H, deuterium, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl; More preferably, Z is R1 and R2 are independently selected from H, deuterium, halogen or C 1-6 Alkyl, preferably H or C 1-4 alkyl.

5. The antibody-drug conjugate according to any one of claims 1 to 4, wherein: E is absent or selected from -NH-C 1-6 Alkylene- or -OC 1-6 Alkylene-, the -NH-C 1-6 Alkylene and -OC 1-6 Alkylene- may optionally be selected from H, halogen, C 1-6 Alkyl and C 1-6 substituted by 1, 2 or 3 substituents in a haloalkyl group; Preferably, E is absent or is -NH-C 1-4 Alkylene-, the -NH-C 1-4 Alkylene- may optionally be selected from H, halogen, C 1-4 Alkyl and C 1-4 substituted by 1, 2 or 3 substituents in a haloalkyl group; More preferably, E is -NH-(CH2) 1-4 -, for example -NH-CH2-.

6. The antibody drug conjugate according to any one of claims 1 to 5, wherein D is selected from metal complexes; glycopeptide antibiotics; DNA topoisomerase inhibitors; drugs that interfere with DNA synthesis; drugs that act on structural proteins; tumor signaling pathway inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor angiogenesis inhibitors; cell cycle protein inhibitors; serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors or histidine kinase inhibitors; and other active substances that inhibit tumor cell growth and promote tumor cell apoptosis or necrosis; Preferably, D is selected from oxaliplatin; bleomycin or bleomycin; camptothecin, camptothecin derivatives (such as 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; maytansine derivatives; calicheamicin derivatives; auristatin derivatives; pyrrolobenzodiazepine dimer derivatives; melphalan; mitomycin C; or chlorambucil; More preferably, D is in, R D absent, or selected from -NH- or -O-; L D Selected from -C 0-6 Alkylene-OC 0-6 Alkylene- or -C 0-6 Alkylene-OC 0-6 Alkylene-C(O)NH-CHR D4 -; R D1 and R D2 independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy; or, R D1 、R D2 Together with the carbon atoms to which they are attached, they form a ring, e.g. 6-10 Aryl, 6-10 membered heteroaryl, C 3-7 Cycloalkyl or 3-7 membered heterocyclic group, preferably 5-6 membered heterocyclic group; R D3 and R D4 independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy; or, R D3 、R D4 Together with the carbon atoms to which they are attached, they form a ring, e.g., C 6-10 Aryl, 5-10 membered heteroaryl, C 5-10 Cycloalkyl or 5-10 membered heterocyclic group, preferably C 5-6 Cycloalkyl; More preferably, D is selected from:

7. The antibody drug conjugate according to any one of claims 1 to 6, wherein X is selected from C 6-10 Aryl or 5-10 membered heteroaryl; X is optionally substituted with 1, 2, 3, 4 or 5 R*; R* is selected from H, deuterium, halogen, CN, NO2, OH, C 1-6 alkyl; Y is -Y1-Y2-Y3-, where Y is connected to X through Y1 and to Z through Y3; in, Y1 is selected from C 2-6 Alkenyl or C 2-6 Alkynyl; Y2 is a bond, or C 1-6 alkylene; Y3 is selected from -C(O)- or -NHC(O)-; Y is optionally substituted with 1, 2, 3, 4 or 5 R#; R# is selected from 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-gly, val-gly-gly, val-gln-gly, val-glu-gly, phe-lys-gly, leu-lys-gly, gly-val-lys-gly, va l-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 above dipeptide, tripeptide, tetrapeptide modified by substitution; E is absent or selected from -NH-C 1-6 Alkylene- or -OC 1-6 Alkylene-, the -NH-C 1-6 Alkylene and -OC 1-6 Alkylene- may optionally be selected from H, halogen, C 1-6 Alkyl and C 1-6 substituted by 1, 2 or 3 substituents in a haloalkyl group; D is selected from metal complexes; glycopeptide antibiotics; DNA topoisomerase inhibitors; drugs that interfere with DNA synthesis; drugs that act on structural proteins; tumor signaling pathway inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor angiogenesis inhibitors; cell cycle protein inhibitors; serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors or histidine kinase inhibitors; and other active substances that inhibit tumor cell growth and promote tumor cell apoptosis or necrosis.

8. The antibody drug conjugate according to any one of claims 1 to 7, wherein X is a 5-6 membered heteroaryl group; X is optionally substituted with 1, 2 or 3 R*; R* is selected from H, deuterium, halogen or C 1-4 alkyl; Y is -Y1-Y2-Y3-, where Y is connected to X through Y1 and to Z through Y3; in, Y1 is selected from C 2-4 Alkenyl or C 2-4 Alkynyl; Y2 is C 1-4 alkylene; Y3 is -C(O)-; Y is optionally substituted with 1, 2 or 3 R#; R# is selected from H, deuterium, halogen or C 1-4 alkyl; Z is E does not exist or is -NH-C 1-4 Alkylene-, the -NH-C 1-4 Alkylene- may optionally be selected from H, halogen, C 1-4 Alkyl and C 1-4 substituted by 1, 2 or 3 substituents in a haloalkyl group; R1 and R2 are independently selected from H, deuterium, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl; D is selected from oxaliplatin; bleomycin or bleomycin; camptothecin, camptothecin derivatives (such as 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; maytansine derivatives; calicheamicin derivatives; auristatin derivatives; pyrrolobenzodiazepine dimer derivatives; melphalan; mitomycin C; or chlorambucil; Preferably, D is in, R D absent, or selected from -NH- or -O-; L D Selected from -C 0-6 Alkylene-OC 0-6 Alkylene- or -C 0-6 Alkylene-OC 0-6 Alkylene-C(O)NH-CHR D4 -; R D1 and R D2 independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy; or, R D1 、R D2 Together with the carbon atoms to which they are attached, they form a ring, e.g. 6-10 Aryl, 5-10 membered heteroaryl, C 3-7 Cycloalkyl or 3-7 membered heterocyclic group, preferably 5-6 membered heterocyclic group; R D3 and R D4 independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy; or, R D3 、R D4 Together with the carbon atoms to which they are attached, they form a ring, e.g., C 6-10 Aryl, 5-10 membered heteroaryl, C 5-10 Cycloalkyl or 5-10 membered heterocyclic group, preferably C 5-6 Cycloalkyl.

9. The antibody drug conjugate according to any one of claims 1 to 8, wherein X is a pyrimidinyl group, preferably Y is Z is R1 and R2 are independently selected from H, deuterium, halogen, C 1-6 Alkyl, preferably H or C 1-4 alkyl; E is -NH-CH2-; D is 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.

10. The antibody drug conjugate according to any one of claims 1 to 9, wherein The antibody-drug conjugate is a compound of formula (II) or a tautomer, stereoisomer or pharmaceutically acceptable salt thereof: in, Ab is anti-MSLN antibody; R1 and R2 are independently selected from C 1-6 Alkyl, preferably C 1-4 alkyl; D is q is an integer selected from 1-10, for example, selected from 4, 5, 6, 7, 8, 9 or 10, preferably 8.

11. The antibody drug conjugate according to any one of claims 1 to 10, wherein the antibody drug conjugate is selected from the following compounds or tautomers, stereoisomers or pharmaceutically acceptable salts thereof: 1) 2) 3) 4) 5) in, Ab is anti-MSLN antibody; q is an integer selected from 1-10, for example, selected from 4, 5, 6, 7, 8, 9 or 10, preferably 8.

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

69.

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

56. Acid sequence; or (19) The VH comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 58, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 59, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 49, and the VL comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 62, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 54, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

56.

14. The antibody drug conjugate of claim 12 or 13, wherein (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 comprising 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 at least 80%, at least 85%, at least 90%, at least 10% or more of the same sequence as SEQ ID NO:

64. the VL comprising 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 that has 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 that has 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.

15. The antibody drug conjugate of any one of claims 12-14, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), and wherein: (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 at least 80%, at least 85%, at least 90%, at least 100% or more of the same SEQ ID NO:

34. the LC comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 40, 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.

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 antibody drug conjugate of any one of claims 12 to 16, wherein the antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, 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 according to any one of claims 1 to 18, wherein the antibody drug conjugate is selected from the following compounds or tautomers, stereoisomers or pharmaceutically acceptable salts thereof: 1) 2) 3) 4) 5) in, Ab is anti-MSLN antibody; q is an integer selected from 1-10, for example selected from 4, 5, 6, 7, 8, 9 or 10, preferably 8; The MSLN antibody comprises a heavy chain (HC) and a light chain (LC), wherein: (1) the HC comprises the amino acid sequence shown in SEQ ID NO: 34, and the LC comprises the amino acid sequence shown in SEQ ID NO: 37; (2) the HC comprises the amino acid sequence shown in SEQ ID NO: 34, and the LC comprises the amino acid sequence shown in SEQ ID NO: 43; (3) the HC comprises the amino acid sequence shown in SEQ ID NO: 70, and the LC comprises the amino acid sequence shown 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.

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

21. The pharmaceutical composition of claim 20, wherein the composition further comprises a second therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody drug, a bi / 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.

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

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

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

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