Anti-CDH6 antibody, conjugate thereof and use thereof

Abstract

Provided in the present disclosure are an anti-CDH6 antibody, a conjugate thereof and the use thereof. In addition, also provided is the use of the anti-CDH6 antibody or the conjugate thereof in treating diseases related to CDH6 abnormal expression.

Description

An anti-CDH6 antibody, its conjugate and its applications

[0001] This application claims priority to Chinese Patent Application No. 2024118856689, filed on December 19, 2024, and Chinese Patent Application No. 2025100331587, filed on January 8, 2025. The full text of the aforementioned Chinese patent applications is incorporated herein by reference. Technical Field

[0002] This disclosure relates to the field of biopharmaceuticals, and more specifically, to antibody molecules that bind to CDH6 or their antigen-binding fragments and their conjugates with deuterated camptothecin compounds and their pharmaceutically acceptable salts, as well as methods for preparing and using these conjugates. This disclosure also relates to pharmaceutical conjugates comprising said anti-CDH6 antibody or its antigen-binding fragment and their related applications in the treatment of cancer. Background Technology

[0003] Cadherins are glycoproteins on the cell membrane surface that function as cell-cell adhesion molecules or as signaling molecules responsible for cell-cell interactions through calcium-dependent binding of their N-terminal extracellular domain. Classical cadherins are single-transmembrane proteins belonging to the cadherin family, composed of three distinct domains: a five-cadherin extracellular domain, a transmembrane domain, and an intracellular domain. Classical cadherins are further classified into type I and type II families based on their amino acid homology.

[0004] cadherin 6 (CDH6) is a type II cadherin composed of 790 amino acids. It shares 75% homology with its family members CDH9 and CDH10. Literature reports that CDH6 plays a role in intercellular adhesion and β-catenin interactions. CDH6 contains an RGD motif that can interact with integrins, promoting cancer cell proliferation, invasion, and metastasis through mechanisms such as promoting EMT and inhibiting autophagy. CDH6 is specifically expressed during developmental stages; its expression is limited in normal adult tissues, but it is highly expressed in renal cell carcinoma (RCC) and ovarian cancer (OVC). It is associated with poor prognosis in renal cell carcinoma. Furthermore, literature and databases show that CDH6 is expressed to varying degrees in thyroid cancer, gastric cancer, and cholangiocarcinoma.

[0005] Renal cell carcinoma (RCC) is a malignant tumor originating from the renal tubular epithelium, accounting for 80%-90% of all kidney malignancies. The etiology of RCC is not fully understood, but its development is associated with factors such as obesity, genetics, and smoking. Treatment options, based on clinical stage and patient tolerance, primarily include surgery, chemotherapy, radiotherapy, and drug therapy. Current clinical drug therapy guidelines mainly include immune checkpoint inhibitors, targeted therapy, and cytokine therapy. Although first-line drug therapy is effective, the median progression-free survival and disease control rate for RCC patients who have failed first-line therapy remain limited, indicating unmet clinical needs.

[0006] Due to the current lack of effective screening and early diagnosis measures for ovarian cancer, the vast majority of patients already have local or distant metastasis at the time of diagnosis, with a 5-year survival rate of approximately 46%. Based on its histopathological characteristics, ovarian cancer is mainly divided into three categories: epithelial ovarian cancer, germ cell tumors, and sex cord-stromal tumors. Ovarian cancer has a high recurrence rate; approximately 70%-80% of patients with advanced ovarian cancer experience disease recurrence after receiving standard platinum-based chemotherapy, and drug resistance is easily developed after recurrence. For recurrent epithelial ovarian cancer, treatment options for platinum-resistant ovarian cancer are currently limited, mainly including single-agent chemotherapy, but the activity of single-agent chemotherapy is limited (ORR, 4%–13%), and these patients do not benefit from existing treatment regimens. 65%-85% of ovarian cancer patients express CDH6, which is associated with poor prognosis.

[0007] Currently, no cancer therapies targeting CDH6 have been approved for marketing, but antibody-drug conjugates (ADCs) targeting the same target have entered clinical trials. Ds-6000a, an ADC product targeting CDH6 developed by Daiichi Sankyo, showed an overall ORR of 46% (23 / 50) in its Phase I clinical trial. Compared with competing products for the same indication, this molecule still demonstrated good clinical efficacy without limiting CDH6 expression levels, indicating the potential of this target for ADC development and validating the effectiveness of the CDH6-Dxd combination. ADCs combine the advantages of precise targeting by monoclonal antibodies and the efficient killing effect of a payload (cytotoxic drugs), demonstrating enormous clinical therapeutic potential. Several ADC drugs have already been approved for marketing. Clinical practice also shows that CDH6 is a well-validated drug target, and CDH6-based monoclonal antibody ADCs may provide more patients with more effective treatment options. Summary of the Invention

[0008] In a first aspect, this disclosure provides a ligand-drug conjugate of Formula I or a pharmaceutically acceptable salt thereof or a stereoisomer thereof:

[0009] Wherein, Ab represents an anti-CDH6 antibody or its antigen-binding fragment; in one embodiment, the anti-CDH6 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the group consisting of: (1) SEQ ID NO: 5, 6, 7, 8, 9, and 10; or (2) SEQ ID NO: 11, 12, 13, 14, 15, and 16; or (3) SEQ ID NO: 10, ... NO:11, 43, 13, 14, 15 and 16; or, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are respectively selected from the heavy chain variable region and light chain variable region HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 sequences of the heavy chain variable region and light chain variable region selected from the group consisting of: (1) SEQ ID NO:19 and 20; or (2) SEQ ID NO:27 and 28; or (3) SEQ ID NO:3 and 4; In one embodiment, the anti-CDH6 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region respectively comprising amino acid sequences selected from the group consisting of: (1) SEQ ID NO:1 and 2; or (2) SEQ ID NO:17 and 18; or (3) SEQ ID NO:19 and 20; or (4) SEQ ID NO:21 and 22; or (5) SEQ ID NO:23 and 24; or (6) SEQ ID NO:25 and 26; or (7) SEQ ID NO:3 and 4; or (8) SEQ ID NO:27 and 28; or (9) SEQ ID NO:29 and 30; or (10) SEQ ID NO:31 and 32; In a preferred embodiment, the anti-CDH6 antibody or its antigen-binding fragment comprises: (1) the heavy chain sequence shown in SEQ ID NO:37 and the light chain sequence shown in SEQ ID NO:38, or, comprises (2) the heavy chain sequence shown in SEQ ID NO:39 and the light chain sequence shown in SEQ ID NO:40;

[0010] L represents the linker connecting Ab and the warhead drug molecule; n is selected from 2-9 or 4-9;

[0011] R1 is selected from H, halogens, OH, SH, NH2, and C. 1-4 Alkyl, C 1-4 Halogenated alkyl or C 1-4 Alkoxy;

[0012] Furthermore, when R1 is a connection site connected to L, R1 is -O-, -S-, -NH-, or C. 1-4 Alkylene;

[0013] R2 is selected from H, halogens, and C. 1-4 Alkyl or C 1-4 Alkoxy;

[0014] Alternatively, R1 and R2 can cyclize to form -O-(CH2). m -O-, where m is selected from 1, 2, and 3;

[0015] R3 and R4 are each independently selected from H and C. 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkyl or C 1-4 Halogenated alkoxy groups;

[0016] Alternatively, R3 and R4 can cyclize to form -(CH2). k - where k is selected from 1, 2, 3, and 4;

[0017] X is selected from H, OH, HO-CH(R5)-(CH2). p -CO-NH- or -N(R6)(R7), where p is selected from 0, 1, and 2;

[0018] Furthermore, when X is a linker site connected to L, X is selected from -O- and -O-CH(R5)-(CH2). p -CO-NH-, -N(R6)- or -N(R7)-, where p is selected from 0, 1, or 2;

[0019] R5 is selected from H, deuterium, CN, halogen, and C. 1-4 Alkyl, C 1-4 Halogenated alkyl, 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl;

[0020] R6 is selected from H and C. 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0021] R7 is selected from H or R8-S(O)2-;

[0022] R8 is selected from C 1-4 Alkyl groups; and

[0023] t is selected from 0, 1, 2, 3, 4 and 5.

[0024] In this disclosure, it is stated that "R1 and R2 cyclize to -O-(CH2)". m "-O-" means that R1 and R2 are connected to form -O-(CH2). m -O-. This indicates that "R3 and R4 cyclize to -(CH2)".k -” refers to the connection of R3 and R4 to form -(CH2). k -

[0025] In any embodiment of the ligand-drug conjugate of Formula I disclosed herein, R1 is C 1-4 Alkyl group, and R2 is a halogen.

[0026] In any embodiment of the ligand-drug conjugate of Formula I disclosed herein, R1 is methyl and R2 is F.

[0027] In any embodiment of the ligand-drug conjugate of Formula I disclosed herein, R1 is H and R2 is H.

[0028] In any embodiment of the formula I ligand-drug conjugate disclosed herein, R1 and R2 are cyclized to -O-CH2-O-.

[0029] In any embodiment of the formula I ligand-drug conjugate disclosed herein, R1 is NH2, and R2 is H or a halogen.

[0030] In any embodiment of the ligand-drug conjugate of Formula I disclosed herein, R1 is NH2 and R2 is H or F.

[0031] In any embodiment of the ligand-drug conjugate of Formula I disclosed herein, R3 is H and R4 is H.

[0032] In any embodiment of the ligand-drug conjugate of Formula I disclosed herein, R3 is H and R4 is C. 1-4 alkyl.

[0033] In any embodiment of the ligand-drug conjugate of Formula I disclosed herein, R3 is H and R4 is methyl.

[0034] In any embodiment of the formula I ligand-drug conjugate disclosed herein, R3 and R4 are cyclized to -CH2-CH2-.

[0035] In any embodiment of the formula I ligand-drug conjugate disclosed herein, X is HO-CH(R5)-(CH2). p -CO-NH-, where p is selected from 0, 1 and 2.

[0036] In any embodiment of the ligand-drug conjugate of Formula I disclosed herein, X is selected from OH or NH2.

[0037] In any embodiment of the ligand-drug conjugate of Formula I disclosed herein, X is H and t is 0.

[0038] In any embodiment of the formula I ligand-drug conjugate disclosed herein, X is -N(R6)(R7).

[0039] In any embodiment of the ligand-drug conjugate of Formula I disclosed herein, L is L1-L2-L3-L4, wherein one end of L1 is connected to Ab, and one end of L4 is connected to the warhead drug molecule D; L1 is selected from the following groups, and the end of L1 marked with an asterisk * is connected to Ab:

[0040] L2 is selected from chemical bonds, -N(R) 10 )-CH2-CO-、 Furthermore, one end of the CO pin of L2 is connected to L3, and the other end is connected to L1; R 10 Selected from C 1-4 Alkyl, C 1-4 Haloalkyl; L3 is selected from polypeptide residues consisting of 2-6 amino acids, and the C-terminus of the polypeptide residue is connected to L4; L4 is selected from chemical bonds, -NH-CH2-, One end of the CH2 group is connected to the warhead drug molecule, and the -NH- end is connected to L3. In one embodiment, L is L1-L2-L3-L4, and L2 is a chemical bond; in another embodiment, L is L1-L2-L3-L4, and L2 is -N(CH3)-CH2-CO-. In yet another embodiment, L is L1-L2-L3-L4, and L3 is selected from the following polypeptide residues: GFG, GGFG (SEQ ID NO:44), GGGFG (SEQ ID NO:45), GGVA (SEQ ID NO:46), V-Cit, And VA. In another embodiment of the ligand-drug conjugate of formula I of this disclosure, L is L1-L2-L3-L4, and L1 is selected from... One end of the asterisk * is connected to Ab. L2 is selected from chemical bonds or -N(CH3)-CH2-CO-, L3 is selected from GFG, GGFG, GGGFG, and L4 is -NH-CH2-.

[0041] In any embodiment of the Formula I ligand-drug conjugate of this disclosure, n is selected from 4.0-7.0 or 7.0-9.0. In one embodiment of the Formula I ligand-drug conjugate of this disclosure, n is selected from 5.0-7.0, or 5.0-6.0, or 5.3-5.9, or 5.5-5.8, or 5.6-5.8; in another embodiment of the Formula I ligand-drug conjugate of this disclosure, n is selected from 7.0-8.5, or 7.2-8.3, or 7.3-8.0, or 7.5-8.0.

[0042] The ligand-drug conjugate of Formula Ia or its pharmaceutically acceptable salt, or its stereoisomer, is shown below:

[0043] Wherein, Ab represents an anti-CDH6 antibody or its antigen-binding fragment; in one embodiment, the anti-CDH6 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the group consisting of: (1) SEQ ID NO: 5, 6, 7, 8, 9, and 10; or (2) SEQ ID NO: 11, 12, 13, 14, 15, and 16; or (3) SEQ ID NO: 10, ... NO:11, 43, 13, 14, 15 and 16; or, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are respectively selected from the heavy chain variable region and light chain variable region HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 sequences of the heavy chain variable region and light chain variable region selected from the group consisting of: (1) SEQ ID NO:19 and 20; or (2) SEQ ID NO:27 and 28; or (3) SEQ ID NO:3 and 4; In one embodiment, the anti-CDH6 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region respectively comprising amino acid sequences selected from the group consisting of: (1) SEQ ID NO:1 and 2; or (2) SEQ ID NO:17 and 18; or (3) SEQ ID NO:19 and 20; or (4) SEQ ID NO:21 and 22; or (5) SEQ ID NO:23 and 24; or (6) SEQ ID NO:25 and 26; or (7) SEQ ID NO:3 and 4; or (8) SEQ ID NO:27 and 28; or (9) SEQ ID NO:29 and 30; or (10) SEQ ID NO:31 and 32; In a preferred embodiment, the anti-CDH6 antibody or its antigen-binding fragment comprises: (1) the heavy chain sequence shown in SEQ ID NO:37 and the light chain sequence shown in SEQ ID NO:38, or comprises, (2) the heavy chain sequence shown in SEQ ID NO:39 and the light chain sequence shown in SEQ ID NO:40;

[0044] L represents the linker connecting Ab and the X1 site of the warhead drug molecule, and n is selected from 2-9;

[0045] R1 is selected from H, halogens, OH, SH, NH2, and C. 1-4 Alkyl, C 1-4 Halogenated alkyl or C 1-4 Alkoxy;

[0046] R2 is selected from H, halogens, and C. 1-4 Alkyl or C 1-4 Alkoxy;

[0047] Alternatively, R1 and R2 can cyclize to form -O-(CH2). m -O-, where m is selected from 1, 2, and 3;

[0048] R3 and R4 are each independently selected from H and C. 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkyl or C 1-4 Halogenated alkoxy groups;

[0049] Alternatively, R3 and R4 can cyclize to form -(CH2). k - where k is selected from 1, 2, 3, and 4;

[0050] X1 is selected from chemical bonds, -O-, -N(R6)-, and -O-CH(R5)-(CH2). p -CO-, where p is selected from 0, 1 and 2, and when X1 is an asymmetric structure, its CO end is connected to NH;

[0051] R5 is selected from H, deuterium, CN, halogen, and C. 1-4 Alkyl, C 1-4 Halogenated alkyl, 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl;

[0052] R6 is selected from H and C. 1-4 Alkyl or C 1-4 Halogenated alkyl groups.

[0053] In one embodiment of the formula I ligand-drug conjugate disclosed herein, it is a formula Ia ligand-drug conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof;

[0054] Wherein, R1, R2, R3, and R4 are as defined in any embodiment of the ligand-drug conjugate of Formula I; X1 is selected from chemical bonds, -O-CH(R5)-(CH2). p -CO-, where the -CO- end is connected to -NH-; p is selected from 0, 1, 2; R5 is selected from H, C 1-4 Alkyl, 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl.

[0055] In any embodiment of the formula Ia ligand-drug conjugate disclosed herein, R1 is selected from methyl or methoxy; or R1 is methyl.

[0056] In any embodiment of the formula Ia ligand-drug conjugate disclosed herein, R2 is selected from F or C1; or R2 is F.

[0057] In any embodiment of the formula Ia ligand-drug conjugate disclosed herein, R1 is methyl and R2 is F.

[0058] In any embodiment of the formula Ia ligand-drug conjugate disclosed herein, R3 and R4 are cyclized to -(CH2). k - where k is 2.

[0059] In any embodiment of the ligand-drug conjugate of formula Ia disclosed herein, R3 is H; and R4 is H.

[0060] In any embodiment of the ligand-drug conjugate of formula Ia disclosed herein, X1 is -O-CH(R5)-(CH2). p -CO-, where the -CO- end is connected to -NH-; where p is selected from 0 or 1, and R5 is selected from H or C. 1-4 Alkyl or 3-6 membered cycloalkyl.

[0061] In any embodiment of the ligand-drug conjugate of formula Ia disclosed herein, X1 is -O-CH(R5)-(CH2). p -CO-, where the -CO- end is connected to -NH-; where p is selected from 0 or 1, and R5 is selected from H, methyl or cyclopropyl.

[0062] In any embodiment of the ligand-drug conjugate of formula Ia disclosed herein, X1 is -O-CH(R5)-CO-, wherein the -CO- end is connected to -NH-; and R5 is selected from H or 3-6 membered cycloalkyl groups.

[0063] In any embodiment of the ligand-drug conjugate of formula Ia disclosed herein, X1 is -O-CH(R5)-CO-, wherein the -CO- end is connected to -NH-; and R5 is selected from H or cyclopropyl.

[0064] In any embodiment of the ligand-drug conjugate of formula Ia disclosed herein, X1 is -O-CH(R5)-CH2-CO-, wherein the -CO- end is connected to -NH-; and R5 is C 1-4 alkyl.

[0065] In any embodiment of the ligand-drug conjugate of formula Ia disclosed herein, X1 is -O-CH(R5)-CH2-CO-, wherein the -CO- end is connected to -NH-; and R5 is methyl.

[0066] In any embodiment of the formula Ia ligand-drug conjugate disclosed herein, X1 is selected from the following groups: The -CO- end is connected to the -NH end.

[0067] In one embodiment of the formula Ia ligand-drug conjugate disclosed herein, L has the same meaning as in any of the schemes of formula I.

[0068] In some embodiments of the ligand-drug conjugate of formula Ia disclosed herein, the ligand-drug conjugate of formula Ia is selected from the ligand-drug conjugates of formula Ia-1 or Ia-2 below, or their pharmaceutically acceptable salts or stereoisomers thereof.

[0069] Ab, L, R1, R2, X1, and n are defined as in any embodiment of the formula Ia ligand-drug conjugate.

[0070] In any embodiment of the formula Ia ligand-drug conjugate disclosed herein, Selected from the following compound fragments:

[0071] in This indicates that the position is connected to the linker L via a chemical bond.

[0072] In any of the technical solutions of formula Ia in this disclosure, it is a ligand-drug conjugate of formula Ia-3 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof:

[0073] Wherein, Ab, L, R5, R1, R2, and n are defined as in any technical solution of formula Ia.

[0074] In any of the technical solutions of formula Ia in this disclosure, it is a ligand-drug conjugate of formula Ia-4 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof:

[0075] Wherein, Ab, L2, L3, L4, R5, and n are as defined in any of the aforementioned technical solutions.

[0076] In one embodiment of the Formula I ligand-drug conjugate disclosed herein, the Formula I ligand-drug conjugate is a Formula Ib ligand-drug conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof.

[0077] Wherein, Ab, L, and n are as defined in any embodiment of the formula I ligand-drug conjugate;

[0078] R1 is selected from H, OH, halogens, NH2, and C. 1-4 Alkyl, C 1-4 Halogenated alkyl or C 1-4 Alkoxy;

[0079] R2 is selected from H, halogens, and C. 1-4 Alkyl or C 1-4 Alkoxy;

[0080] Alternatively, R1 and R2 can cyclize to form -O-(CH2). m -O-, where m is selected from 1, 2, and 3;

[0081] R3 and R4 are each independently selected from H and C. 1-4 Alkyl or C 1-4 Alkoxy;

[0082] Alternatively, R3 and R4 can cyclize to form -(CH2). k - where k is selected from 1, 2, and 3;

[0083] X2 is selected from O or -N(R6)-;

[0084] R6 is selected from H or C. 1-4 alkyl;

[0085] q is selected from 0, 1, 2, 3 and 4.

[0086] In any embodiment of the formula Ib ligand-drug conjugate disclosed herein, R1 is selected from methyl, and R2 is selected from H, Cl, and F.

[0087] In any embodiment of the formula Ib ligand-drug conjugate disclosed herein, R1 and R2 are cyclized to -O-(CH2). m -O-, where m is selected from 1 or 2.

[0088] In any embodiment of the Ib ligand-drug conjugate disclosed herein, both R3 and R4 are H.

[0089] In any embodiment of the formula Ib ligand-drug conjugate disclosed herein, R3 is H and R4 is methyl.

[0090] In any embodiment of the formula Ib ligand-drug conjugate disclosed herein, X2 is selected from -O- or -NH-.

[0091] In any embodiment of the formula Ib ligand-drug conjugate disclosed herein, X2 is selected from -O- or -NH-, and q is 0, 1, 2, 3, or 4.

[0092] In any embodiment of the ligand-drug conjugate of formula Ib disclosed herein, X2 is selected from -O- or -NH-, q is 1, 2, 3 or 4; R3 and R4 are both H; R1 is selected from methyl and R2 is selected from H, Cl or F, or R1 and R2 are cyclized to -O-(CH2). m -O-, where m is selected from 1 or 2.

[0093] In some embodiments of the formula Ib ligand-drug conjugate disclosed herein, the formula Ib compound is a formula Ib-1 compound.

[0094] In formula Ib-1 ligand-drug conjugates, Ab, L, n, X2, and q are as defined in any embodiment of formula Ib ligand-drug conjugates.

[0095] In any implementation of formula Ib, Selected from the following compound fragments;

[0096] in This indicates that the position is connected to the linker L via a chemical bond.

[0097] In one embodiment of the ligand-drug conjugate of formula I disclosed herein, the ligand-drug conjugate of formula I is a ligand-drug conjugate of formula Ic:

[0098] Wherein, X3 is selected from NH, O and S; Ab, L, n, R2, R3 and R4 are defined as in any embodiment of the ligand-drug conjugate of Formula I of this disclosure.

[0099] In any embodiment of the formula Ic ligand-drug conjugate disclosed herein, R2 is selected from H or halogen, or R2 is selected from H or F.

[0100] In any embodiment of the formula Ic ligand-drug conjugate disclosed herein, R3 is H and R4 is C. 1-4 Alkyl; or, R3 is selected from H and R4 is selected from methyl.

[0101] In any embodiment of the formula Ic ligand-drug conjugate disclosed herein, R3 and R4 are cyclized to -(CH2). k -, k is selected from 1, 2 and 3.

[0102] In any embodiment of the Ic ligand-drug conjugate disclosed herein, R3 and R4 are cyclized to -(CH2)2-.

[0103] In any embodiment of the Ic ligand-drug conjugate disclosed herein, X3 is selected from NH or O; or X3 is selected from NH, or X3 is selected from O;

[0104] In any embodiment of the formula Ic ligand-drug conjugate disclosed herein, Selected from the following compound fragments:

[0105] In one embodiment of the formula I ligand-drug conjugate disclosed herein, the formula I ligand-drug conjugate is a ligand-drug conjugate of formula Id or a pharmaceutically acceptable salt thereof:

[0106] Wherein, R1, R2, R3, R4, R6 and R7 are defined as in any embodiment of the Formula I ligand-drug conjugate of this disclosure, Ab, L, n are defined as in any embodiment of the Formula I ligand-drug conjugate of this disclosure, and r is selected from 0, 1, 2 and 3.

[0107] In any embodiment of the formula Id ligand-drug conjugate disclosed herein, both R1 and R2 are H.

[0108] In any embodiment of the formula Id ligand-drug conjugate disclosed herein, R3 and R4 are both H.

[0109] In any embodiment of the formula Id ligand-drug conjugate disclosed herein, r is 1.

[0110] In any embodiment of the ligand-drug conjugate of formula Id disclosed herein, R6 is selected from methyl, ethyl, and isopropyl; or, R6 is isopropyl.

[0111] In any embodiment of the formula Id ligand-drug conjugate disclosed herein, R7 is selected from H.

[0112] In any embodiment of the ligand-drug conjugate of formula Id disclosed herein, R7 is selected from R8-S(O)2-, wherein R8 is selected from methyl and ethyl; or, R8 is selected from methyl.

[0113] In any embodiment of the formula Id ligand-drug conjugate disclosed herein, Selected from the following compound fragments:

[0114] In any embodiment of the ligand-drug conjugate of formula I, Ia, Ia-1, Ia-2, Ib, Ib-1, Ic, or Id disclosed herein, L is selected from the following fragments:

[0115] In this context, "*" indicates the position where the linker connects to the warhead drug molecule, while the other end indicates the position where the linker connects to Ab.

[0116] In any embodiment of the Formula I ligand-drug conjugate disclosed herein, Selected from the following structural fragment:

[0117] In any embodiment of the formula Ia ligand-drug conjugate disclosed herein, Selected from the following structural fragment:

[0118] In one embodiment of this disclosure, the ligand-drug conjugate is selected from the following ligand-drug conjugates or their pharmaceutically acceptable salts, stereoisomers, etc.:

[0119] Wherein, n has the same meaning as in Equation I; for example, n is selected from 7.0-9.0, or n is selected from 7.0-8.5 or 7.2-8.3 or 7.3-8.0 or 7.5-8.0.

[0120] In any embodiment of this disclosure, n is selected from 4.0-7.0 or 7.0-9.0.

[0121] In any embodiment of this disclosure, n is selected from 5.0-7.0, or 5.0-6.0, or 5.3-5.9, or 5.5-5.8, or 5.6-5.8; in another embodiment of the ligand-drug conjugate of Formula I of this disclosure, n is selected from 7.0-8.5, or 7.2-8.3, or 7.3-8.0, or 7.5-8.0.

[0122] In any of the foregoing technical solutions of this disclosure, the Ab is an anti-CDH6 antibody or its antigen-binding fragment, wherein the heavy chain variable region contains HCDR1, HCDR2, and HCDR3 identical to those in the heavy chain variable region shown in SEQ ID NO:19, and / or the light chain variable region contains LCDR1, LCDR2, and LCDR3 identical to those in the light chain variable region shown in SEQ ID NO:20; or, the heavy chain variable region contains HCDR1, HCDR2, and HCDR3 identical to those in the heavy chain variable region shown in SEQ ID NO:27, and / or the light chain variable region contains LCDR1, LCDR2, and LCDR3 identical to those in the light chain variable region shown in SEQ ID NO:28; or, the heavy chain variable region contains LCDR1, LCDR2, and LCDR3 identical to those in the light chain variable region shown in SEQ ID NO:28. The heavy chain variable regions shown in NO:3 contain the same HCDR1, HCDR2, and HCDR3 as the HCDR1, HCDR2, and HCDR3 of the heavy chain variable regions, and / or the light chain variable regions contain the same LCDR1, LCDR2, and LCDR3 as the light chain variable regions shown in SEQ ID NO:4.

[0123] In any of the foregoing technical solutions of this disclosure, the Ab is an anti-CDH6 antibody or its antigen-binding fragment. The heavy chain variable region contains HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and / or the light chain variable region contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; or, the heavy chain variable region contains HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, and / or the light chain variable region contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively; or, the heavy chain variable region contains HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:11, SEQ ID NO:43, and SEQ ID NO:13, and / or the light chain variable region contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively; or, the heavy chain variable region contains HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:11, SEQ ID NO:43, and SEQ ID NO:13, and / or the light chain variable region contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:15, SEQ ID NO:16, respectively. LCDR1, LCDR2, and LCDR3 are shown in NO:14, SEQ ID NO:15, and SEQ ID NO:16.

[0124] In any of the foregoing technical solutions disclosed herein, the anti-CDH6 antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0125] In any of the foregoing technical solutions of this disclosure, the Ab is an anti-CDH6 antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:19 or SEQ ID NO:27, or the amino acid sequence of the heavy chain variable region has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:19 or SEQ ID NO:27, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:20 or SEQ ID NO:28, or the amino acid sequence of the light chain variable region has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:20 or SEQ ID NO:28. In a preferred embodiment, the anti-CDH6 antibody or its antigen-binding fragment comprises a heavy chain variable region as shown in SEQ ID NO:19 and a light chain variable region as shown in SEQ ID NO:20, or the anti-CDH6 antibody or its antigen-binding fragment comprises a heavy chain variable region as shown in SEQ ID NO:27 and a light chain variable region as shown in SEQ ID NO:28.

[0126] In any of the foregoing technical solutions disclosed herein, the Ab is an anti-CDH6 antibody or its antigen-binding fragment, which comprises a heavy chain as shown in SEQ ID NO:37 and a light chain as shown in SEQ ID NO:38, or the anti-CDH6 antibody or its antigen-binding fragment comprises a heavy chain as shown in SEQ ID NO:39 and a light chain as shown in SEQ ID NO:40.

[0127] In any of the foregoing technical solutions disclosed herein, the Ab is an anti-CDH6 antibody or its antigen-binding fragment, which comprises a heavy chain as shown in SEQ ID NO:37 and a light chain as shown in SEQ ID NO:38; and n is selected from 7.0-8.5 or 7.2-8.3 or 7.3-8.0 or 7.5-8.0.

[0128] In any of the foregoing technical solutions disclosed herein, the Ab is an anti-CDH6 antibody or its antigen-binding fragment, which comprises a heavy chain as shown in SEQ ID NO:39 and a light chain as shown in SEQ ID NO:40, and n is selected from 7.0-8.5 or 7.2-8.3 or 7.3-8.0 or 7.5-8.0.

[0129] A second aspect of this disclosure provides pharmaceutical compositions comprising a ligand-drug conjugate of the formula I, Ia, Ib, Ic, Id, Ia-1, Ia-2 or Ib-1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0130] A third aspect of this disclosure provides the use of the ligand-drug conjugates, stereoisomers thereof, or pharmaceutically acceptable salts thereof of formulas I, Ia, Ib, Ic, Id, Ia-1, Ia-2, Ia-3, Ia-4, or Ib-1 in the preparation of a medicament for treating a disease. In a preferred embodiment, the disease is a disease associated with abnormal CDH6 expression; more preferably, the disease associated with abnormal CDH6 expression is a tumor, specifically a solid tumor such as cervical cancer, endometrial cancer, urothelial carcinoma, bladder cancer, bile duct cancer, renal cancer, or ovarian cancer.

[0131] The fourth aspect of this disclosure provides an anti-CDH6 antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof specifically binding to CDH6, and in a preferred embodiment, said antibody or antigen-binding fragment thereof binding to CDH6 and having internalization activity.

[0132] In one embodiment, CDH6 is a polypeptide having the sequence shown in SEQ ID NO:33 (Uniprot ID:P55285).

[0133] In one embodiment, this disclosure provides an anti-CDH6 antibody or its antigen-binding fragment thereof, the anti-CDH6 antibody or its antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising LCDR1, LCDR2, and LCDR3, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 being as follows:

[0134] (1) SEQ ID NO: 5, 6, 7, 8, 9 and 10;

[0135] (2) SEQ ID NO: 11, 12, 13, 14, 15 and 16; or

[0136] (3)SEQ ID NO: 11, 43, 13, 14, 15 and 16.

[0137] In one embodiment, the anti-CDH6 antibody or its antigen-binding fragment, wherein the heavy chain variable region and the light chain variable region each comprise an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the following groups of heavy chain variable regions and light chain variable regions:

[0138] (1) SEQ ID NO:1 and 2; (2) SEQ ID NO:17 and 18; (3) SEQ ID NO:19 and 20; (4) SEQ ID NO:21 and 22; (5) SEQ ID NO:23 and 24; (6) SEQ ID NO:25 and 26; (7) SEQ ID NO:3 and 4; (8) SEQ ID NO:27 and 28; (9) SEQ ID NO:29 and 30; or (10) SEQ ID NO:31 and 32.

[0139] In one embodiment, the anti-CDH6 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3. The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region and the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are selected from the following sequences: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region and LCDR1, LCDR2, and LCDR3 in the light chain variable region.

[0140] (1) SEQ ID NO:1 and 2; (2) SEQ ID NO:17 and 18; (3) SEQ ID NO:19 and 20; (4) SEQ ID NO:21 and 22; (5) SEQ ID NO:23 and 24; (6) SEQ ID NO:25 and 26; (7) SEQ ID NO:3 and 4; (8) SEQ ID NO:27 and 28; (9) SEQ ID NO:29 and 30; or (10) SEQ ID NO:31 and 32.

[0141] In a preferred embodiment, the anti-CDH6 antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region. Preferably, the heavy chain constant region includes a natural Fc or a modified variant Fc. More preferably, the Fc is derived from a mouse or a human.

[0142] In a preferred embodiment, the anti-CDH6 antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

[0143] In a preferred embodiment, the heavy chain constant region comprises natural Fc or modified variant Fc.

[0144] In a preferred embodiment, Fc is derived from mice or humans.

[0145] In a preferred embodiment, the anti-CDH6 antibody or its antigen-binding fragment described in this disclosure is a murine antibody, a chimeric antibody, a fully human antibody, or a humanized antibody.

[0146] In a preferred embodiment, the anti-CDH6 antibody or its antigen-binding fragment is a monoclonal antibody or its antigen-binding fragment, a bispecific antibody or its antigen-binding fragment, or a multispecific antibody or its antigen-binding fragment.

[0147] In a preferred embodiment, the anti-CDH6 antibody is a full-length antibody.

[0148] In a preferred embodiment, the anti-CDH6 antibody or its antigen-binding fragment described in this disclosure is in the form of IgG1, IgG2, IgG3 or IgG4.

[0149] In a preferred embodiment, the antigen-binding fragments against CDH6 described in this disclosure are Fab, Fv, scFv, F(ab')2, linear antibodies, and single-domain antibodies.

[0150] In one embodiment, this disclosure provides a conjugate formed by conjugating the aforementioned anti-CDH6 antibody or its antigen-binding fragment with a capture marker or detection marker. The detection marker includes, but is not limited to, radionuclides, luminescent substances (e.g., fluorescein), colored substances, or enzymes.

[0151] In one embodiment, this disclosure provides a fusion protein, wherein one fused portion comprises an anti-CDH6 antibody of this disclosure or an antigen-binding fragment thereof.

[0152] In one embodiment, this disclosure provides a bispecific antibody or a multispecific antibody, or an antigen-binding fragment thereof, wherein one antigen-binding domain comprises the anti-CDH6 antibody or an antigen-binding fragment thereof disclosed herein.

[0153] In one embodiment, this disclosure provides a nucleic acid encoding any of the aforementioned anti-CDH6 antibodies or antigen-binding fragments thereof. In one embodiment, this disclosure also provides a recombinant vector comprising said nucleic acid.

[0154] In one embodiment, this disclosure provides a host cell comprising the recombinant vector described herein or a nucleic acid containing an antibody encoding the anti-CDH6 antibody or an antigen-binding fragment thereof. In some preferred embodiments, the host cell may be a prokaryotic cell, such as *Escherichia coli*; or a eukaryotic cell, such as yeast; or a mammalian cell, such as CHO cells or HEK293 cells.

[0155] In one embodiment, this disclosure provides a method for preparing the anti-CDH6 antibody or an antigen-binding fragment thereof, comprising: culturing the host cells of this disclosure under suitable conditions and purifying the expression product from the cells.

[0156] In one embodiment, this disclosure provides the use of the antibody against CDH6 or an antigen-binding fragment thereof for the detection, diagnosis, or treatment of diseases associated with CDH6 expression.

[0157] In one embodiment, this disclosure provides a method for detecting CDH6 in a sample, comprising: contacting the sample with the aforementioned anti-CDH6 antibody or its antigen-binding fragment; detecting the formation of a complex of the anti-CDH6 antibody or its antigen-binding fragment with CDH6; optionally, the anti-CDH6 antibody or its antigen-binding fragment is detectably labeled.

[0158] In one embodiment, this disclosure provides a pharmaceutical composition comprising an effective amount of the disclosed anti-CDH6 antibody or its antigen-binding fragment, or an effective amount of nucleic acid encoding the anti-CDH6 antibody or its antigen-binding fragment, or an effective amount of a recombinant vector containing encoding nucleic acid, or an effective amount of a host cell containing encoding nucleic acid, or an effective amount of the disclosed fusion protein, or an effective amount of the disclosed bispecific or multispecific antibody. In some more preferred embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0159] In one specific embodiment, the pharmaceutical composition further comprises one or more additional therapeutic agents. The additional therapeutic agents are pharmaceutical formulations that can be administered to a subject in combination with the pharmaceutical compositions of this disclosure.

[0160] In one embodiment, this disclosure provides a method for treating a subject with a CDH6-related disease, the method comprising administering a pharmaceutical composition of this disclosure to a subject in need. In a preferred embodiment, the disease is a tumor, preferably, the tumor is associated with CDH6 expression. In another preferred embodiment, the disease is a tumor, more preferably, the tumor is a solid tumor, such as cervical cancer, endometrial cancer, urothelial carcinoma, bladder cancer, bile duct cancer, renal cancer, or ovarian cancer. In a more preferred embodiment, the method further comprises administering additional therapeutic agents to the subject.

[0161] A fifth aspect of this disclosure provides an antibody-drug conjugate (ADC) comprising an anti-CDH6 antibody or its antigen-binding fragment as described in any of the technical solutions of the fourth aspect of this disclosure. In one embodiment, the antibody-drug conjugate is formed by conjugating the aforementioned anti-CDH6 antibody or its antigen-binding fragment to other bioactive molecules. Preferably, the anti-CDH6 antibody or its antigen-binding fragment is conjugated to other bioactive molecules via a linker. Preferably, the other bioactive molecules are small molecule drugs, such as antitumor drugs, and more preferably, the antitumor drug is an antitumor compound. In one embodiment, this disclosure also provides a pharmaceutical composition and its use in treating a subject with a CDH6-related disease, the pharmaceutical composition comprising the antibody-drug conjugate of this disclosure. The use includes administering the pharmaceutical composition of this disclosure to a subject in need. In a preferred embodiment, the disease is a tumor, preferably one associated with CDH6 expression. In another preferred embodiment, the disease is a tumor, such as cervical cancer, endometrial cancer, urothelial carcinoma, bladder cancer, bile duct cancer, kidney cancer, or ovarian cancer. In a more preferred embodiment, the method further includes administering additional therapeutic agents to the subject. Preferably, the pharmaceutical composition further comprises one or more additional therapeutic agents, said additional therapeutic agents being pharmaceutical formulations that can be administered to the subject in combination with the pharmaceutical compositions of this disclosure.

[0162] The sixth aspect of this disclosure provides a method of treating tumors, comprising administering to a patient in need an effective amount of the ligand-drug conjugate of formula I, Ia, Ib, Ic, Id, Ia-1, Ia-2, Ia-3, Ia-4 or Ib-1, its stereoisomers or pharmaceutically acceptable salts thereof, or administering to a patient in need an effective amount of the pharmaceutical composition of the second aspect of this disclosure; or administering to a patient in need an effective amount of the anti-CDH6 antibody or its antigen-binding fragment of any of the technical solutions of the fourth aspect of this disclosure, or administering to a patient in need an effective amount of the antibody-drug conjugate of the fifth aspect of this disclosure.

[0163] The seventh aspect of this disclosure provides ligand-drug conjugates of the formulas I, Ia, Ib, Ic, Id, Ia-1, Ia-2, Ia-3, Ia-4, or Ib-1, stereoisomers thereof, or pharmaceutically acceptable salts thereof for treating tumors, or pharmaceutical compositions of the second aspect of this disclosure, or anti-CDH6 antibodies or antigen-binding fragments thereof of the fourth aspect of this disclosure, or antibody-drug conjugates of the fifth aspect of this disclosure.

[0164] The eighth aspect of this disclosure provides the use of the ligand-drug conjugates of formula I, Ia, Ib, Ic, Id, Ia-1, Ia-2, Ia-3, Ia-4 or Ib-1, their stereoisomers or pharmaceutically acceptable salts thereof, the pharmaceutical compositions of the second aspect of this disclosure, or the anti-CDH6 antibody or its antigen-binding fragment of the fourth aspect of this disclosure, or the antibody-drug conjugate of the fifth aspect of this disclosure in the treatment of tumors.

[0165] The loaded compound (i.e., the warhead drug molecule) in the ligand-drug conjugates of formulas I, Ia, Ib, Ic, Id, Ia-1, Ia-2, Ia-3, Ia-4, or Ib-1 of this disclosure exhibits better stability in plasma, a shorter half-life in vivo, and lower in vivo exposure. Therefore, the loaded compound in the ligand-drug conjugates of this disclosure is more easily eliminated after detachment in vivo, resulting in lower toxicity. Furthermore, toxicity tests of this disclosure have demonstrated that the ligand-drug conjugates of this disclosure exhibit lower in vivo and hematologic toxicity, demonstrating better safety. Attached Figure Description

[0166] The accompanying drawings further illustrate the novel features disclosed herein. A better understanding of the features and advantages disclosed herein will be achieved by referring to these drawings; however, it should be understood that these drawings are for illustrating specific embodiments of the principles disclosed herein and are not intended to limit the scope of the appended claims.

[0167] Figure 1A shows the binding of the mouse-derived anti-CDH6 chimeric antibody of this disclosure to CDH6-overexpressing cells (OVCAR3).

[0168] Figure 1B shows the binding of the mouse-derived anti-CDH6 chimeric antibody of this disclosure to cells with low CDH6 expression (PA1).

[0169] Figure 1C shows the binding of the mouse-derived anti-CDH6 chimeric antibody of this disclosure to cyno CDH6-expressing cells (CHOK1-cyno CDH6).

[0170] Figure 2A shows the internalization of the mouse-derived anti-CDH6 chimeric antibody of this disclosure in CDH6-overexpressing cells (OVCAR3).

[0171] Figure 2B shows the internalization of the mouse-derived anti-CDH6 chimeric antibody of this disclosure in a CDH6-low-expressing tumor cell line (PA-1).

[0172] Figure 3A shows the binding of the mouse-derived anti-CDH6 chimeric antibody of this disclosure to the family-related protein CDH9.

[0173] Figure 3B shows the binding of the mouse-derived anti-CDH6 chimeric antibody of this disclosure to the family-related protein CDH10.

[0174] Figures 4A and 4B show the binding of the mouse-derived anti-CDH6 humanized antibody of this disclosure to CDH6-overexpressing cells (OVCAR3).

[0175] Figures 4C and 4D show the binding of the mouse-derived anti-CDH6 humanized antibody of this disclosure to cells with low CDH6 expression (PA1).

[0176] Figures 4E and 4F show the binding of the mouse-derived anti-CDH6 humanized antibody of this disclosure to cyno CDH6-expressing cells (CHOK1-cyno CDH6).

[0177] Figures 5A and 5B show the internalization of the mouse-derived anti-CDH6 humanized antibody of this disclosure in CDH6-overexpressing cells (OVCAR3).

[0178] Figures 5C and 5D show the internalization of the mouse-derived anti-CDH6 humanized antibody of this disclosure in low-expressing CDH6 cells (PA-1).

[0179] Figure 6A shows the binding level of the mouse-derived anti-CDH6 humanized antibody of this disclosure to the mouseCDH6 protein.

[0180] Figure 6B shows the binding level of the mouse-derived anti-CDH6 humanized antibody of this disclosure to the RatCDH6 protein.

[0181] Figure 7A shows the binding level of the mouse-derived anti-CDH6 humanized antibody of this disclosure to the CDH9 protein, a member of the same family.

[0182] Figure 7B shows the binding level of the mouse-derived anti-CDH6 humanized antibody of this disclosure to the CDH10 protein, a member of the same family. Detailed Implementation

[0183] Terms and Explanations

[0184] Unless otherwise stated, the terms used herein have their general meanings within the technical field. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0185] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if specifically and individually indicated that each individual publication, patent or patent application is incorporated by reference.

[0186] Before this disclosure is described in detail below, it should be understood that this disclosure is not limited to the specific methodologies, procedures, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0187] When referring to measurable values ​​such as quantities, temporary durations, etc., the term “about” means a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0188] The three-letter and single-letter codes for amino acids used in this article are as described in J. BIOL. CHEM, 243, P3558 (1968).

[0189] As used in this article, the term "antibody" typically refers to a Y-type tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Natural IgG antibodies possess this structure. Each light chain contains a light chain variable domain (VL) and a light chain constant domain (CL). Each heavy chain contains a heavy chain variable domain (VH) and a heavy chain constant domain (CH), or heavy chain constant region (CH).

[0190] The term "antibody" in this article may include complete antibodies (e.g., full-length monoclonal antibodies) and any antigen-binding fragment (i.e., antigen-binding part) or its single chain, and may also include products with antigen-specific binding ability formed by modifying complete antibodies or their antigen-binding fragments or their single chains (e.g., linking other peptides, rearranging functional units, etc.).

[0191] Five main classes of antibodies are known in this art: IgA, IgD, IgE, IgG, and IgM, with their corresponding heavy chain constant domains referred to as α, δ, ε, γ, and μ, respectively. IgG and IgA can be further subdivided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA into IgA1 and IgA2. The light chain of antibodies from any vertebrate species can be identified as one of two distinct types based on the amino acid sequence of their constant domains, termed κ and λ.

[0192] In the case of IgG, IgA, and IgD antibodies, this heavy chain constant region contains three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA, and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a variable-length segment rich in proline and cysteine. Each class of antibody further contains interchain and intrachain disulfide bonds formed by paired cysteine ​​residues.

[0193] As used herein, the terms "anti-CDH6 antibody" or "CDH6-binding antibody" refer to antibodies that are able to bind to CDH6 with sufficient affinity.

[0194] The term "variable region" or "variable domain" indicates a significant change in the amino acid composition from one antibody to another and is primarily responsible for antigen recognition and binding. The variable region of each light / heavy chain pair forms the antigen-binding site, giving the complete IgG antibody two binding sites (i.e., it is bivalent). The variable region (VH) of the heavy chain and the variable region (VL) of the light chain each contain three regions with extreme variability, referred to as hypervariable regions (HVR), or more commonly, complementarity-determining regions (CDR). Each VH and VL has four backbone regions (FR), denoted as FR1, FR2, FR3, and FR4, respectively. Therefore, the CDR and FR sequences typically appear in the following sequence of the heavy chain variable domain (VH) (or light chain variable domain (VL)): FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0195] The term "Fc" is used herein to define the C-terminal region of the immunoglobulin heavy chain, which comprises at least a portion of the constant region. This term includes both native sequence Fc regions and variant Fc regions. Unless otherwise stated, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0196] As used herein, the broad category of "antibody" may include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primate-derived antibodies, CDR-grafted antibodies, human antibodies (including recombinant human antibodies), recombinant antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-individual genotype antibodies, synthetic antibodies (including mutant proteins and their variants), etc.

[0197] The terms “full-length antibody,” “complete antibody,” and “intact antibody” may be used interchangeably in this document to refer to antibodies whose structure is substantially similar to that of natural antibodies or that contain the FC region.

[0198] The term "monoclonal antibody" (or "mAb") refers to a substantially homogeneous antibody produced from a single cell clone that targets only a specific antigenic epitope. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, synthetic technology, or combinations of the above.

[0199] The term "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0200] The term "humanized antibody" refers to a hybrid immunoglobulin, immunoglobulin chain, or fragment thereof containing a minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies) where residues of the receptor's core sequence (CDR) are replaced by residues of a CDR from a non-human species (donor antibody) possessing the desired specificity, affinity, and performance, such as mice, rats, rabbits, or primates. In some cases, framework region residues of the human immunoglobulin are replaced by corresponding non-human residues. In certain circumstances, "reversion mutations" can be introduced into humanized antibodies where residues in one or more frame regions (FRs) of the variable region of the recipient human antibody are replaced by corresponding residues from a non-human species donor antibody. Such reversion mutations can help maintain the appropriate three-dimensional conformation of one or more grafted CDRs and thus improve affinity and antibody stability. Antibodies from a variety of donor species can be used, including but not limited to mice, rats, rabbits, or non-human primates. Additionally, humanized antibodies may contain novel residues not found in the recipient antibody or the donor antibody to further improve antibody performance.

[0201] It should be noted that the division of the CDR and FR in the variable region of the monoclonal antibody disclosed herein is determined according to the Kabat definition. Other nomenclature and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, humanized antibodies containing one or more CDRs derived from any nomenclature system based on the monoclonal antibody sequence of this disclosure are explicitly kept within the scope of this disclosure.

[0202] The terms "sequence identity," "sequence similarity," or "sequence homology" refer to the percentage of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence after aligning the sequences (and, where necessary, introducing gaps) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine the percentage amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0203] The term "antibody fragment" includes at least a portion of a complete antibody. As used herein, a "fraction" of an antibody molecule includes an "antigen-binding fragment" of the antibody, and the term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that specifically binds to or reacts with a selected antigen or its epitope, or a fusion protein product further derived from such fragment, or a product conjugated with other compounds, such as a single-chain antibody, an extracellular binding region in a chimeric antigen receptor, etc. Exemplary antibody fragments or their antigen-binding fragments include, but are not limited to: variable light chain fragments (VL), variable heavy chain fragments (VH), Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-domain antibodies (VHH), linear antibodies, single-chain antibodies (scFv), and bispecific or multispecific antibodies formed from antibody fragments, etc.

[0204] The term "Fab" or "Fab fragment" includes the variable regions of the heavy chain and the light chain, and also includes the constant region of the light chain and the first constant region CH1 of the heavy chain, which is a monovalent antibody fragment. The term "F(ab')2 fragment" contains two Fab fragments and a hinge region, which is a bivalent antibody fragment.

[0205] The term "Fd fragment" generally includes the heavy chain variable region and the constant region CH1; the term "Fv fragment" contains the antibody heavy chain variable region and the light chain variable region, but no constant region, and is the smallest antibody fragment with all antigen binding sites.

[0206] The term "scFv" refers to a fusion protein comprising at least one antibody fragment including a variable region of a light chain and at least one antibody fragment including a variable region of a heavy chain, wherein the light and heavy chain variable regions are adjacent (e.g., via a synthetic linker, such as a short, flexible peptide linker) and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it originates. Unless otherwise specified, the scFv may have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0207] The term "fusion protein" refers to a larger molecule formed by linking different polypeptides / proteins together through genetic recombination or chemical methods. Linkers can be used for this linking, or not.

[0208] The term "multispecific antibody" refers to a novel antibody construct formed by functionally linking an antibody to one or more other binding molecules (e.g., chemical conjugation, gene fusion, non-covalent binding, or other methods) to bind to two or more different sites and / or targets. Among these, "bispecific antibody" is more commonly used, specifically referring to an antibody construct that is specific to two different antigens. Typically, bispecific or multispecific antibodies include at least two antigen-binding domains.

[0209] The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or its antigen-binding fragment. In a broad sense, an antigen can include any immunogenic fragment or determinant of a selected target, including single epitopes, multiple epitopes, single domains, multiple domains, or intact extracellular domains (ECDs) or proteins. Peptides, proteins, glycoproteins, polysaccharides, and lipids, as well as portions thereof, can constitute antigens. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, etc. "Antigen" can also refer to a molecule that elicits an immune response. Any form of antigen, or cells or preparations containing that antigen, can be used to generate antibodies specific to the antigenic determinant. An antigen can be an isolated full-length protein, a cell surface protein (e.g., used for immunization with cells expressing at least a portion of the antigen on their surface), or a soluble protein (e.g., used for immunization with only the ECD portion of the protein), or a protein construct (e.g., an Fc antigen). The antigen can be produced in genetically modified cells. Any of the foregoing antigens can be used alone or in combination with one or more immunogenic adjuvants known in the art. The DNA encoding the antigen can be genomic or non-genomic (e.g., cDNA) and can encode at least a portion of the ECD sufficient to elicit an immunogenic response. Any vector can be used to transform cells expressing the antigen, including but not limited to adenoviral vectors, lentiviral vectors, plasmids, and non-viral vectors such as cationic lipids.

[0210] The term "epitope," also known as an "antigenic determinant," refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or from non-adjacent amino acids arranged side-by-side through the ternary folding of a protein. Epitopes formed from adjacent amino acids are typically retained after exposure to denaturing solvents, while epitopes formed through ternary folding are typically lost after treatment with denaturing solvents. Epitopes typically consist of 3–15 amino acid residues. Methods for determining the epitope bound to a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0211] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. Polymers may be linear, cyclic, or branched, may contain modified amino acids, particularly conserved modified amino acids, and may be interrupted by non-amino acid components. The term also includes amino acid polymers that have been modified, for example, by glycosylation, esterification, acetylation, phosphorylation, methylation, or any other manipulation such as conjugation with a labeled component. As used herein, the term “amino acid” refers to natural and / or non-natural or synthetic amino acids, including glycine and its D or L optical isomers, as well as amino acid analogs and peptide mimics. “Derived from” a specified protein refers to the source of the polypeptide. The term also includes polypeptides expressed by a specified nucleic acid sequence.

[0212] The term "amino acid modification" (or "modified amino acid") includes amino acid substitutions, insertions, and / or deletions in a polypeptide sequence. "Amino acid substitution" or "replacement" means replacing an amino acid at a specific position in the parent polypeptide sequence with another amino acid. For example, substitution of S32A means that the serine at position 32 is replaced by alanine.

[0213] The sequence identity or homology between the variable region of the humanized antibody and the variable region of the human receptor can be determined as discussed herein, and when such a determination is made, preferably at least 60% or 65% sequence identity will be shared, more preferably at least 70%, 75%, 80%, 85%, or 90% sequence identity, and even more preferably at least 93%, 95%, 98%, or 99% sequence identity. Preferably, the different residue positions are due to conserved amino acid substitutions. A “conserved substitution” is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue with a side chain (R group) having similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions do not substantially alter the functional properties of the protein. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region or frame region of the disclosed antibody can be replaced with amino acid residues of other similar side chains. In cases where two or more amino acid sequences differ from each other due to conserved substitutions, the sequence identity percentage or degree of similarity can be adjusted upwards to correct for the conservatism of the substitution.

[0214] During monoclonal antibody production, various physicochemical factors can easily generate post-translational modification (PTM) variants, such as glycosylation, oxidation, glycation, deamidation, isomerization, and terminal cyclization. These PTMs can cause changes in the physicochemical properties of antibodies, alter their interaction with the antibody Fc receptor, and affect their binding activity to the target antigen. Some PTMs can even reduce antibody stability and induce immunogenicity (JARASCH et al., JOURNAL OF PHARMACEUTICAL SCIENCES, 2015). The negative effects of PTMs can be eliminated by modifying the amino acid sites, such as through conserved substitutions. Amino acid substitutions of antibody CDRs for the purpose of modifying PTMs are also explicitly kept within the scope of this disclosure.

[0215] The antibodies disclosed herein may also include substitutions or modifications to constant regions (e.g., Fc), including but not limited to amino acid residue substitutions, mutations, and / or modifications, which produce compounds having preferred characteristics, including but not limited to: altered pharmacokinetics, increased serum half-life, increased binding affinity, decreased immunogenicity, increased yield, altered binding to Fc receptors (FcRs), enhanced or weakened ADCC or CDC, altered glycosylation and / or disulfide bonds, and modified binding specificity.

[0216] The term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The term "KD" refers to the dissociation constant of a specific antibody-antigen interaction. Binding affinity can be determined using a variety of techniques known in the art, such as surface plasmon resonance, biolayer interferometry, bipolar interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry.

[0217] The term "pharmaceutical composition" refers to a formulation or combination of formulations containing one, two, or more active ingredients, wherein the active ingredients contained herein are present in a biologically effective form and do not contain any additional ingredients that would have unacceptable toxicity to a subject administering the formulation. When a "pharmaceutical composition" exists as a combination of individual formulations containing two or more different active ingredients, it can be administered simultaneously, sequentially, separately, or at intervals, with the aim of exerting the biological activity of multiple active ingredients together for the treatment of a disease.

[0218] The term "ligand-drug conjugate" refers to a ligand covalently coupled to a bioactive molecule, preferably an antibody.

[0219] The term "antibody-drug conjugate" (ADC) refers to an antibody covalently conjugated to a bioactive molecule, such as a therapeutic active substance or active pharmaceutical ingredient (API), so that the therapeutic active substance or active pharmaceutical ingredient (API) can target the antibody's binding target to exhibit its pharmacological function. The therapeutic active substance or active pharmaceutical ingredient can be a cytotoxic agent capable of killing cells targeted by the ADC, preferably malignant or cancerous cells. The covalent conjugation of the therapeutic active substance, active pharmaceutical ingredient, or cytotoxic agent can be performed in a non-site-specific manner using standard chemical linkers that conjugate the payload to lysine or cysteine ​​residues, or preferably, the conjugation is performed in a site-specific manner, which allows complete control over the conjugation site and the drug-to-antibody ratio of the resulting ADC. The ADCs described herein can be used to deliver cytotoxic agents or other payloads to target sites (e.g., tumorigenic cells and / or cells expressing CDH6). As used herein, the terms "drug" or "warhead" are used interchangeably and refer to a biologically active or detectable molecule or compound, including anticancer agents. A "payload" can comprise a drug or warhead in combination with an optional linker compound. The warhead can contain peptides, polypeptides, proteins, precursor drugs that are metabolized into active agents in the body, polymers, nucleic acid molecules, small molecules, binders, mimics, synthetic drugs, inorganic molecules, organic molecules, and radioactive isotopes.

[0220] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0221] The term “pharmaceutical carrier” or “pharmaceuticalally acceptable carrier” refers to a diluent, adjuvant (e.g., Freund’s adjuvant (complete and incomplete)), excipient, or medium that is administered with a therapeutic agent.

[0222] The term "effective dose" refers to a dose of a pharmaceutical composition comprising an antibody or its antigen-binding fragment or ligand-drug conjugate disclosed herein, which, when administered to a patient in a single or multiple doses, produces the intended effect in the treated patient. The effective dose can be readily determined by an attending physician skilled in the art by considering a variety of factors, such as: racial differences; weight, age, and health status; the specific disease involved; the severity of the disease; the individual patient's response; the specific antibody administered; the administration modality; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies.

[0223] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include the primary transformed cells and their progeny, regardless of the number of passages. Progeny may not be identical to the parent cells in terms of nucleic acid content and may contain mutations. This document includes mutant progeny that have the same function or biological activity as those screened or selected in the initially transformed cells.

[0224] As used in this article, the term "transfection" refers to the introduction of exogenous nucleic acids into eukaryotic cells. Transfection can be achieved through a variety of techniques known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipid transfection, protoplast fusion, retroviral infection, and biolistics.

[0225] The term "stable transfection" or "stable transformation" refers to the introduction and integration of exogenous nucleic acids, DNA, or RNA into the genome of transfected cells. The term "stable transfectant" refers to a cell in which foreign DNA is stably integrated into the genomic DNA.

[0226] The terms "isolated polynucleotide" or "isolated nucleic acid" refer to nucleic acid molecules, DNA, or RNA that have been removed from their natural environment. For example, for the purposes of this disclosure, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell or (partially or substantially) purified polynucleotides in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that normally contain such polynucleotide molecules, but which are present outside the chromosome or at a chromosomal location other than their natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of this disclosure, as well as positive-stranded, negative-stranded, and double-stranded forms.

[0227] The terms "nucleic acid molecule encoding," "encoding DNA sequence," and "encoding DNA" refer to the sequence of deoxyribonucleotides along a deoxyribonucleic acid (DNA) chain. This sequence of deoxyribonucleotides determines the sequence of amino acids along a polypeptide (protein) chain. Therefore, a nucleic acid sequence encodes an amino acid sequence.

[0228] Methods for producing and purifying antibodies and antigen-binding fragments are well-known and available in the prior art, such as in Cold Spring Harbor's *Antibody Laboratory Techniques Guide*, Chapters 5-8 and 15. The antibodies or antigen-binding fragments described in this invention are genetically engineered to add one or more human FR regions to a non-human CDR region. Human FR germline sequences are available from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr or from the journal *Immunoglobulins*, (2001) ISBN: 012441351.

[0229] The engineered antibodies or antigen-binding fragments thereof disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. Cultures secreting antibodies can be purified and collected using conventional techniques. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving and ion exchange.

[0230] As used herein, the terms “individual” or “subject” refer to any animal, such as a mammal or marsupial. Individuals disclosed herein include, but are not limited to, humans, non-human primates (such as cynomolgus monkeys or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any kind of poultry.

[0231] As used herein, the terms “disease,” “symptom,” or “disorder,” etc., refer to any alteration or dysregulation that impairs or interferes with the normal function of cells, tissues, or organs. For example, “disease” includes, but is not limited to: tumors, pathogen infections, autoimmune diseases, T-cell dysfunction disorders, or deficiencies in immune tolerance (such as transplant rejection).

[0232] As used in this article, the term "tumor" refers to a disease characterized by the pathological proliferation of cells or tissues, and their subsequent migration or invasion into other tissues or organs. Tumor growth is typically uncontrolled and progressive, neither inducing nor inhibiting the proliferation of normal cells.

[0233] As used in this article, the term "treatment" refers to a clinical intervention in an attempt to alter an individual's or treat a disease caused by cells, which can be preventative or intervention in a clinicopathological process. Treatment effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the condition, and alleviating or improving prognosis.

[0234] As used herein, the term "combination" refers to a treatment regimen that provides two or more different therapies to achieve a specified therapeutic effect. These therapies can be physical, such as radiation therapy, or chemical, such as administering a drug to the subject, including combination drugs. "Combination drugs" refers to a combination of two or more pharmaceutical preparations, each containing an active ingredient, that are administered to a subject in combination. The active ingredients may be mixed together to form a single dosing unit or may be administered separately as independent dosing units; during administration, the different pharmaceutical preparations may be administered substantially synchronously, simultaneously, or sequentially.

[0235] In any embodiment of the compound of formula I disclosed herein, when R1 and R2 are cyclized to -O-(CH2) m When -O-, the compound of formula I is the compound of formula I-1 as follows. In the compound of formula I disclosed herein, or in any embodiment thereof, when R3 and R4 are cyclized to -(CH2) k - When, it is presented in the form of I-2 as follows.

[0236] In the formula I ligand-drug conjugates disclosed herein, This indicates that the linker L can be chemically bonded to any connectable site of the warhead drug molecule D in Formula I, provided that it is chemically feasible and yields a robust drug.

[0237] Those skilled in the art will understand that in Formula I, Ia, Ib, Ic, Id, Ia-1, Ia-2, Ia-3, Ia-4, or Ib-1, or their specific ligand-drug conjugates, the linker L or its corresponding specific structure is connected to Ab via a sulfur atom from Ab. This sulfur atom S may be embodied in the structural or chemical formula of L or L1, or it may be omitted, hidden, or not shown. For example... It can also be written as Both represent the same structure and meaning; It can also be written as It can be written as It can also be written as Other L1 segments can be understood in the same way.

[0238] The term "warhead drug molecule," also known as "load," "loaded drug," or simply "payload," refers to a substance with preventative or therapeutic effects against diseases. In antibody-drug conjugates, the drug typically refers to a cytotoxic drug, a chemical molecule that can strongly disrupt the normal growth of tumor cells.

[0239] The term "linker" refers to a chemical structural segment or bond that is connected to a ligand at one end and to a drug at the other end. It can also be connected to other linkers before being linked to a drug.

[0240] The term "drug-linker conjugate" is also called a load-linker conjugate or a linker-load conjugate, and has the same meaning in this disclosure.

[0241] The terms “optional” or “optionally” mean that the event or condition described below may or may not occur, including both the occurrence and non-occurrence of the event or condition. For example, the term “optionally” means that the group may be unsubstituted or substituted with a specified substituent; whether substituted or not, it falls within the scope of “optionally”.

[0242] The terms "independently selected" or "each independently selected" mean that each substituent in the preceding term can choose different optional variables from a specified range of choices, unaffected by the selection results of other substituents. When a substituent that can be "independently selected" exists as multiple identical substituents in the general chemical formula, it can still choose the same or different optional variables.

[0243] The term "halogen" or "halogen atom" refers to fluorine, chlorine, bromine, and iodine.

[0244] The term "halogenated" refers to a group formed when one or more hydrogen atoms in a substituent are replaced by halogen atoms.

[0245] The term "alkyl" refers to a straight-chain or branched hydrocarbon group in which carbon atoms are linked by single bonds. Alkyl groups are preferably C14-245 ... 1-4 Or C 1-6 Alkyl group. "C" 1-4 "Alkyl" refers to a straight-chain or branched alkyl group having 1, 2, 3, or 4 carbon atoms. C 1-4 Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl. 1-6 "Alkyl" refers to a straight-chain or branched alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms. C 1-6 Examples of alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, and n-hexyl.

[0246] The term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by halogen atoms. 1-4 Examples of alkyl halogens include, but are not limited to, trifluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl and 2,2,2-trichloroethyl.

[0247] The term "alkoxy" refers to an alkyl-O- group, wherein the alkyl group, as defined above, includes C0. 1-4 Alkoxy or C 1-6 Alkyl group. C 1-4 Alkoxy can be understood as "C 1-4 Alkyl-O-", examples of which include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, and tert-butoxy. C 1-6 Alkoxy can be understood as "C 1-6 Alkyl-O-”, examples of which include the aforementioned C 1-4 In addition to specific examples of alkoxy groups, other examples include, but are not limited to, n-pentoxy, neopentoxy, and n-hexyloxy.

[0248] The term "haloalkoxy" refers to an alkoxy group in which one or more hydrogen atoms are replaced by halogen atoms. 1-4 Examples of haloalkoxy groups include, but are not limited to, trifluoromethoxy, trichloromethoxy, 2,2,2-trifluoroethoxy, and 2,2,2-trichloroethoxy.

[0249] The term "cycloalkyl" refers to a cyclic, saturated monocyclic or polycyclic hydrocarbon group formed by single bonds between carbon atoms. The hydrogen atom of the ring carbon atom is optionally oxidized, i.e., the "-CH2-" in the ring is optionally oxidized to form "-C(O)-". 3- to 6-membered cycloalkyl refers to 3, 4, 5, or 6-membered cycloalkyl groups, specific examples of which include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0250] The term "heterocyclic alkyl" refers to a cycloalkyl group in which one or more (e.g., 2, 3, or 4) cyclic carbon atoms are replaced by heteroatoms or heteroatom groups (i.e., groups containing heteroatoms). The cyclic carbon atoms are the carbon atoms that form the cyclic framework; the heteroatoms are atoms in an organic compound other than C and H, such as nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), or boron (B), and the heteroatoms, such as nitrogen and sulfur atoms, can be oxidized, and the nitrogen atom can be quaternized; examples of heteroatom groups include, but are not limited to, -S(=O)2-, -S(=O)-, and optionally substituted -NH-, -S(=O)(=NH)-, -C(=O)NH-, -C(=NH)-, -S(=O)2NH-, S(=O)NH-, or -NHC(=O)NH-, etc. 3-6 membered heterocyclic alkyl refers to 3, 4, 5 or 6 membered heterocyclic alkyl, and specific examples include, but are not limited to, azirropropane, oxacyclopropane, thiohexanepropane, azirrobutyl, oxacyclobutyl, thiohexanebutyl, azirropentane, oxacyclopentane, thiohexanepentane, piperazinyl, piperidinyl, oxacyclohexyl, morpholinyl and 1,4-dioxanecyclo.

[0251] In this disclosure, the letters G, F, V, A, and Cit used to represent amino acids or amino acid residues represent glycine, phenylalanine, valine, alanine, and citrulline, respectively.

[0252] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of an acid or base, including salts formed by a compound with an inorganic or organic acid, and salts formed by a compound with an inorganic or organic base.

[0253] In this disclosure, the substituents in This indicates the connection point of the substituent to the parent structure or other segments. A dash "-" in the substituent structure indicates the connection point of the substituent; for example, -CH3 indicates that the group is connected to the parent structure or other segments through a carbon atom. and The absolute configuration representing the center of a solid, i.e., the R or S configuration.

[0254] In this disclosure, chemical bonds are depicted by solid and dashed lines. Whether a chemical bond is a single or double bond can be determined based on the valence of the atoms at both ends of the bond, within the range known to those skilled in the art.

[0255] In this disclosure, the term "isomer" includes geometric isomers and stereoisomers, such as blocked trans isomers, cis-trans isomers, enantiomers, diastereomers, tautomers, racemic mixtures thereof, and other mixtures, all of which are within the scope of this disclosure. The term "enantiomer" refers to stereoisomers that are mirror images of each other. The term "tautomer" refers to a functional group isomer that has different hydrogen bonding sites through one or more double bond shifts; for example, a ketone and its enol form are keto-enol tautomers. The term "diastereomer" refers to stereoisomers of molecules having two or more chiral centers and being non-mirror images of each other. The term "cis-trans isomer" refers to different spatial configurations of a molecule where double bonds or single bonds of cyclic carbon atoms cannot rotate freely. The term "blocked trans isomer" refers to a stereoisomer that can be separated due to impeded or very slow rotation of single bonds.

[0256] The stereoisomers of the compounds disclosed herein can be prepared by chiral synthesis or using chiral reagents or other conventional techniques. For example, an enantiomer of a compound disclosed herein can be prepared by asymmetric catalysis or chiral derivative derivatization. Alternatively, a single stereoisomer can be obtained from a mixture using chiral resolution techniques. Alternatively, it can be prepared directly from chiral starting materials. The separation of optically pure compounds in this disclosure is typically accomplished using preparative chromatography, employing chiral chromatographic columns to achieve the separation of chiral compounds.

[0257] In this disclosure, the unit of solution concentration M represents mol / L, mM represents mmol / L, and nM represents nmol / L. The unit of solution concentration N represents equivalent concentration, which is expressed as the number of gram equivalents of solute contained in 1 liter of solution, denoted by the symbol N. For example, if 1 liter of concentrated hydrochloric acid contains 12.0 gram equivalents of hydrochloric acid (HCl), then the concentration is 12.0N. Equivalent concentration = number of gram equivalents of solute / solution volume (liters). In in vivo drug activity experiments, the unit of administered dose mpk refers to mg / kg.

[0258] The chemical abbreviations used in this disclosure and the chemical names they refer to are as follows:

[0259] Example

[0260] The present disclosure is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions (such as those described in "J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002") or as recommended by the manufacturer.

[0261] The present disclosure is further described in detail below through specific preparation examples and biological experiments. However, it should be understood that these examples and biological experiments are for illustrative purposes only and should not be construed as limiting the present disclosure in any way. Those skilled in the art will understand that, unless otherwise specified, the materials used below are well-known in the art and can be obtained commercially or by those skilled in the art based on published literature or conventional methods. Unless otherwise stated, all compound preparation reactions of the present disclosure are carried out under continuous magnetic stirring in a dry nitrogen or argon atmosphere, with a dry solvent, wherein: (i) the temperature is expressed in degrees Celsius (°C), and the operation is carried out at room temperature, generally 15-35°C, preferably 20-30°C, more preferably 20-25°C; (ii) solvent removal is performed by rotary evaporator under reduced pressure, with a bath temperature not exceeding 60°C; (iii) the reaction process is monitored by thin-layer chromatography (TLC); (iv) the final product has a satisfactory proton nuclear magnetic resonance spectrum (NMR). 1 H-NMR and / or mass spectrometry (MS) data.

[0262] The negative control antibody in the examples is an irrelevant antibody and is used only as an IgG control.

[0263] Example 1: Preparation of compound LK1-1

[0264] Step 1:

[0265] Compound 1-2 (160 g, 1.0 eq) was added to a 10 L four-necked flask, followed by 2.4 L of ethylene glycol dimethyl ether. The mixture was mechanically stirred, then compound 1-1 (123.37 mL, 2.0 eq) was added. The mixture was stirred at 0 °C, and 10 mol / L sodium hydroxide (43.43 mL, 1.0 eq) was added dropwise using a constant pressure dropping funnel. The mixture was stirred for 1 h. TLC monitoring was performed. After the reaction was complete, acetic acid (12.42 mL) was added, and the mixture was stirred for 1 h. Water (660 mL) was added, and the mixture was stirred for 1 h. Distilled water (1.54 L) was added, and the mixture was stirred for 1 h. The mixture was filtered, and the filter cake was washed with 50% (v / v) ethylene glycol dimethyl ether aqueous solution (640 mL). The mixture was then dried in an oven at 40 °C to obtain 204.3 g of compound 1-3, with a yield of 99%. MS (ESI) m / z: 475.3 [M+H] + .

[0266] 1H NMR (400MHz, DMSO-d6) δ8.74(t,J=6.6Hz,1H),7.90(d,J=7.5Hz,2H),7.72(d,J=7.4Hz,2H),7.59(t,J=5.9Hz,1H ),7.49–7.21(m,9H),5.14(s,2H),4.63(d,J=6.7Hz,2H),4.34–4.18(m,3H),4.15(s,2H),3.63(d,J=6.0Hz,2H).

[0267] Step 2:

[0268] Compound 1-3 (180 g, 90%, 1.0 eq) was added to a 10 L four-necked flask, followed by 5.4 L of acetonitrile. The mixture was mechanically stirred, and then DBU (25.5 mL, 0.5 eq) was added. The mixture was stirred at room temperature for 4 h, and then HOBT (101.5 g, 2.2 eq) was added. The mixture was stirred at room temperature for 0.5 h, then cooled to 0 °C and stirred overnight. The mixture was filtered, and the filter cake was washed with acetonitrile (800 mL) and dried in an oven at 40 °C to give 119.0 g of the HOBT salt of compound 1-4, with a yield of 81%. MS (ESI) m / z: 253.4 [M+H] + .

[0269] 1 H NMR(400MHz, DMSO-d6)δ9.09(t,J=6.2Hz,1H),7.80–7.70(m,1H),7.60–7.27(m,7H) ),7.22–7.13(m,2H),5.16(s,2H),4.68(d,J=6.4Hz,2H),4.18(s,2H),3.45(s,2H).

[0270] Step 3:

[0271] Compound 1-5 (55 g, 1.0 eq) was added to a 5 L three-necked flask, dissolved in 600 mL acetonitrile and 100 mL water. Compound 1-4 (50 g, 1.0 eq) was then added, and EDCI (24 g, 1.0 eq) was added under stirring at 0 °C for 4 h. After the reaction was complete as monitored by TLC, 500 mL ethanol and 750 mL water were added, and the mixture was stirred overnight at room temperature. Then, 1450 mL water was added, and the mixture was stirred for 2 h. The mixture was filtered, and the filter cake was dried in an oven at 40 °C to give 80.0 g of compound 1-6, with a yield of 96%. MS (ESI) m / z: 648.4 [M+H] + .

[0272] Step 4:

[0273] Under nitrogen protection, 840 mL of tetrahydrofuran and 540 mL of water were added to compounds 1-6 (40.0 g, 1.0 eq), followed by the addition of Pd (5%) / C (8.8 g, 0.07 eq) to displace hydrogen. The mixture was stirred overnight at room temperature. After the reaction was complete as monitored by HPLC, Pd / C was filtered off using diatomaceous earth. The Pd / C was washed with 300 mL of water, and the filtrates were combined. The filtrate was concentrated under reduced pressure, and then concentrated again under reduced pressure with 400 mL of ethanol. This process was repeated three times. Subsequently, 800 mL of ethanol was added, and the mixture was magnetically stirred and filtered. The filter cake was dried in an oven at 40 °C to give 19.6 g of compounds 1-7, with a yield of 75%. MS (ESI) m / z: 424.2 [M+H] + .

[0274] 1 H NMR(400MHz,DMSO-d6)δ7.44–7.21(m,5H),4.75–4.53(m,3H),3.95(s,2H),3 .94–3.73(m,6H),3.15(dd,J=13.8,7.0Hz,1H),3.04(dd,J=13.7,8.4Hz,1H).

[0275] Step 5:

[0276] Compound 1-8 (5.4 g, 1.5 eq) of 6-(maleimide)hexanoic acid succinimide was added to a 250 mL single-necked flask and dissolved in 45 mL of acetonitrile. Compound 1-7 (5.0 g, 1.0 eq), 105 mL of water, and DIPEA (1.56 mL, 0.8 eq) were then added, and the mixture was stirred overnight at room temperature. After the reaction was monitored by HPLC to be complete, 50 mL of isopropyl acetate, 10 g of anhydrous sodium dihydrogen phosphate, and 0.65 g of disodium hydrogen phosphate were added to the reaction mixture, and the mixture was stirred for 0.5 h. The organic phase was removed by separation. Another 50 mL of isopropyl acetate was added, and the organic phase was removed by separation. In the aqueous phase, 25 mL of ethylene glycol dimethyl ether, 25 mL of ethyl acetate, 2.5 mL of acetonitrile, and 40 g of anhydrous sodium dihydrogen phosphate were added, and the mixture was stirred for 1 h before separating the aqueous phase. The organic phase was added twice, with 75 mL of acetonitrile, 11.3 mL of water, 3 g of sodium chloride, and 750 mg of anhydrous sodium dihydrogen phosphate. After stirring, the aqueous phase was separated. The organic phase was concentrated to 50 mL under reduced pressure, and 75 mL of ethylene glycol dimethyl ether was added to concentrate it to 50 mL. Then, 1 mL of water and 100 mL of ethylene glycol dimethyl ether were added, and the mixture was stirred overnight at room temperature. The mixture was then filtered, and the filter cake was added to 200 mL of ethylene glycol dimethyl ether and 6.5 mL of water. The mixture was stirred at 45 °C for 0.5 h, washed with 15 mL of a mixture of ethylene glycol dimethyl ether and water (v / v: 97 / 3), and the combined filtrates were concentrated to 100 mL. 25 mL of ethylene glycol dimethyl ether was added, and the mixture was stirred overnight at room temperature. The precipitated solid was washed with 25 mL of ethylene glycol dimethyl ether and dried under reduced pressure at 25 °C to give the 1,2-dimethoxyethane adducts of compounds 1-9, in 58% yield. MS (ESI) m / z: 617.3 [M+H] + .

[0277] 1 H NMR (400MHz, DMSO-d6) δ12.57(s,1H),8.57(s,1H),8.32(s,1H),8.14(d,J=7.1Hz,1H) ,8.08(s,1H),8.02(s,1H),7.21(d,J=22.0Hz,5H),7.00(s,2H),4.61(s,2H),4.50(s, 1H),3.98(s,2H),3.80–3.59(m,6H),3.43(s,4H),3.37(s,2H),3.24(s,6H),3.06(d,J =13.3Hz, 1H), 2.81 (t, J = 11.5Hz, 1H), 2.11 (s, 2H), 1.47 (d, J = 5.8Hz, 4H), 1.19 (s, 2H).

[0278] Step 6:

[0279] In a 10 mL single-necked flask, anhydrous sodium sulfate (60 mg, 2.25 eq), ethyl 2-oxime cyanoacetate (60 mg, 2.25 eq), compounds 1-9 (20 mg, 1.45 eq), 0.8 mL water, and 0.6 mL tetrahydrofuran were added. The mixture was stirred at room temperature for 30 min. Then, compound 1 (prepared according to the method disclosed in PCT / CN2024 / 091220, 100 mg, 1.0 eq), 0.3 mL water, and 0.5 mL tetrahydrofuran were added. After stirring for 15 min, N-methylmorpholine (23 μL, 1.1 eq) and 0.3 mL tetrahydrofuran were added. After stirring for 15 min, EDCI (72 mg, 2.0 eq), 0.5 mL water, and 0.5 mL tetrahydrofuran were added. The mixture was stirred at room temperature for 3 h, and the reaction was monitored by HPLC until complete. Extracted with dichloromethane, dried, and the combined organic phases were evaporated to dryness. Then, reversed-phase preparative separation was performed (Sante C18 column (20 g), 35-40% acetonitrile / water) to give 99 mg of compound LK1-1, yield 51%. MS (ESI) m / z: 1036.5 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.63(t,J=6.4Hz,1H),8.51(d,J=8.9Hz,1H),8.31(d,J=5.9Hz,1H),8.13(d,J=8.0Hz,1H),8.07(d,J=5.9Hz,1H), 8.01(d,J=5.8Hz,1H),7.77(d,J=10.9Hz,1H),7.30(s,1H),7.28–7.11(m,5H),6.99(s,2H),6.52(s,1H),5.60(d,J=7.5Hz,1H),5.19(s,2H ),4.63(d,J=6.5Hz,2H),4.47(s,1H),4.02(s,2H),3.81–3.53(m,6H),3.29–3.08(m,2H),3.02(d,J=14.3Hz,1H),2.77(t,J=12.1Hz,1H),2 .38(s,3H),2.25–2.14(m,2H),2.08(t,J=7.5Hz,2H),1.89–1.82(m,2H),1.46(d,J=10.0Hz,4H),1.20–1.08(m,2H),0.87(t,J=7.4Hz,3H).

[0280] Example 2: Preparation of compound LK1-2

[0281] Following the preparation method of Example 1, compound LK1-2 was prepared by replacing compound 1-1 (i.e., benzyl glycolate) with (R)-2-cyclopropyl-2-hydroxyacetic acid benzyl ester. MS (ESI) m / z: 1076.0 [M+H] + .

[0282] 1 H NMR (400MHz, DMSO-d6) δ8.62(t,J=6.6Hz,1H),8.51(d,J=8.8Hz,1H),8.30(t,J=5.9Hz,1H),8.12(d,J=8.0Hz,1H),8.07(t,J=5.8 Hz,1H),8.01(t,J=5.7Hz,1H),7.75(d,J=10.9Hz,1H),7.30(s,1H),7.27–7.12(m,5H),6.99(s,2H),6.52(s,1H),5.60(q,J=7.0H z,1H),5.25(d,J=19.1Hz,1H),5.12(d,J=19.0Hz,1H),4.68(dd,J=10.0,6.6Hz,1H),4.52–4.44(m,2H),3.76–3.51(m,7H),3.28– 3.07(m,3H),3.01(dd,J=13.8,4.5Hz,1H),2.81–2.69(m,1H),2.36(s,3H),2.19(q,J=7.9,7.0Hz,2H),2.08(t,J=7.5Hz,2H),1.90 -1.82(m,2H),1.50–1.41(m,4H),1.27–1.15(m,4H),0.87(t,J=7.3Hz,3H),0.65–0.39(m,4H).

[0283] Example 3: Preparation of compound LK1-3

[0284] Following the preparation method of Example 1, compound LK1-3 was prepared by replacing compound 1-1 (i.e., benzyl glycolate) with (S)-2-cyclopropyl-2-hydroxyacetic acid benzyl ester. MS (ESI) m / z: 1076.0 [M+H] + .

[0285] 1H NMR (400MHz, DMSO-d6) δ8.66(t,J=6.7Hz,1H),8.55(d,J=8.7Hz,1H),8.35–8.21(m,1H),8.16–8.04(m,2H),8.00(t,J=5.7Hz,1H),7.77(d,J=10. 8Hz,1H),7.32(s,1H),7.26–7.15(m,5H),6.99(s,2H),6.53(s,1H),5.54 (q,J=6.2,5.6Hz,1H),5.24(q,J=19.0Hz,2H),4.74(t,J=8.5Hz,1H),4.5 6(dd,J=10.3,6.6Hz,1H),4.47(t,J=10.1Hz,1H),3.71–3.54(m,7H),3. 25–3.00(m,4H),2.77(dd,J=13.9,9.8Hz,1H),2.37(s,3H),2.18(q,J=6. 8Hz,2H),2.09(t,J=7.5Hz,2H),1.85(q,J=6.6Hz,2H),1.50–1.41(m,4H) ,1.16(dt,J=13.6,8.0Hz,4H),0.87(t,J=7.4Hz,3H),0.51–0.41(m,4H).

[0286] Example 4: Preparation of compound LK1-4

[0287] Following the preparation method of Example 1, compound LK1-4 was prepared by replacing compound 1-1 (i.e., benzyl glycolate) with (R)-3-hydroxybutyrate benzyl ester. MS (ESI) m / z: 1064.0 [M+H] + .

[0288] 1H NMR (400MHz, DMSO-d6) δ8.49(t,J=7.9Hz,2H),8.29(t,J=5.8Hz,1H),8.16–8.04(m,2H),7.99(t,J=5.7Hz,1H),7.80(d,J =10.9Hz,1H),7.33(s,1H),7.28–7.08(m,5H),6.99(s,2H),6.54(s,1H),5.64–5.50(m,1H),5.22(q,J=19.2Hz,2H),4.73 –4.46(m,3H),4.03(q,J=6.5Hz,1H),3.76–3.55(m,6H),3.16(s,2H),3.02(dd,J=13.9,4.4Hz,1H),2.77(dd,J=13.9,9.7 Hz,1H),2.40(s,4H),2.26–2.08(m,5H),1.91-1.80(m,2H),1.51–1.42(m,4H),1.27–1.09(m,6H),0.87(t,J=7.3Hz,3H).

[0289] Example 5: Preparation of compound LK1-5

[0290] Following the preparation method of Example 1, compound LK1-5 was prepared by replacing compound 1-1 (i.e., benzyl glycolate) with (S)-3-hydroxybutyrate benzyl ester. MS (ESI) m / z: 1064.6 [M+H] + .

[0291] 1H NMR (400MHz, DMSO-d6) δ8.48(d,J=8.5Hz,2H),8.29(t,J=5.5Hz,1H),8.13(d,J=8.0Hz,1H),8.07(t,J=5.8Hz,1H),8.01(t,J=5.7 Hz,1H),7.79(d,J=10.9Hz,1H),7.30(s,1H),7.28–7.15(m,5H),6.99(d,J=1.8Hz,2H),6.52(s,1H),5.56(dd,J=8.9,4.7Hz,1H),5 .28–5.07(m,2H),4.56(d,J=6.5Hz,2H),4.49(q,J=8.4Hz,1H),4.00(p,J=6.4Hz,1H),3.77-3.56(m,6H),3.17(d,J=5.1Hz,2H),3. 04(dd,J=14.0,4.4Hz,1H),2.79(dd,J=13.9,9.7Hz,1H),2.46-2.39(m,4H),2.23(dd,J=14.1,6.4Hz,1H),2.18–2.05(m,4H),1.91 -1.80(m,2H),1.51-1.42(m,4H),1.25-1.13(m,6H),0.87(t,J=7.2Hz,3H).

[0292] Example 6: Preparation of compound LK1a-1

[0293] Step 1:

[0294] Glycine (3 g, 1.00 eq) was added to a 250 mL four-necked flask, followed by 90 mL of tetrahydrofuran and 30 mL of purified water. Sodium bicarbonate (3.70 g, 1.10 eq) was then added, and the mixture was mechanically stirred until the solution became clear. Compound 6-1 (12.8 g, 1.00 eq) was dissolved in 30 mL of DME and slowly added using a constant-pressure dropping funnel. The mixture was then stirred overnight at room temperature. 100 mL of saturated sodium bicarbonate and 100 mL of purified water were added, and the mixture was washed three times with 100 mL of ethyl acetate. The pH was adjusted to 4 by slowly adding 12 M hydrochloric acid. The mixture was filtered, the filter cake was washed with water, and dried to give 10.1 g of compound 6-2, with a yield of 98%. MS (ESI) m / z: 281.1 [M+H] + .

[0295] Step 2:

[0296] In a 500 mL four-necked flask, L-phenylalanine tert-butyl hydrochloride (6.5 g, 1.00 eq) and compound 6-2 (7.8 g, 1.1 eq) were added to 200 mL of N,N-dimethylformamide, followed by triethylamine (3.5 mL, 1.00 eq) and DMTMM (8.9 g, 1.20 eq). The mixture was stirred at room temperature for 2 hours. The reaction solution was then added to 1 L of purified water, filtered, and the filter cake was washed with water to give 11 g of compound 6-3, with a yield of 90%. MS (ESI) m / z: 484.2 [M+H] + .

[0297] Step 3:

[0298] Compound 6-3 (10 g, 1.0 eq), 100 mL of dichloromethane, and trifluoroacetic acid (50 mL, 30.00 eq) were added to a 250 mL four-necked flask and stirred at room temperature for 2 hours. The reaction solution was concentrated, slurried with MTBE, filtered, and the filter cake was washed with water to give 8.5 g of compound 6-4, with a yield of 96%. MS (ESI) m / z: 428.2 [M+H] + .

[0299] Step 4:

[0300] Following the preparation method of Example 1, N-(benzyloxy)carbonylglycylglycyl-L-phenylalanine (i.e., compound 6-4) was used to replace N-(benzyloxy)carbonylglycylglycyl-L-phenylalanine (i.e., compound 1-5) to prepare LK1a-1. MS (ESI) m / z: 1050.4 [M+H] + .

[0301] 1H NMR (400MHz, DMSO-d6) δ8.65(dt,J=12.6,6.7Hz,1H),8.52(dd,J=9.0,4.6Hz,1H),8.35(dt,J=20.4,5.8Hz,1H),8.19(dd,J=17.6,7.5Hz,1H),8.11(d ,J=7.8Hz,1H),8.04(t,J=5.7Hz,1H),7.78(d,J=10.9Hz,1H),7.31(s,1H), 7.26–7.17(m,5H),6.99(d,J=5.1Hz,2H),5.65–5.54(m,1H),5.20(s,2H),4 .63(d,J=6.5Hz,2H),4.54–4.42(m,1H),4.02(s,2H),3.80–3.63(m,6H),3. 47(t,J=5.3Hz,2H),3.18(d,J=17.9Hz,2H),3.02(dd,J=13.9,4.5Hz,1H),2 .92(s,2H),2.39(s,3H),2.27(t,J=7.4Hz,1H),2.19–2.13(m,3H),1.91–1. 80(m,2H),1.45(d,J=8.9Hz,4H),1.19–1.13(m,2H),0.85(d,J=6.8Hz,3H).

[0302] Example 7: Preparation of compound LK1b-1

[0303] Step 1:

[0304] Compound 7-1 (10.0 g, 1.0 eq) and N-hydroxysuccinimide (5.15 g, 1.2 eq) were added to a 250 mL single-necked flask and dissolved in 100 mL of dichloromethane. Then, N,N'-dicyclohexylcarbodiimide (10.0 g, 1.3 eq) was added, and the mixture was stirred overnight at room temperature. After the reaction was complete as monitored by TLC, the mixture was filtered, and the filtrate was concentrated under reduced pressure to give 14.0 g of compound 7-2. No further purification was required; it was used directly in the next reaction. MS (ESI) m / z: 366.4 [M+H] + .

[0305] In a 250 mL single-necked flask, 13.6 g (1.0 eq) of compound 7-2 was added and dissolved in 135 mL of acetonitrile. Then, 18.9 g (1.2 eq) of compound 7-3, 270 mL of water, and 5.3 mL (0.8 eq) of DIPEA were added, and the mixture was stirred overnight at room temperature. After the reaction was monitored by HPLC to be complete, the reaction solution was extracted three times with 300 mL of ethyl acetate. 10 g of anhydrous sodium dihydrogen phosphate was added to the aqueous phase, and the aqueous phase was extracted three times with 300 mL of ethylene glycol dimethyl ether. The ethylene glycol dimethyl ethers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the primary product. The crude product was separated using reverse-phase preparative chromatography (acetonitrile / water) to give 18.6 g of compound 7-4, with a yield of 74%.

[0306] In a 250 mL single-necked flask, compound 7-4 (8.3 g, 1.4 eq), compound 1 (prepared according to the method disclosed in PCT / CN2024 / 091220, 4.9 g, 1.0 eq), and 175 mL of DMF were added. The mixture was stirred at room temperature, and then triethylamine (1.9 mL, 1.6 eq) and DMTMM (3.87 g, 1.5 eq) were added. The mixture was stirred at room temperature for 1 hour, and the reaction was monitored to be complete by HPLC. The reaction solution was added to 500 mL of water, filtered, and dried to obtain 12 g of crude product. The crude product was purified by column chromatography to obtain 8.9 g of compound LK1b-1, yield: 85%. MS (ESI) m / z: 1093.6 [M+H] + .

[0307] 1H NMR (400MHz, DMSO-d6) δ9.10(s,2H),8.64(t,J=6.3Hz,1H),8.51(d,J=8.9Hz,1H),8.31(t,J=5.3Hz,1H),8.18(t,J=5.4Hz,1H),8.13(d,J= 7.8Hz,1H),8.06(t,J=5.1Hz,1H),7.76(d,J=10.9Hz,1H),7.30(s,1H),7.28–7.11(m,5H),6.52(s,1H),5.60(d,J=6.5Hz,1H),5.18(s,2H) ,4.64(d,J=6.2Hz,2H),4.47(q,J=8.8Hz,1H),4.02(s,2H),3.81–3.54(m,6H),3.41(s,3H),3.26–3.11(m,2H),3.02(dd,J=13.0,3.2Hz,1H ), 2.77(dd,J=13.2,10.1Hz,1H),2.56(t,J=7.0Hz,2H),2.41–2.29(m,5H),2.17(t,J=8.5Hz,2H),1.92–1.75(m,4H),0.87(t,J=7.2Hz,3H).

[0308] Example 8: Preparation of compound LK1c-1

[0309] Following the preparation method of Example 1, compounds 1-8 were replaced with 2,5-dioxopyrrolidone-1-yl (6-(2,5-dioxo-2,5-dihydro-1H-pyrrolidone-1-yl)hexanoyl)glycine ester (CAS No.: 1956326-21-8) to prepare LK1c-1. MS (ESI) m / z: 1093.5 [M+H] + .

[0310] 1H NMR (400MHz, DMSO-d6) δ8.66(t,J=6.7Hz,1H),8.52(d,J=8.9Hz,1H),8.31(t,J=5.9Hz,1H),8.11(d,J=7.1Hz,2H),8.05(dt,J=11.8,5.8Hz,2H),7.78 (d,J=10.9Hz,1H),7.31(s,1H),7.26–7.15(m,5H),6.99(s,2H),6.52(s,1H ),5.60(q,J=6.1Hz,1H),5.20(s,2H),4.64(d,J=6.6Hz,2H),4.47(td,J=8. 9,4.7Hz,1H),4.02(s,2H),3.78–3.65(m,7H),3.58(dd,J=17.1,5.2Hz,1H ),3.54–3.45(m,1H),3.23–3.12(m,2H),3.09–2.98(m,2H),2.82–2.72(m,1 H),2.38(s,3H),2.19(q,J=7.3Hz,2H),2.09(t,J=7.5Hz,2H),1.87-1.80(m ,2H), 1.47(d,J=11.8Hz,4H), 1.24(d,J=6.3Hz,2H), 0.87(t,J=7.2Hz,3H).

[0311] Example 9: Preparation of compound LK1d-1

[0312] Following the preparation method of Example 1, LK1d-1 was prepared by replacing compounds 1-8 with 2,5-dioxo-1-pyrrolidinyl 3-[2-[2-(2,5-dihydro-2,5-dioxo-1H-pyrrolidinyl)ethoxy]ethoxy]propionate (CAS: 1433997-01-3). MS (ESI) m / z: 1082.5 [M+H] + .

[0313] 1H NMR (400MHz, DMSO-d6) δ8.64(t,J=6.6Hz,1H),8.51(d,J=8.9Hz,1H),8.30(t,J=5.9Hz,1H),8.21–8.08(m,2H),8.00(t,J=5.7 Hz,1H),7.77(d,J=10.9Hz,1H),7.30(s,1H),7.26–7.14(m,5H),7.01(d,J=1.1Hz,2H),6.52(s,1H),5.60(q,J=6.0Hz,1H),5.1 9(s,2H),4.64(d,J=6.6Hz,2H),4.47(td,J=8.9,4.6Hz,1H),4.02(s,2H),3.78–3.64(m,5H),3.61–3.53(m,5H),3.50–3.39(m, 6H),3.23–3.11(m,2H),3.02(dd,J=13.9,4.5Hz,1H),2.76(dd,J=14.1,9.8Hz,1H),2.41–2.29(m,5H),2.21–2.15(m,2H),1.91 -1.81(m,2H),0.87(t,J=7.3Hz,3H).

[0314] Example 10: Human CDH6 and cynomolgus monkey CDH6 antigen information

[0315] The full-length amino acid sequence of human CDH6 used in the examples (SEQ ID NO:33) (Uniprot ID: P55285) is shown below and was purchased from Acro Bio (catalog number CA6-H5229).

[0316] Note: Double underlined parts are signal peptides (1-18); underlined parts are the extracellular region of CDH6 (19-615); dotted underlined parts are transmembrane regions (616-636); italicized parts are intracellular regions (637-790).

[0317] The full-length amino acid sequence (SEQ ID NO:34) (Uniprot ID: A0A2K5TW62_MACFA) of the cynomolgus monkey CDH6 (cyno-CDH6) used in the examples is shown below. It was purchased from Acro Bio (catalog number CA6-C52H3).

[0318] Note: Double underlined parts are signal peptides (1-21); underlined parts are the extracellular region of CDH6 (22-614); dotted underlined parts are transmembrane regions (615-636); italicized parts are intracellular regions (637-790).

[0319] Example 11: Antigen information of family members CDH9 and CDH10

[0320] The full-length amino acid sequence of human CDH9 used in the examples (SEQ ID NO:35) (Uniprot ID: Q9ULB4) is shown below, which was purchased from Acro Bio (catalog number CA9-H52H6).

[0321] Note: Double underlined parts are signal peptides (1-21); underlined parts are the extracellular region of CDH9 (22-615); dotted underlined parts are transmembrane regions (616-636); italicized parts are intracellular regions (636-789).

[0322] The full-length amino acid sequence of human CDH10 used in the examples (SEQ ID NO:36) (Uniprot ID: Q9Y6N8) is shown below, and it was purchased from Acro Bio (catalog number CA0-H52H5).

[0323] Note: Double underlined parts are signal peptides (1-22); underlined parts are CDH10 extracellular regions (23-613); dotted underlined parts are transmembrane regions (614-634); italicized parts are intracellular regions (634-788).

[0324] Example 12: Obtaining anti-CDH6 antibodies derived from mouse hybridomas

[0325] Anti-CDH6 monoclonal antibodies were produced by immunizing mice. Female SJL mice (Shanghai Lingchang Biotechnology Co., Ltd.) were used in the experiment. The mice were housed in SPF-grade environments. After purchase, the mice were housed in a laboratory environment for one week with a 12 / 12-hour light / dark cycle, temperature 20-25℃, and humidity 40-60%. Immunization was performed using both gene and cell methods, with vaccinations on days 0, 14, 28, 42, 56, and 70, and a booster immunization 3 days before spleen cell fusion. During this period, mouse serum antibody titers were measured using ELISA and FACS methods. After the sixth immunization, mice with high and plateauing antibody titers in their serum were selected for spleen cell fusion. An optimized electrofusion procedure was used to fuse spleen lymphocytes with myeloma Sp2 / 0 cells (…). CRL-8287 TM Hybridoma cells are obtained by fusing them together.

[0326] After culturing hybridoma cells for 7-14 days, the culture supernatant was collected. ELISA was performed using CDH6 recombinant protein to screen for antibodies in the hybridoma supernatant. Positive antibody strains were further screened using HEK-293 cells stably expressing CDH6, compared with blank HEK293 cells to exclude non-specific antibody-binding hybridoma strains. Flow cytometry was used to select hybridomas that bound both the recombinant protein and the cell-expressed antigen. Hybridoma cells in the logarithmic growth phase were collected, and RNA was extracted using Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript). TM Reverse Transcriptase (Takara#2680A). The cDNA obtained by reverse transcription was amplified by PCR using a mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) and then sequenced to obtain the amino acid sequences of the variable regions of the two monoclonal antibodies disclosed in this invention, as shown in Table 1A.

[0327] Table 1A shows the amino acid sequences of the variable regions of two monoclonal antibodies against CDH6 derived from mouse hybridomas.

[0328] Based on the above amino acid sequence, the CDR and FR of the antibody variable region were divided using the Kabat / IMGT numbering rule. The composition of the 6 CDR sequences of each antibody is shown in Table 1B below.

[0329] Table 1B CDR sequences of two monoclonal antibodies against CDH6 derived from mouse hybridoma.

[0330] Example 13: Construction of mouse hybridoma-derived anti-CDH6 chimeric antibody, its transient transfection expression in eukaryotic cells, and in vitro cell binding verification.

[0331] The target gene fragments generated by splicing the coding nucleic acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody of this disclosure, which had been sequenced, with the coding nucleic acid sequences of the IgG1 heavy chain constant region and κ light chain constant region, respectively, were cloned into the pTT5 expression vector to prepare transfection-grade expression plasmids. Expi293F cells were cultured in serum-free medium. TMCells (Thermo Fisher Scientific) were seeded in shake flasks (Corning Inc.) and cultured on a shaker at 37°C, 8% CO2, and 125 rpm. Cell density was adjusted, and the recombinant expression vector containing the target gene fragment and PEI transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flasks. Feeding was performed on days 1 and 3 post-transfection. After 6 days of cell culture, the expression supernatant was collected, centrifuged at high speed to remove cell debris, and purified using a Protein A column for affinity purification. The column was washed with PBS until the A280 reading returned to baseline. The target protein was eluted with acidic elution buffer (pH 3.0-3.5) and neutralized with 1 M Tris-HCl (pH 8.0-9.0). The eluted sample was appropriately concentrated, replaced with PBS, filtered, sterilized, and dispensed for use. The final purified chimeric antibody was analyzed for purity and A280 concentration by SDS-PAGE and HPLC.

[0332] OVCAR3, PA-1, and CHOK1-cyno CDH6 cells were cultured. OVCAR3 cells were cultured in RPMI 1640 + 10% FBS + Insulin (0.01 mg / ml). PA-1 cells were cultured in MEM + 10% FBS + 1% NEAA + 1% NAP. CHOK1-cyno CDH6 cells were cultured in F12K + 10% FBS + 200 μg / ml Hygromycin. All cells were cultured in T75 cell culture flasks at 37°C in a 5% CO2 incubator. Before use, cells were washed with sterile DPBS, digested with 0.25% trypsin and EDTA for approximately 5 minutes, and then the culture was stopped with complete culture medium.

[0333] Centrifuge the digested cells at 1000 rpm at room temperature for 5 minutes, discard the supernatant, and rehydrate with 100 μL of 5% FBS (in DPBS with Ca). 2+ Resuspend cells. Count cells and adjust the cell density to 1E6 / mL. Spread cells into 96-well round-bottom plates (corning 3799), centrifuge at 1500 rpm for 5 minutes at 4°C, discard the supernatant, and store at 4°C for later use. Incubate with 5% FBS (in DPBS with Ca). 2+ Dilute the antibody sample to be tested, starting at a concentration of 100 nM, and then dilute 4-fold down to 8 concentrations. Resuspend the cells in the diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm at 4°C for 5 minutes and discard the supernatant. 160 μL of 5% FBS (in DPBS with Ca) 2+ Resuspend and wash, centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Use 5% FBS (in DPBS with Ca) 2+Dilute the secondary antibody (goat anti-human IgG Fc APC) 1:500 according to the instructions, resuspend the cells in the diluted secondary antibody at 100 μL / well, and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Add 160 μL of 5% FBS (in DPBS with Ca) to each well. 2+ Resuspend and wash, centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Add 100 μL of 5% FBS (in DPBS with Ca) to the solution. 2+ Cells were resuspended, filtered through 300-mesh gauze, and the average fluorescence intensity of the APC channel was detected by flow cytometry.

[0334] Export the FCS file from the flow cytometer, and analyze the mean fluorescence intensity (MFI) of the APC channel for each sample using software. Based on the obtained mean fluorescence intensity, further analyze the half-maximal binding concentration (EC) of the antibody to cells. 50 The results of the highest average fluorescence intensity (Top MFI) are shown in Table 2 and Figures 1A-1C.

[0335] Table 2. Binding of mouse hybridoma-derived anti-CDH6 chimeric antibodies to CDH6-expressing cells.

[0336] Example 14: Internalization of mouse hybridoma-derived anti-CDH6 chimeric antibody

[0337] PA-1 and OVCAR3 cells in good growth condition were collected by centrifugation and seeded into 96-well white transparent plates at a certain cell density for overnight culture. The candidate antibody and positive control (H01L02) disclosed herein were mixed with DT3C at a certain ratio and incubated at 37°C for 30 minutes to form mAb-DT3C conjugates. The mAb-DT3C conjugates were serially diluted. The diluted mAb-DT3C conjugates were added to 96-well white transparent plates and incubated at 37°C with 5% CO2 for 3 days. After incubation, the 96-well plates were removed and equilibrated at room temperature for 30 minutes. A certain amount of CellTiter-GloReagent was added to each well, and the plates were incubated with shaking at room temperature for 10 minutes. The luminescence signal (RLU) was detected using a microplate reader. The internalization ability of each candidate antibody was calculated using the following formula. The results are shown in Table 3 and Figures 2A and 2B. Internalization rate (%) = (1 - RLU) 实验组 / RLU 对照组 )×100%

[0338] Table 3. Internalization of anti-CDH6 chimeric antibodies derived from mouse hybridomas in CDH6-expressing cells.

[0339] Example 15: Binding experiment of mouse hybridoma-derived anti-CDH6 chimeric antibody to family proteins CDH9 and CDH10

[0340] Dissolve the antigen protein human CDH9 (ACRO, catalog number CA0-H52H6) or human CDH10 (ACRO, catalog number CA0-H52H5) in 1X DPBS (with Ca2+). 2+ The antigen was added to a high-affinity ELISA plate (Biolegend, catalog number 423501) at a concentration of 1 μg / mL. The plate was then incubated overnight at 4°C. The antigen was washed three times with 300 μL of DPBS (infused with 0.05% Tween). The plate was then blocked with 200 μL of 2% BSA (in DPBS) and incubated at 37°C for 1.5 hours.

[0341] Dilute the antibody to be tested with 2% BSA (in DPBS) to an initial concentration of 100 nM, and then dilute 4-fold down to 8 concentrations. Wash the plate with DPBS (with 0.05% Tween added), 300 μL / well, three times. Add the diluted antibody to the ELISA plate, 100 μL / well, and incubate at room temperature for 2 hours. Wash the antibody with DPBS (with 0.05% Tween added), 300 μL / well, three times. Dilute the secondary antibody (Goat Anti-Human IgG Fc(HRP) for CDH9) 1:20000 with 2% BSA (in DPBS). Add 100 μL of the diluted secondary antibody to each ELISA plate and incubate at room temperature for 1 hour. Wash the plate 6 times with 300 μL of DPBS (with 0.05% Tween added). Prepare the chromogenic solution (TMA and TMB mixed 1:1) and add 100 μL of the solution to each well, incubating in the dark for 5 minutes. Add 50 μL of ELISA stop solution to the plate and mix well.

[0342] Read OD on envision 450 , using OD 450 Plot the results on GraphPad. The results are shown in Figures 3A and 3B, indicating that the tested antibody did not bind nonspecifically to either CDH9 or CDH10, which are proteins of the same family.

[0343] Example 16: Humanization of anti-CDH6 antibody derived from mouse hybridoma

[0344] After expression purification tests, cell-level binding and internalization tests, the two chimeric antibodies were designed to be humanized.

[0345] Humanization of murine anti-human CDH6 monoclonal antibodies was performed using methods published in numerous publications in the field. In short, a human constant domain was used to replace the parental (murine antibody) constant domain, and human antibody sequences were selected based on the homology between the murine and human antibodies. Based on the obtained typical VH / VL CDR structure of the murine antibody, the heavy and light chain variable region sequences were compared with human antibody germline databases to obtain highly homologous human germline templates.

[0346] The CDR region of the murine antibody was transplanted onto a selected humanized template, replacing the humanized variable region, and then recombined with the IgG constant region (preferably IgG1 for the heavy chain and κ for the light chain). Based on the three-dimensional structure of the murine antibody, reverse mutations were performed on embedded residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of VL and VH. Antibodies composed of the following humanized light and heavy chain variable region sequences were designed: 5 humanized antibodies were designed using 32G-12A10 (H1L1 / H2L1 / H3L1 / H4L1 / H5L1), and 3 humanized antibodies were designed using 26B-5F7 (H6L2 / H7L3 / H8L4). The heavy and light chain variable regions are shown in Table 4.

[0347] Table 4. Amino acid sequences of the variable regions of eight humanized antibodies derived from mouse hybridomas. Note: The underlined part is the CDR region determined according to the Kabat / IMGT numbering rules. The amino acid sequence of the heavy chain variable region HCDR2 of H6L2, H7L3 and H8L4 is QIYPGDGDTNYQGKFKG (SEQ ID NO:43).

[0348] Example 17: Preparation of humanized anti-CDH6 antibody derived from hybridoma

[0349] Referring to Example 13, the target gene fragment generated by splicing the variable region of the humanized antibody with the constant region of human IgG1 was cloned into the pTT5 expression vector to prepare a transfection-grade expression plasmid.

[0350] Expi293F cultured in serum-free medium TM Cells (Thermo Fisher Scientific) were seeded in shake flasks (Corning Inc.) and cultured on a shaker at 37°C with 8% CO2. Cell density was adjusted, and the recombinant expression vector containing the target gene fragment and PEI transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flasks. After 6 days of cell culture, the expression supernatant was collected, centrifuged at high speed to remove cell debris, and then purified using a Protein A column for affinity purification. The column was washed with PBS until A... 280The reading dropped to baseline. The target protein was eluted with acidic elution buffer (pH 3.0-3.5) and neutralized with 1M Tris-HCl (pH 8.0-9.0). After appropriate concentration, the eluted sample was transferred to PBS for aliquoting. The final purified humanized antibody was analyzed for purity by SDS-PAGE and HPLC. 280 Concentration determination.

[0351] Example 18: In vitro cell binding verification of mouse hybridoma-derived anti-CDH6 humanized antibody

[0352] OVCAR3, PA-1, and CHOK1-cyno CDH6 cells were cultured in T75 cell culture flasks at 37°C in a 5% CO2 incubator. Before use, cells were washed with sterile DPBS, digested with 0.25% trypsin and EDTA for approximately 5 minutes, and then the culture was stopped with complete culture medium.

[0353] Centrifuge the digested cells at 1000 rpm at room temperature for 5 minutes, discard the supernatant, and rehydrate with 100 μL of 5% FBS (in DPBS with Ca). 2+ Resuspend cells. Count cells and adjust the cell density to 1E6 / mL. Plate cells into 96-well round-bottom plates (corning 3799), centrifuge at 1500 rpm for 5 minutes at 4°C, discard the supernatant, and store at 4°C for later use. Dilute the antibody sample to be tested with 5% FBS (in DPBS with Ca2+), starting at 50 nM, and then dilute 4-fold down to 7 concentrations. Resuspend cells in the diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm for 5 minutes at 4°C, discard the supernatant. 160 μL of 5% FBS (in DPBS with Ca2+) is added to each well. 2+ Resuspend and wash, centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Use 5% FBS (in DPBS with Ca) 2+ Dilute the secondary antibody (goat anti-human IgG Fc APC) 1:500 according to the instructions, resuspend the cells in the diluted secondary antibody at 100 μL / well, and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Add 160 μL of 5% FBS (in DPBS with Ca) to each well. 2+ Resuspend and wash, centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Add 30 μL of 5% FBS (in DPBS with Ca) 2+ Resuspend cells, flow cytometry 3. Detect the average fluorescence intensity of the APC channel.

[0354] The average fluorescence intensity obtained from the analysis was imported into Graphpad to analyze the half-maximal binding concentration (EC50) of the antibody to cells. 50 The results of the highest average fluorescence intensity (Top MFI) are shown in Table 5 and Figures 4A-4F.

[0355] Table 5. Binding of mouse hybridoma-derived anti-CDH6 humanized antibodies to CDH6-expressing cells.

[0356] Example 19: Verification of the internalization of anti-CDH6 humanized antibody derived from mouse hybridoma

[0357] PA-1 and OVCAR3 cells in good growth condition were collected by centrifugation and seeded into 96-well white transparent plates at a certain cell density for overnight culture. The candidate antibody and positive control (H01L02) disclosed herein were mixed with DT3C at a certain ratio and incubated at 37°C for 30 minutes to form mAb-DT3C conjugates. The mAb-DT3C conjugates were serially diluted. The diluted mAb-DT3C conjugates were added to 96-well white transparent plates and incubated at 37°C with 5% CO2 for 3 days. After incubation, the 96-well plates were removed and equilibrated at room temperature for 30 minutes. A certain amount of CellTiter-GloReagent was added to each well, and the plates were incubated with shaking at room temperature for 10 minutes. The luminescence signal (RLU) was detected using a microplate reader. The internalization ability of each candidate antibody was calculated. The results are shown in Table 6 and Figures 5A-5D. Internalization rate (%) = (1-RLU) 实验组 / RLU 对照组 )×100%

[0358] Table 6. Internalization of anti-CDH6 humanized antibodies derived from mouse hybridomas in CDH6-expressing cells.

[0359] Example 20: Biacore affinity assay for mouse hybridoma-derived anti-CDH6 humanized antibody

[0360] The affinity and kinetic properties of the anti-CDH6 humanized antibody with human CDH6 were analyzed using a Biacore 8K instrument. The CM5 chip was first activated with EDC and NHS, then immobilized with a mouse monoclonal antibody against human Fc, and finally blocked with ethanolamine.

[0361] To determine the affinity and kinetic properties with human CDH6, the humanized CDH6 antibody was diluted to 10 μg / mL with HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM CaCl2) buffer and captured at a flow rate of 10 μL / min for 60 s. Human CDH6 was serially diluted two-fold to a series of concentrations (400 nM - 1.56 nM), bound at a flow rate of 30 μL / min for 120 s, and dissociated for 600 s.

[0362] After each round of experiments, the chip was washed with 3M MgCl2 solution at a flow rate of 30 μL / min for 30 s to remove the captured antibodies along with the antigens, thus completing chip regeneration. The raw data were analyzed using software and fitted with a (1:1) Langmuir model. The results are shown in Tables 7A and 7B.

[0363] Table 7A shows the affinity assay results between mouse hybridoma-derived anti-CDH6 humanized antibodies and human CDH6 antigen protein.

[0364] Table 7B shows the affinity assay results between the mouse hybridoma-derived anti-CDH6 humanized antibody and the CynoCDH6 antigen protein.

[0365] Example 21: Binding experiment of mouse hybridoma-derived anti-CDH6 humanized antibody with Mouse and RatCDH6

[0366] Dissolve the antigen protein Mouse CDH6 (ACRO, catalog number CA6-M52H8) or Rat CDH6 (Sino, catalog number 80277-R08H) in 1X DPBS (with Ca2+). 2+ The antigen was added to a high-affinity ELISA plate (Biolegend, catalog number 423501) at a concentration of 1 μg / mL. The plate was then incubated overnight at 4°C. The antigen was washed three times with 300 μL of DPBS (infused with 0.05% Tween). The plate was then blocked with 200 μL of 2% BSA (in DPBS) and incubated at 37°C for 1.5 hours.

[0367] Dilute the antibody to be tested with 2% BSA (in DPBS) to an initial concentration of 200 nM, and then dilute 4-fold down to 10 concentrations. Wash the plate with DPBS (with 0.05% Tween added), 300 μL / well, three times. Add the diluted antibody to the ELISA plate, 100 μL / well, and incubate at room temperature for 2 hours. Wash the antibody with DPBS (with 0.05% Tween added), 300 μL / well, three times. Dilute the secondary antibody (Goat Anti-Human IgG Fc(HRP) for CDH9) 1:20000 with 2% BSA (in DPBS). Add 100 μL of the diluted secondary antibody to each ELISA plate and incubate at room temperature for 1 hour. Wash the plate 6 times with 300 μL of DPBS (with 0.05% Tween added). Prepare the chromogenic solution (TMA and TMB mixed 1:1) and add 100 μL of the solution to each well, incubating in the dark for 5 minutes. Add 50 μL of ELISA stop solution to the plate and mix well.

[0368] Read OD on envision 450 , using OD 450 Plotting the results on GraphPad. The results are shown in Figures 6A and 6B, demonstrating that the tested antibodies specifically bind to both MouseCDH6 and RatCDH6.

[0369] Example 22: Binding experiment of mouse hybridoma-derived anti-CDH6 humanized antibody with family proteins CDH9 and CDH10

[0370] Dissolve the antigen protein human CDH9 (ACRO, catalog number CA0-H52H6) or human CDH10 (ACRO, catalog number CA0-H52H5) in 1X DPBS (with Ca2+). 2+ The antigen was added to a high-affinity ELISA plate (Biolegend, catalog number 423501) at a concentration of 1 μg / mL. The plate was then incubated overnight at 4°C. The antigen was washed three times with 300 μL of DPBS (infused with 0.05% Tween). The plate was then blocked with 200 μL of 2% BSA (in DPBS) and incubated at 37°C for 1.5 hours. Subsequent experimental procedures were performed as described in Example 15. The results were plotted on a GraphPad. Figures 7A and 7B show that the antibody did not specifically bind to either CDH9 or CDH10, both members of the same family.

[0371] Example 23: Drug Metabolism Study in Rats

[0372] The in vivo metabolism of the chimeric antibody 32G-12A10 derived from mouse hybridomas and the humanized antibodies H2L1, H3L1, and H4L1 was determined. SD rats with an average weight of approximately 150g were selected as experimental animals, and four rats (half male and half female) were administered each antibody. The administration was a single, single dose of 5 mg / kg, administered in a volume of 5 mL / kg. Serum samples were collected and separated at different time points after administration (0, 5 min, 1 h, 2 h, 4 h, 6 h, 24 h, 72 h, 120 h, 144 h, 240 h, 336 h, 504 h, and 672 h). The data are shown in Table 8.

[0373] Table 8. Data from PK studies in rats.

[0374] Example 24: Preparation of CDH6-resistant ADC

[0375] Example 24-1: ADC Analysis Method

[0376] 1. Determine the ADC DAR value using reversed-phase chromatography (RP-HPLC). Sample preparation: Sample concentration 0.5–1.5 mg / mL, completely reduced with excess DTT, centrifuged and the supernatant was injected; Instrumentation: Waters e2695 HPLC System; Column: Agilent PLRP-S (8 μm, 2.1 × 150 mm, Mobile phase A: 0.1% TFA - water; Mobile phase B: 0.1% TFA - acetonitrile; Flow rate: 0.3 mL / min; Detection wavelength: 214 nm, 280 nm, 360 nm; Column temperature: 60-90℃; Sample loading: 5-20 μg; RP chromatography elution method: Under equilibrium conditions, phase B increases from ~30% to ~45% within 23 minutes.

[0377] RP-HPLC data analysis: By comparing the spectra of the sample with those of the bare antibody, the positions of the light and heavy chains are distinguished. Then, the spectra of the sample are integrated to calculate the DAR value.

[0378] The calculation formula is as follows:

[0379] Total LC peak area = LC peak area + LC+1 peak area

[0380] Total HC peak area = HC peak area + HC+1 peak area + HC+2 peak area + HC+3 peak area

[0381] LC DAR = Σ(number of linked drugs * percentage of peak area) / total LC peak area

[0382] HC DAR = Σ(number of linked drugs * percentage of peak area) / total HC peak area

[0383] DAR = LC DAR + HC DAR

[0384] Where LC represents a light chain; LC+1 represents a light chain with one connector-load; HC represents a heavy chain; HC+1 represents a heavy chain with one connector-load; HC+2 represents a heavy chain with two connector-loads; HC+3 represents a heavy chain with three connector-loads.

[0385] 2. Determination of ADC purity by size exclusion chromatography-HPLC: Sample preparation: Sample concentration 1.0-5.0 mg / mL, centrifuge and collect supernatant for loading; Experimental instrument: Waters e2695 HPLC System; Chromatographic column: TOSOH, TSKgel G3000SWxL, 5μm, 7.8mm×300mm; Mobile phase: 150mmol / L K3PO4, 200mmol / L KCl, 10% isopropanol pH 7.0; Flow rate: 0.5-1mL / min; Detection wavelength: 214nm, 280nm, 360nm; Column temperature: 30℃; Loading amount: 30μg; SEC elution method: isocratic elution for 35min.

[0386] In this disclosure, PB refers to sodium phosphate buffer solution with disodium hydrogen phosphate (DHP) as the main component. DHP buffer solutions with different pH values ​​are typically prepared using solutions of sodium hydrogen phosphate and disodium dihydrogen phosphate at the same concentration.

[0387] Example 24-2: Preparation of Drug Conjugates (ADCs)

[0388] Antibodies 32G-H2L1 and 26B-H6L2 were replaced with 1×PBS buffer (pH 7.4 ± 0.2) by desalting chromatography or ultrafiltration. Antibody concentration was determined using a UV method. EDTA was added to the antibody solution to a final concentration of 5 mmol / L. The sample protein concentration was diluted to 4-10 g / L with 10 mmol / L phosphate + 137 mmol / L sodium chloride + 5 mmol / L EDTA buffer. 10 mM TCEP solution (6-10 equivalents) was added, and the mixture was incubated at 37°C for 3 hours. The reaction solution was then cooled to 15-25°C, and 10 mM linker-loaded LK1b-1 dimethyl sulfoxide solution (10-20 equivalents) was added. The mixture was incubated for 1.5-2.5 hours. After the coupling reaction was completed, 100 mM N-acetyl-cysteine ​​solution was added at a quenching ratio of 20:1 (quencher: antibody), and the mixture was shaken well and quenched at room temperature for 20 min to terminate the coupling reaction. The ADC buffer was replaced with 20 mM His-HAc + 9% sucrose buffer at pH 5.5 using desalting chromatography or ultrafiltration. The buffer was then filtered through a 0.22 μm PES filter to obtain the ADC stock solution, which was aliquoted according to experimental requirements and stored at -20℃ or -40℃. The absorbance was measured at 280 nm and 370 nm using UV chromatography to calculate the antibody concentration. The DAR value was detected using RP-HPLC, and the ADC purity was determined using SEC-HPLC. The obtained products are shown in Table 9 below. The amino acid sequences of antibodies 32G-H2L1 and 26B-H6L2 are shown in Table 10.

[0389] Table 9. ADC molecules and their DAR values ​​obtained in this publication.

[0390] Table 10 Full-length amino acid sequence of humanized antibodies against CDH6

[0391] Example 24-3: Preparation of control ADC Ds-6000a

[0392] Positive control molecules, antibodies H01L02 and ADC Ds-6000a were prepared according to CN 110651045 A (Example 7), with a DAR value of 7.8. The heavy chain amino acid sequence of the antibody is as follows:

[0393] The amino acid sequence of its antibody light chain is as follows:

[0394] Its connector-load structure is as follows:

[0395] Bioactivity test

[0396] Test Example 1: ADC Protein Level Binding Experiment

[0397] Experimental objective: To assess the affinity of the disclosed ADC for human CDH6 protein at the protein level using ELISA.

[0398] Experimental method: Dilute human CDH6 antigen (purchased from: SinoBiological) to 1 μg / ml with coating buffer, and use D-PBS (containing Ca) 2+ and Mg 2+ Dilute the antigen and add 100 μL to each well of a 96-well plate, incubate overnight at 4°C. Wash three times with PBST and block with 2% BSA for 2 hours. Prepare the test antibody: Dilute the test antibody to a specific concentration (50 nM) according to the dilution list, performing 4-fold serial dilutions for a total of 11 dilutions. After blocking, wash three times with PBST, add 50 μL of the diluted antibody to each well. Incubate at room temperature for 2 hours. Discard the liquid in each well, wash three times with PBST, add 50 μL of enzyme-labeled secondary antibody diluted to a specific concentration to each well. Incubate at room temperature for 1 hour. Discard the liquid in each well, wash five times with PBST. Invert the 96-well plate and gently tap on absorbent paper to remove residual liquid. Add 100 μL of TMB chromogenic solution to each well and incubate at room temperature in the dark for 10 minutes. Determine the stop time based on the color intensity. Add 50 μL of stop solution to each well. Measure the absorbance at 450 nm using a microplate reader within 30 minutes of adding the stop solution. The results were calculated using a 4-parameter curve fitting method. The binding activity of ADC protein levels is shown in Table 11.

[0399] Table 11 Affinity of ADC to human CDH6 protein level

[0400] Conclusion: At the protein level, the ADC1 disclosed in this paper has a slightly stronger binding ability to human CDH6 than Ds-6000a.

[0401] Test Example 2: Detection of in vitro antitumor activity of ADC

[0402] Experimental objective: To investigate the in vitro killing ability of the ADC molecule disclosed in this patent in tumor cell lines expressing different CDH6.

[0403] Experimental Methods: Log-phase tumor cells OVCAR-3 (CDH6-high expressing, human ovarian adenocarcinoma cells, purchased from Nanjing Kebai Biotechnology Co., Ltd.) and PA-1 (CDH6-low expressing, human ovarian teratoma cells, purchased from Nanjing Kebai Biotechnology Co., Ltd.) were added to cell culture plates at a density of 1500 cells / well (OVCAR-3) and 800 cells / well (PA-1). The cell culture plates were incubated overnight at 37°C in a 5% CO2 cell culture incubator. 50 μl of sample (starting at 4 μg / ml for OVCAR-3, 5-fold dilution, 9 concentrations; starting at 200 μg / ml for PA-1, 5-fold dilution, 9 concentrations) was added to each well, gently mixed, and incubated. After 6 days of incubation, 100 μL of CellTiter-Glo was added. TM (Promega, catalog number: G7571) Working solution, gently shake to lyse cells, and read the plate on a microplate reader. The cell proliferation inhibition rate is calculated as follows: Cell proliferation inhibition rate = (RLU cell control - RLU sample / RLU cell control - RLU culture medium control) × 100%. Plot the sample concentration (Log value) on the x-axis and Cytotoxicity% on the y-axis, and analyze the data to obtain the IC50 for each sample. 50 The values ​​are shown in Table 12.

[0404] Table 12 Results of in vitro activity tests of the loaded compounds disclosed herein

[0405] Conclusion: The ADC1 disclosed herein exhibits in vitro killing activity comparable to the control molecule Ds-6000a in OVCAR3 and PA-1 cell lines.

[0406] Test Example 3: The In Vitro Bystander Effect of ADCs

[0407] Experimental objective: To detect the in vitro bystander effect of the ADC compound disclosed herein using the OVCAR3 / MDA-MB-468 tumor cell system via mediator transfer method.

[0408] Experimental Methods: MDA-MB-468 (human breast cancer cells, purchased from the Cell Bank of the Chinese Academy of Sciences). CDH6+ cell seeding: OVCAR3 cells were collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1-2 ml of complete culture medium (RPMI 1640 + 20% FBS). An equal volume of trypan blue staining solution was mixed with the cell suspension, and the cells were counted using a Countstar counter. The cell density was adjusted to 5 × 10^6 cells / ml with complete culture medium. 4Cells / mL. Following the "Sample Layout," add 100.0 μl / well to the corresponding 96-well plate and incubate overnight. Dilute the test sample to 3× working solution using complete culture medium, starting at 100 μg / ml, with 5-fold gradients and 9 concentrations. Add 50.0 μl / well of the prepared test sample to the corresponding 96-well plate. After administration, gently tap the culture plate to mix. Incubate the 96-well plate in an incubator for 6 days. CDH6-cell seeding: Collect MDA-MB-468 cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in 1-2 ml of complete culture medium (complete culture medium: RPMI 1640 + 10% FBS). Mix the cell suspension with an equal volume of trypan blue staining solution, count the cells using a Countstar counter, and adjust the cell density to 4×10^9 cells using complete culture medium. 4 Cells / mL. Following the "Sample Layout," add 50.0 μl / well of the adjusted cells to the corresponding 96-well plate and incubate overnight. Dilute the test sample to 2× working solution using complete culture medium, starting at 100 μg / mL, with a 5-fold gradient and 9 concentrations. Add 50.0 μl / well of the prepared test sample to the corresponding 96-well plate. After administration, gently tap the culture plate to mix. Simultaneously, set up an MDA-MB-468 administration plate (100 μl OVCAR3 cell supernatant). Media transfer method: Discard all MDA-MB-468 culture medium and transfer 100 μl of the supernatant from the CDH6+ cell administration plate to the MDA-MB-468 administration plate per well. Incubate in an incubator for 6 days. After incubation, the 96-well cell culture plate was removed and allowed to stand at room temperature for 30 min. 100.0 μl of CellTiter-Glo luminescent dye was added to each well, and the plate was shaken at 200 rpm for 10 min at room temperature. All these procedures were performed under dark conditions. The experimental results are shown in Table 13.

[0409] Table 13 In vitro bystander test results of the disclosed ADC compounds

[0410] Conclusion: The ADC1 disclosed herein exhibits in vitro bystander killing capability comparable to that of the control molecule.

[0411] Test Example 4: Stability of ADCs in various plasmas in vitro

[0412] Experimental objective: To co-incubate the disclosed ADC with human / monkey / mouse / rat plasma in vitro, and to evaluate the toxin shedding rate and SEC purity change rate by LC-MS / MS.

[0413] Experimental Methods: ADC samples, healthy human plasma (donated by colleagues at Qilu Pharmaceutical Staff Hospital), cynomolgus monkey plasma (Nanjing Senbeijia Biotechnology Co., Ltd.), SD rat plasma (Shandong Xinbo Pharmaceutical Research Co., Ltd.), and CD1 mouse plasma (Beijing Huizhi Taikang Pharmaceutical Technology Co., Ltd.) were sterilized by filtration through a 0.22 μm filter. ADC samples were added to the sterile plasma at a final concentration of 100 ug / ml and incubated in a 37℃ cell culture incubator. The day of incubation was recorded as day 0. Samples were then collected on day 21 for free toxin detection.

[0414] Free toxin detection method: Add 15 μL of sample and 60 μL of acetonitrile to a centrifuge tube, vortex to mix, and centrifuge at 13000 rpm for 15 minutes; add 30 μL of supernatant and 30 μL of deionized water to a new centrifuge tube, vortex to mix, and perform LC-MS / MS analysis with 2 μL (Shimadzu LC-MS 8050 triple quadrupole LC-MS). The experimental results are shown in Table 14.

[0415] SEC purity determination method: The sample was centrifuged at 12,000 rpm for 10 minutes, and 3 μL of the supernatant was analyzed by SEC-FLR (Waters ACQUITY Arc liquid chromatograph - Waters 2475 fluorescence detector). The test results are shown in Table 15.

[0416] Table 14 Free toxin release rate in plasma of various genera

[0417] Table 15: Decrease rate of SEC purity at different time points during co-incubation of human plasma.

[0418] Conclusion: The ADC1 disclosed in this patent exhibits significantly better stability than the control ADC in plasma of various genera.

[0419] Test Example 5: Pharmacokinetics, concomitant hematologic toxicity, and in vivo stability of ADCs in cynomolgus monkeys

[0420] Experimental objective: Using cynomolgus monkeys as the relevant species, this study investigates the pharmacokinetic characteristics, associated hematologic toxicity, and in vivo stability of the disclosed ADC and control molecules after single-injection administration in cynomolgus monkeys.

[0421] Experimental Methods: Two cynomolgus monkeys (Hainan Jingang Biotechnology Co., Ltd., and Guangxi Fangchenggang Changchun Biotechnology Development Co., Ltd.) were randomly assigned to each group (half male and half female). A single injection of 80 MPa (prepared with an appropriate volume of physiological saline) was administered. Blood samples were collected before administration and at 5 minutes, 1 hour, 4 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, 120 hours, 168 hours, 240 hours, 336 hours, 504 hours, and 672 hours after administration. The concentrations of total antibody and conjugated antibody (ADC concentration) in plasma samples were detected using ELISA. The concentration of free toxin in plasma samples was detected using LC-MS / MS, and the DAR value of the ADC was determined using LC-HRMS. Pharmacokinetic parameters for different detection methods were calculated. Specific pharmacokinetic results are shown in Table 16, and the changes in the DAR value of the ADC in cynomolgus monkeys are shown in Table 17. Blood samples were collected 1 day before administration, 2 days after administration, 7 days after administration (blood routine test only), and 23 days after administration to test blood routine, blood coagulation and blood biochemistry. The test results are shown in Table 18, where ADC refers to the coupled mode concentration.

[0422] Table 16 Pharmacokinetic characteristics of ADCs in cynomolgus monkeys

[0423] Conclusion: In cynomolgus monkeys, at the same dose, the in vivo exposure of the ADC disclosed herein was higher than that of the control ADC, while the in vivo clearance rate was lower than that of the control ADC.

[0424] Table 17. Decrease rate of in vivo DAR value of ADC in cynomolgus monkeys

[0425] Conclusion: During the in vivo circulation of cynomolgus monkeys, the ADC disclosed in this study exhibited excellent in vivo DAR value stability, with no significant change in DAR value after 21 days of in vivo circulation, while the control molecule showed poor in vivo circulation stability.

[0426] Table 18 Summary of blood routine data in cynomolgus monkeys before and on day 7 after drug administration.

[0427] Conclusion: At a dose of 80 mpk, the control molecule showed significant reductions in white blood cell count, neutrophil count, reticulocyte count, and thrombocytopenia on day 7 post-administration, while the ADC1 disclosed in this study had significantly weaker effects on white blood cell and erythrocyte markers than the control molecule. Test Example 6: In vivo efficacy of ADC in an OVCAR3 xenograft model

[0428] Experimental objective: To evaluate the in vivo antitumor activity of the ADC disclosed herein in a CDH6-positive ovarian cancer OVCAR3 xenograft model.

[0429] Experimental Methods: BALB / c Nude mice (purchased from Vital River Pharmaceuticals, Beijing) were used as test animals to evaluate the efficacy of CDH6-ADC administered via tail vein injection in a human ovarian cancer cell OVCAR3 xenograft model. OVCAR3 cells (source: Nanjing Kebai Biotechnology Co., Ltd.) were subcutaneously inoculated into the right axilla of mice (5×10⁻⁶ cells). 6 (each tumor, with 50% artificial basement membrane), grew for 44 days, reaching an average tumor volume of 150 mm. 3 Animals were randomly divided into groups of 6 based on tumor volume. Treatment was administered via tail vein injection, once a week for a total of two weeks. Tumor volume and body weight were measured twice weekly and recorded. Tumor inhibition rate (TGI) (%) = [1 - (T... 36 -T0) / (V 36 -V0)]×100,T 36 T0 and V represent the tumor volume of the experimental group on day 36 after administration and on the day of administration, respectively. 36 V0 and Vb represent the tumor volumes of the blank control group (Vehicle, PBS) on day 36 after drug administration and on the day of drug administration, respectively. The experiment ended on day 36 after the start of drug administration. Detailed experimental results are shown in Table 19.

[0430] Table 19. In vivo efficacy evaluation of the disclosed ADC compounds.

[0431] Conclusion: The ADC disclosed in this patent exhibits antitumor activity comparable to the control molecule in the OVCAR3 xenograft model.

[0432] Test Example 7: In vivo efficacy of ADC in PA-1 xenograft model

[0433] Experimental objective: To evaluate the in vivo antitumor activity of the ADC disclosed herein in a CDH6-positive ovarian cancer PA-1 xenograft model.

[0434] Experimental Methods: BALB / c Nude mice (purchased from Vital River Pharmaceuticals, Beijing) were used as test animals to evaluate the efficacy of CDH6-ADC administered via tail vein injection in a human ovarian cancer cell PA-1 xenograft model. PA-1 cells (human ovarian teratoma cells, source: Nanjing Kebai Biotechnology Co., Ltd.) (5×10⁻⁶ cells) were subcutaneously inoculated into the right axilla of mice. 6 (each tumor, with 50% artificial basement membrane), grew for 49 days, reaching an average tumor volume of 260 mm. 3Animals were randomly divided into groups of 7 based on tumor volume. Treatment was administered via tail vein injection, once a week for a total of two weeks. Tumor volume and body weight were measured twice weekly and recorded. Tumor inhibition rate (TGI) (%) = [1 - (T... 21 -T0) / (V 21 -V0)]×100,T 21 T0 and V represent the tumor volume of the experimental group on day 21 after administration and day of administration, respectively. 21 V0 and V0 represent the tumor volumes of the blank control group (Vehicle, PBS) on day 21 after drug administration and on the day of drug administration, respectively. The experiment ended on day 21 after the start of drug administration. Detailed experimental results are shown in Table 20.

[0435] Table 20: In vivo efficacy evaluation of the disclosed ADC compounds

[0436] Conclusion: The ADC disclosed in this patent exhibits antitumor activity comparable to the control molecule in the PA-1 xenograft model.

[0437] Test Example 8: In vivo efficacy of ADC in the 786-O xenograft model

[0438] Experimental objective: To evaluate the in vivo antitumor activity of the ADC disclosed herein in a CDH6-positive human renal clear cell adenocarcinoma 786-O xenograft model.

[0439] Experimental Methods: BALB / c Nude mice (purchased from Vital River Pharmaceuticals, Beijing) were used as test animals to evaluate the efficacy of ADC administration via tail vein injection in a human renal clear cell adenocarcinoma 786-O xenograft model. Mice were subcutaneously inoculated with 786-O cell line (source: Nanjing Kebai Biotechnology Co., Ltd.) (5×10⁻⁶ cells). 6 (each tumor, with 50% artificial basement membrane), grew for 27 days, reaching an average tumor volume of 140 mm. 3 Animals were randomly divided into groups of 7 based on tumor volume. Treatment was administered via tail vein injection, once a week for a total of two weeks. Tumor volume and body weight were measured twice weekly and recorded. Tumor inhibition rate (TGI) (%) = [1 - (T... 42 -T0) / (V 42 -V0)]×100,T 42 T0 and V represent the tumor volume of the experimental group on day 42 after administration and on the day of administration, respectively. 42 V0 and V0 represent the tumor volumes of the blank control group (Vehicle, PBS) on day 42 after drug administration and on the day of drug administration, respectively. The experiment ended on day 42 after the start of drug administration. Detailed experimental results are shown in Table 21.

[0440] Table 21. In vivo efficacy evaluation of the disclosed ADC compounds.

[0441] Conclusion: The ADC disclosed in this patent exhibits significantly superior antitumor activity compared to the control molecule in the 786-O xenograft model.

[0442] Test Example 9: In vivo efficacy of ADC in the OV-90 xenograft model

[0443] Experimental objective: To evaluate the in vivo antitumor activity of the ADC disclosed herein in a CDH6-positive human ovarian cancer OV-90 xenograft model.

[0444] Experimental Methods: Female BALB / c Nude mice (purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.) were used as test animals to evaluate the efficacy of CDH6-ADC administered via tail vein injection in a human ovarian cancer cell OV-90 xenograft model. OV-90 cells (source: ATCC) were subcutaneously inoculated into the right axilla of mice (5×10⁻⁶ cells). 6 (each tumor, with 50% artificial basement membrane), grew for 10 days, reaching an average tumor volume of 130 mm. 3 Animals were randomly divided into groups of 8 based on tumor volume. Treatment was administered via tail vein injection, once weekly for a total of 3 weeks. Tumor volume and body weight were measured twice weekly and recorded. Tumor inhibition rate (TGI) (%) = [1 - (T... 35 -T0) / (V 35 -V0)]×100,T 35 T0 and V represent the tumor volume of the experimental group on day 35 after administration and on the day of administration, respectively. 35 V0 and Vb represent the tumor volumes of the blank control group (Vehicle, PBS) on day 35 after drug administration and on the day of drug administration, respectively. The experiment ended on day 35 after the start of drug administration. Detailed experimental results are shown in Table 22.

[0445] Table 22 In vivo efficacy evaluation of the disclosed ADC compounds

[0446] Conclusion: The ADCs disclosed in this patent exhibited antitumor activity comparable to the control molecule in the OV-90 xenograft model. At low doses (1 mpk), ADC1 and ADC2 showed superior efficacy compared to the control molecule.

[0447] The embodiments described above are merely exemplary, and any person skilled in the art will recognize or be able to identify numerous equivalents of specific compounds, materials, and operations without the need for extraordinary experimentation. All such equivalents are within the scope of this disclosure and are encompassed by the claims.

Claims

1. A ligand-drug conjugate of formula Ia or a pharmaceutically acceptable salt thereof, or its stereoisomer: in, Ab represents an anti-CDH6 antibody or its antigen-binding fragment, wherein the anti-CDH6 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the heavy chain variable region comprises the same HCDR1, HCDR2, and HCDR3 sequences as those in the heavy chain variable region shown in SEQ ID NO:19, and / or the light chain variable region comprises the same LCDR1, LCDR2, and LCDR3 sequences as those in the light chain variable region shown in SEQ ID NO:20; or, the heavy chain variable region comprises the same HCDR1, HCDR2, and HCDR3 sequences as those in the heavy chain variable region shown in SEQ ID NO:27, and / or the light chain variable region comprises the same LCDR1, HCDR2, and LCDR3 sequences as those in the heavy chain variable region shown in SEQ ID NO:

27. The LCDR1, LCDR2, and LCDR3 sequences of the light chain variable region shown in NO:28 are identical to those of LCDR1, LCDR2, and LCDR3; or, the heavy chain variable region contains the same HCDR1, HCDR2, and HCDR3 sequences as those of the heavy chain variable region shown in SEQ ID NO:3, and / or the light chain variable region contains the same LCDR1, LCDR2, and LCDR3 sequences as those of the light chain variable region shown in SEQ ID NO:4; L represents the linker connecting Ab and the X1 site of the warhead drug molecule; n is selected from 2-9; R1 is selected from H, halogens, OH, SH, NH2, and C. 1-4 Alkyl, C 1-4 Halogenated alkyl or C 1-4 Alkoxy; R2 is selected from H, halogens, and C. 1-4 Alkyl or C 1-4 Alkoxy; Alternatively, R1 and R2 can cyclize to form -O-(CH2). m -O-, where m is selected from 1, 2, and 3; R3 and R4 are each independently selected from H and C. 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkyl or C 1-4 Halogenated alkoxy groups; Alternatively, R3 and R4 can cyclize to form -(CH2). k - where k is selected from 1, 2, 3, and 4; X1 is selected from chemical bonds, -O-, -N(R6)-, and -O-CH(R5)-(CH2). p -CO-, where p is selected from 0, 1 and 2, and when X1 is an asymmetric structure, its CO end is connected to NH; R5 is selected from H, deuterium, CN, halogen, and C. 1-4 Alkyl, C 1-4 Halogenated alkyl, 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl; R6 is selected from H and C. 1-4 Alkyl or C 1-4 Halogenated alkyl groups.

2. The ligand-drug conjugate or its pharmaceutically acceptable salt, or its stereoisomer, according to claim 1, wherein R1 is C 1- 4 alkyl groups, and R2 is a halogen; or R1 is methyl and R2 is F; or R1 is H and R2 is H; or R1 and R2 are cyclized to form -O-CH2-O-; or R1 is NH2 and R2 is H or a halogen; or R1 is NH2 and R2 is H or F.

3. The ligand-drug conjugate or its pharmaceutically acceptable salt, or its stereoisomer, according to claim 1 or 2, wherein R3 is H and R4 is H; or R3 is H and R4 is C. 1-4 Alkyl; or, R3 is H and R4 is methyl; or R3 and R4 are cyclized to form -CH2-CH2-.

4. The ligand-drug conjugate or its pharmaceutically acceptable salt, or its stereoisomer, according to any one of claims 1-3, wherein n is selected from 7.0-9.0 or 7.0-8.5 or 7.2-8.3 or 7.3-8.0 or 7.5-8.

0.

5. The ligand-drug conjugate or its pharmaceutically acceptable salt, or its stereoisomer, according to any one of claims 1-4, wherein X1 is -O-CH(R5)-(CH2). p -CO-, p is selected from 0 or 1, R5 is selected from H, C 1-4 Alkyl, C 1-4 Haloalkyl, 3-6 membered cycloalkyl; or, X1 is -O-CH(R5)-CO-, R5 is selected from H or 3-6 membered cycloalkyl; or, X1 is -O-CH(R5)-CO-, R5 is selected from H or cyclopropyl; or, X1 is -O-CH(R5)-CH2-CO-; R5 is C 1-4 Alkyl group, or X1 is -O-CH(R5)-CH2-CO-, where R5 is methyl; or X1 is selected from the following groups: in, The -CO- end of X1 is connected to the NH phase in formula Ia.

6. The ligand-drug conjugate or its pharmaceutically acceptable salt, or its stereoisomer, according to any one of claims 1-5, wherein... Selected from the following compound fragments: in This indicates the position where the linker L is connected via a chemical bond.

7. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, according to any one of claims 1-5, wherein the conjugate is a compound of formula Ia-2 or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof; in, Ab, L, R1, R2, X1, n are as defined in any one of claims 1-5.

8. The ligand-drug conjugate or its pharmaceutically acceptable salt, or its stereoisomer, according to any one of claims 1-5, having the structure shown in formulas Ia-3: in, Ab, L, R5, R1, R2, n are as defined in any one of claims 1-5.

9. The ligand-drug conjugate or its pharmaceutically acceptable salt, or its stereoisomer, according to any one of claims 1-8, wherein L is -L1-L2-L3-L4-, wherein, One end of L1 is connected to the Ab, and one end of L4 is connected to the warhead drug molecule; L1 is selected from the following groups, and the end of L1 marked with an asterisk * is connected to the Ab: L2 is selected from chemical bonds, -N(R) 10 )-CH2-CO-、 Furthermore, one end of the CO pin of L2 is connected to L3, and the other end is connected to L1; R 10 Selected from C 1-4 Alkyl, C 1- 4. Haloalkyl; L3 is selected from polypeptide residues consisting of 2-6 amino acids, wherein the C-terminus of the polypeptide residue is connected to L4; and L4 is selected from chemical bonds, -NH-CH2-, One end of the CH2 component is connected to the warhead drug molecule, and the -NH- end is connected to L3.

10. The ligand-drug conjugate of claim 9 or a pharmaceutically acceptable salt thereof, or its stereoisomer, wherein L2 is a chemical bond; or, L2 is -N(CH3)-CH2-CO-; and / or L3 is selected from the following polypeptide residues: GFG, GGFG, GGGFG, GGVA, V-Cit, VA; or, L1 is selected from L2 is selected from chemical bonds or -N(CH3)-CH2-CO-, and L3 is selected from GFG, GGFG, GGGFG, and L4 is -NH-CH2-.

11. The ligand-drug conjugate or its pharmaceutically acceptable salt, or its stereoisomer, according to any one of claims 8-10, having the structure shown in formulas Ia-4: Wherein Ab, R5, and n are defined as in any one of claims 1-10; L2, L3, and L4 are defined as in claim 9 or 10.

12. The ligand-drug conjugate or its pharmaceutically acceptable salt, or its stereoisomer, according to any one of claims 1-11, wherein the anti-CDH6 antibody or its antigen-binding fragment comprises, respectively, HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and the light chain variable region comprises, respectively, LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; or the heavy chain variable region comprises, respectively, HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, and the light chain variable region comprises, respectively, LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16; or the heavy chain variable region comprises, respectively, LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:11, SEQ ID NO:43, and SEQ ID NO:

16. The light chain variable region includes HCDR1, HCDR2 and HCDR3 as shown in NO:13, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16 respectively.

13. The ligand-drug conjugate or its pharmaceutically acceptable salt, or its stereoisomer, according to any one of claims 1-12, wherein the anti-CDH6 antibody or its antigen-binding fragment comprises, respectively, an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the following groups of heavy chain and light chain variable regions: (1) SEQ ID NO:1 and 2; (2) SEQ ID NO:17 and 18; (3) SEQ ID NO:19 and 20; (4) SEQ ID NO:21 and 22; (5) SEQ ID NO:23 and 24; (6) SEQ ID NO:25 and 26; (7) SEQ ID NO:3 and 4; (8) SEQ ID NO:27 and 28; (9) SEQ ID NO:29 and 30; or (10) SEQ ID NO:31 and 32.

14. The ligand-drug conjugate or its pharmaceutically acceptable salt, or its stereoisomer, according to any one of claims 1-13, wherein the anti-CDH6 antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

15. The ligand-drug conjugate or its pharmaceutically acceptable salt, or its stereoisomer, according to any one of claims 1-14, wherein the anti-CDH6 antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:19 or SEQ ID NO:27 or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO:19 or SEQ ID NO:27, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:20 or SEQ ID NO:28 or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO:20 or SEQ ID NO:

28.

16. The ligand-drug conjugate or its pharmaceutically acceptable salt, or its stereoisomer, according to any one of claims 1-15, wherein, The anti-CDH6 antibody or its antigen-binding fragment comprises a heavy chain as shown in SEQ ID NO:37 and a light chain as shown in SEQ ID NO:38, or the anti-CDH6 antibody or its antigen-binding fragment comprises a heavy chain as shown in SEQ ID NO:39 and a light chain as shown in SEQ ID NO:

40.

17. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, according to any one of claims 1-16, wherein L is selected from the following fragments: in, The asterisk (*) indicates the position where the connector is connected to the warhead drug molecule, while the other end indicates the position where the connector is connected to the Ab.

18. The ligand-drug conjugate or its pharmaceutically acceptable salt, or its stereoisomer, according to any one of claims 1-17. in, Selected from the following structural fragment:

19. The ligand-drug conjugate or its pharmaceutically acceptable salt, or its stereoisomer, according to any one of claims 1-18, wherein the ligand-drug conjugate or its pharmaceutically acceptable salt, or its stereoisomer, is selected from the following: in, n is selected from 2-9 or 7.0-9.0 or 7.0-8.5 or 7.2-8.3 or 7.3-8.0 or 7.5-8.

0.

20. A pharmaceutical composition comprising a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, a stereoisomer thereof, and a pharmaceutically acceptable carrier, preferably, the pharmaceutical composition further comprising one or more other therapeutic agents.

21. Use of the ligand-drug conjugate or its pharmaceutically acceptable salt, stereoisomer, or the pharmaceutical composition according to any one of claims 1-19 in the preparation of a medicament for treating a disease, preferably, the disease being a disease associated with abnormal CDH6 expression, more preferably, the disease associated with abnormal CDH6 expression being a tumor, and even more preferably, the tumor being a solid tumor, such as cervical cancer, endometrial cancer, urothelial carcinoma, bladder cancer, bile duct cancer, kidney cancer, or ovarian cancer.

22. A method of treating a disease, comprising administering to a patient in need an effective amount of a ligand-drug conjugate as described in any one of claims 1-19 or a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a pharmaceutical composition as described in claim 20, preferably, the disease being a disease associated with abnormal CDH6 expression, more preferably, the disease associated with abnormal CDH6 expression being a tumor, and even more preferably, the tumor being a solid tumor, such as cervical cancer, endometrial cancer, urothelial carcinoma, bladder cancer, bile duct cancer, renal cancer, or ovarian cancer.

23. A ligand-drug conjugate according to any one of claims 1-19 or a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a pharmaceutical composition according to claim 20 for treating a disease, preferably, the disease being a tumor associated with abnormal CDH6 expression, more preferably, the disease associated with abnormal CDH6 expression being a tumor, and even more preferably, the tumor being a solid tumor, such as cervical cancer, endometrial cancer, urothelial carcinoma, bladder cancer, bile duct cancer, kidney cancer, or ovarian cancer.

24. An antibody against CDH6 or an antigen-binding fragment thereof, said antibody or antigen-binding fragment specifically binding to CDH6, comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, said light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein the amino acid sequences of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are respectively selected from the group consisting of: (1) SEQ ID NO: 5, 6, 7, 8, 9 and 10; (2) SEQ ID NO: 11, 12, 13, 14, 15 and 16; or (3) SEQ ID NO: 11, 43, 13, 14, 15 and 16; Alternatively, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are respectively selected from the heavy chain variable region and light chain variable region HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, and LCDR3 sequences of the group consisting of: (1) SEQ ID NO:1 and 2; (2) SEQ ID NO:17 and 18; (3) SEQ ID NO:19 and 20; (4) SEQ ID NO:21 and 22; (5) SEQ ID NO:23 and 24; (6) SEQ ID NO:25 and 26; (7) SEQ ID NO:3 and 4; (8) SEQ ID NO:27 and 28; (9) SEQ ID NO:29 and 30; or (10) SEQ ID NO:31 and 32.

25. The antibody or antigen-binding fragment thereof according to claim 24, wherein, The heavy chain variable region and the light chain variable region each comprise an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the following groups of heavy chain variable regions and light chain variable regions: (1) SEQ ID NO:1 and 2; (2) SEQ ID NO:17 and 18; (3) SEQ ID NO:19 and 20; (4) SEQ ID NO:21 and 22; (5) SEQ ID NO:23 and 24; (6) SEQ ID NO:25 and 26; (7) SEQ ID NO:3 and 4; (8) SEQ ID NO:27 and 28; (9) SEQ ID NO:29 and 30; or (10) SEQ ID NO:31 and 32.

26. The antibody or antigen-binding fragment thereof as described in claim 24 or 25, further comprising one or more of the following characteristics: (1) It also includes a heavy chain constant region and / or a light chain constant region; preferably, the heavy chain constant region contains Fc; and / or, Fc is derived from mice or humans; and / or, the sequence of Fc is a natural or modified variant; (2) It is a mouse-derived antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; (3) It is a monoclonal antibody, or a full-length antibody, or its antigen-binding fragment is Fab, Fv, scFv, F(ab')2, linear antibody, or single-domain antibody; (4) It is in the form of IgG1, IgG2, IgG3 or IgG4.

27. An antibody-drug conjugate formed by conjugating an anti-CDH6 antibody or its antigen-binding fragment as described in any one of claims 24-26 with other bioactive molecules, preferably, the other bioactive molecules being small molecule drugs, such as antitumor drugs, preferably antitumor compounds, and preferably, the anti-CDH6 antibody or its antigen-binding fragment and other bioactive molecules are linked by a linker.

28. A fusion protein, wherein, The fusion protein comprises an anti-CDH6 antibody or an antigen-binding fragment thereof as described in any one of claims 24-26.

29. A bispecific antibody or a multispecific antibody, wherein, The bispecific or multispecific antibody comprises at least two antigen-binding domains; one of the antigen-binding domains comprises an anti-CDH6 antibody or an antigen-binding fragment thereof as described in any one of claims 24-26.

30. A nucleic acid encoding an antibody against CDH6 as described in any one of claims 24-26, or an antigen-binding fragment thereof, a fusion protein as described in claim 28, or a bispecific or multispecific antibody as described in claim 29.

31. A recombinant vector comprising the nucleic acid as described in claim 30.

32. A host cell comprising the recombinant vector as claimed in claim 31 or the nucleic acid as claimed in claim 30.

33. The host cell according to claim 32, wherein it is a prokaryotic cell, such as Escherichia coli; or a eukaryotic cell, such as yeast or a mammalian cell, wherein the mammalian cell is preferably a CHO cell or a HEK293 cell.

34. A method for preparing an anti-CDH6 antibody or its antigen-binding fragment as described in any one of claims 24-26, a fusion protein as described in claim 28, or a bispecific or multispecific antibody as described in claim 29, comprising: The host cells of claim 32 or 33 are cultured under suitable conditions, and the expression product is purified from said cells.

35. A method for detecting CDH6 in a sample, comprising: (1) Contact the sample with the anti-CDH6 antibody or its antigen-binding fragment as described in any one of claims 24-26, the fusion protein as described in claim 28, or the bispecific antibody or multispecific antibody as described in claim 29; (2) Detect the formation of a complex of an anti-CDH6 antibody or its antigen-binding fragment, fusion protein, bispecific antibody or multispecific antibody with CDH6; optionally, the anti-CDH6 antibody or its antigen-binding fragment, fusion protein, bispecific antibody or multispecific antibody is detectably labeled.

36. A pharmaceutical composition comprising an anti-CDH6 antibody or an antigen-binding fragment thereof as described in any one of claims 24-26, or an antibody-drug conjugate as described in claim 27, or a fusion protein as described in claim 28, or a bispecific or multispecific antibody as described in claim 29, or a nucleic acid as described in claim 30, or a recombinant vector as described in claim 31, or a host cell as described in claim 32 or 33.

37. The pharmaceutical composition of claim 36, further comprising a pharmaceutically acceptable carrier.

38. The pharmaceutical composition of claim 36 or 37 further comprises one or more other therapeutic agents.

39. A method for treating a disease associated with abnormal CDH6 expression in a subject, the method comprising administering to a subject in need an effective amount of the pharmaceutical composition as described in any one of claims 36-38.

40. The method of claim 39, wherein the disease is a tumor, preferably a solid tumor, such as cervical cancer, endometrial cancer, urothelial carcinoma, bladder cancer, bile duct cancer, kidney cancer, or ovarian cancer.

41. The method of claim 39 or 40, further comprising administering additional therapeutic agents to the subject.

42. Use of the pharmaceutical composition according to any one of claims 36-38 in the preparation of a medicament for treating a disease, preferably, the disease is a disease associated with abnormal CDH6 expression, more preferably, the disease associated with abnormal CDH6 expression is a tumor, and even more preferably, the tumor is a solid tumor, such as cervical cancer, endometrial cancer, urothelial carcinoma, bladder cancer, bile duct cancer, kidney cancer, or ovarian cancer.

43. A pharmaceutical composition according to any one of claims 36-38 for treating a disease, preferably, the disease being a tumor associated with abnormal CDH6 expression, more preferably, the disease associated with abnormal CDH6 expression being a tumor, and even more preferably, the tumor being a solid tumor, such as cervical cancer, endometrial cancer, urothelial carcinoma, bladder cancer, bile duct cancer, kidney cancer, or ovarian cancer.

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