Camptothecin glycan-specific drug conjugate, preparation method therefor, and use thereof

Abstract

The present disclosure provides a class of novel camptothecin linker payloads that can be used for glycan-specific conjugation, a preparation method therefor, and use thereof. The class of camptothecin glycan-specific drug conjugates has uniform DAR values, excellent in vivo efficacy, and excellent pharmacokinetic characteristics, and is suitable for the preparation of drugs for treating tumor-related diseases.

Description

A camptothecin-based sugar-based site-directed drug conjugate, its preparation method and application

[0001] This application claims priority to Chinese patent application 2025103609113, filed on March 25, 2025; Chinese patent application 2026100436776, filed on January 13, 2026; and Chinese patent application 2026103287650, filed on March 17, 2026. The full text of the aforementioned Chinese patent applications is incorporated herein by reference. Technical Field

[0002] This disclosure belongs to the field of medicinal chemistry, specifically relating to a class of camptothecin-based sugar-based site-directed drug conjugates, their preparation methods, and applications, as well as a novel class of camptothecin-based linker loads that can be used for sugar-based site-directed conjugates, their preparation methods, and applications. These camptothecin-based sugar-based site-directed drug conjugates exhibit uniform DAR values, excellent in vivo efficacy and pharmacokinetic characteristics, and are suitable for preparing drugs for the treatment of tumor-related diseases. Background Technology

[0003] Antibody-drug conjugates (ADCs) are a class of biotechnology drugs composed of antibodies, cytotoxins, and linkers. ADCs combine the targeting ability of antibodies with the killing ability of cytotoxins, controlling the release of cytotoxins through linkers to achieve targeted killing of tumor cells. Compared to traditional chemotherapy drugs, ADCs have lower systemic toxicity and a wider therapeutic window; compared to traditional antibody drugs, ADCs have a more direct and less immune system-dependent tumor-killing ability. Currently marketed ADCs mainly rely on naturally occurring lysine (e.g., Trastuzumab emtansine) or cysteine ​​(e.g., Enfortumab vedotin) in antibodies. These ADCs generated using random conjugation methods of natural amino acids have poor homogeneity and unstable conjugation sites, resulting in poor drug safety and a narrow therapeutic window. Currently, various methods exist for preparing site-directed ADCs, including exogenous cysteine ​​insertion technology, non-natural amino acid insertion technology, enzyme-catalyzed conjugation technology, and glycosylation site-directed conjugation technology.

[0004] Natural antibodies possess a conserved N-glycosylation site in their Fc domain. This site has been utilized to develop various site-directed conjugation techniques. Kathrin Zuberbühler et al. used sodium periodate to oxidize fucose, introducing an aldehyde group for payload loading. Qun Zhou et al. used glycosyltransferases beta-1,4-Gal-T1 and sialyltransferase (Sia T) to treat antibodies with homogeneous sialic acid ions at the ends, followed by sodium periodate oxidation to introduce an aldehyde group for conjugation. Wild-type glycosidases can be used to truncate and homogenize antibodies into a shorter, more uniform form. Building on this, Laixi Wang et al. used mutant glycosidases (e.g., Endo SD233Q, Endo S2 D184M) to transfer oxazoline substrates with bioorthogonal reactive groups to the N-glycoterminus, subsequently achieving payload conjugation via bioorthogonal reactions. Based on truncated glycosyl antibodies, Pradman K. Qasba utilized the beta-1,4-galactosyltransferase1-Y289L mutant to achieve the transfer of galactose and N-acetylgalactosamine with bioorthogonal reactive groups, thus realizing payload conjugation. Huang Wei's team and Laixi Wang's team respectively reported the preparation of glycoengineered antibodies and ADCs using wild-type glycosidases Endo S and Endo S2 as substrates with disaccharide linkers. Currently, site-specific glycoconjugation is gradually being applied to ADC clinical research.

[0005] Due to the toxicity and water solubility limitations of eczemab, the impact of different DAR values ​​on pharmacokinetic characteristics, safety, and efficacy must be considered when preparing ADCs. Random conjugation of DAR6 antibody-drug conjugates has certain limitations, such as uneven DAR value distribution, significant batch-to-batch variability, the need for subsequent HIC purification steps, and low yield. Therefore, there is an urgent need to develop efficient and stable camptothecin-based linkers-loaders suitable for sugar-targeted conjugation to achieve efficient and specific conjugation, thereby improving product properties and therapeutic efficacy. Summary of the Invention

[0006] The purpose of this disclosure is to provide a novel antibody-drug conjugate.

[0007] In a first aspect, this disclosure provides the antibody-drug conjugate of formula (I), its isomers, or pharmaceutically acceptable salts thereof:

[0008] Ab-[YX-(Z-L1-L2-L3-D) m ] n (I)

[0009] Wherein, Ab is an antibody or antigen-binding fragment;

[0010] Y is Among them, the wavy line α represents being connected to Ab, and α represents being connected to X; X is a sugar or a sugar derivative.

[0011] Z is the segment connecting X and L1;

[0012] L1 is a chemical bond, or L1 is selected from... Furthermore, the -C(O)- terminal of L1 is connected to L2;

[0013] Where k is selected from integers from 0 to 8, j is selected from integers from 0 to 20, and p is selected from 0, 1, 2, 3, and 4;

[0014] L2 is selected from a peptide group composed of 2-10 amino acid residues; the amino acid is selected from natural amino acids or non-natural amino acids;

[0015] L3 is the segment connecting L2 and D;

[0016] D is a cytotoxic drug;

[0017] t is selected from 0 and 1;

[0018] m is selected from 1 to 3, and m is a decimal or an integer;

[0019] n is selected from 1 to 2, and n is a decimal or an integer.

[0020] In some embodiments of this disclosure, X is selected from disaccharide derivatives; preferably, X is selected from... Among them, wavy lines α represents being connected to Y, and α represents being connected to Z.

[0021] In some embodiments of this disclosure, -YX- is Among them, wavy lines * represents being connected to Ab, and * represents being connected to Z.

[0022] In some embodiments of this disclosure, Z is selected from:

[0023] Among them, the wavy line α represents being connected to X, and α represents being connected to L1.

[0024] In some embodiments of this disclosure, k is any integer from 3 to 5, for example, 4.

[0025] In some embodiments of this disclosure, j is any integer from 5 to 20, preferably any integer from 7 to 15, such as 7, 11 or 15.

[0026] In some embodiments of this disclosure, p is 2, 3, or 4, for example, 3.

[0027] In some embodiments of this disclosure, L1 is Furthermore, the -C(O)- terminal of L1 is connected to L2.

[0028] In some embodiments of this disclosure, L1 is selected from chemical bonds, Furthermore, the -C(O)- terminal of L1 is connected to L2.

[0029] In some embodiments of this disclosure, L1 is Furthermore, the -C(O)- terminal of L1 is connected to L2.

[0030] In some embodiments of this disclosure, L2 is a polypeptide fragment composed of 2-4 amino acids selected from the following: glycine, phenylalanine, valine, alanine, asparagine, citrulline, and the -NH- terminus of L2 is connected to L1.

[0031] In some embodiments of this disclosure, L2 is a polypeptide fragment composed of 2-4 amino acids selected from the following: glycine, L-phenylalanine, L-valine, L-alanine, L-asparagine, and L-citrulline, and the -NH-terminus of L2 is connected to L1.

[0032] In some embodiments of this disclosure, L2 is a polypeptide fragment composed of 2-4 amino acids selected from the following: glycine, phenylalanine, valine, alanine, and citrulline, and the -NH-terminus of L2 is connected to L1.

[0033] In some embodiments of this disclosure, L2 is a polypeptide fragment composed of 2-4 amino acids selected from the following: glycine, L-phenylalanine, L-valine, L-alanine, and L-citrulline, and the -NH-terminus of L2 is connected to L1.

[0034] In some embodiments of this disclosure, L2 is selected from -Val-Ala-, -Val-Cit-, -Gly-Gly-Phe-Gly- (SEQ ID NO:44), wherein the -NH- end of L2 is connected to L1.

[0035] In some embodiments of this disclosure, L2 is -L-Val-L-Ala-, -L-Val-L-Cit-, or -Gly-Gly-L-Phe-Gly-, wherein the -NH- end of L2 is connected to L1.

[0036] In some embodiments of this disclosure, L3 is selected from α represents being connected to D.

[0037] In some embodiments of this disclosure, D is a camptothecin derivative, for example... Where R 1 C 1-6 Alkyl (preferably C) 1- 4 alkyl groups, such as methyl), R 2 It is a halogen (preferably F, Cl or Br, for example F).

[0038] In some embodiments of this disclosure, D is selected from

[0039] In some embodiments of this disclosure, m is selected from 1 to 3 decimals or integers; preferably, m is selected from 2 to 3 decimals or integers.

[0040] In some embodiments of this disclosure, m is 2 or 3, for example, 3.

[0041] In some embodiments of this disclosure, n is selected from 1 to 2 as a decimal or an integer.

[0042] In some embodiments of this disclosure, n is 1 or 2, for example 2.

[0043] In some embodiments of this disclosure, the Ab is selected from anti-LIV1 antibody or its antigen-binding fragment, and anti-Her2 antibody or its antigen-binding fragment.

[0044] In some embodiments of this disclosure, the Ab is selected from anti-LIV1 antibodies or antigen-binding fragments thereof.

[0045] In some embodiments of this disclosure, the anti-LIV1 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 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:8, and SEQ ID NO:9, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, respectively. LCDR1, LCDR2, and LCDR3 as shown in NO:17 and SEQ ID NO:18; or the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively.

[0046] In some embodiments of this disclosure, the anti-LIV1 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region each comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the following groups of heavy chain variable regions and light chain variable regions:

[0047] (1) Heavy chain variable region SEQ ID NO:25 and light chain variable region SEQ ID NO:26;

[0048] (2) Heavy chain variable region SEQ ID NO:27 and light chain variable region SEQ ID NO:28;

[0049] (3) Heavy chain variable region SEQ ID NO:29 and light chain variable region SEQ ID NO:30; or

[0050] (4) Heavy chain variable region SEQ ID NO:31 and light chain variable region SEQ ID NO:32.

[0051] In some embodiments of this disclosure, the anti-LIV1 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region as shown in any of the following groups:

[0052] (1) Heavy chain variable region SEQ ID NO:25 and light chain variable region SEQ ID NO:26;

[0053] (2) Heavy chain variable region SEQ ID NO:27 and light chain variable region SEQ ID NO:28;

[0054] (3) Heavy chain variable region SEQ ID NO:29 and light chain variable region SEQ ID NO:30; or

[0055] (4) Heavy chain variable region SEQ ID NO:31 and light chain variable region SEQ ID NO:32.

[0056] In some embodiments of this disclosure, the anti-LIV1 antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0057] In some embodiments of this disclosure, the anti-LIV1 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:31 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:31, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:26 or SEQ ID NO:28 or SEQ ID NO:30 or SEQ ID NO:32 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:31, and / or the sequence shown in SEQ ID NO:26 or SEQ ID NO:28 or SEQ ID NO:30 or SEQ ID NO:32. The sequence shown in NO:32 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity.

[0058] In some embodiments of this disclosure, the anti-LIV1 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:31, and / or, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:26 or SEQ ID NO:28 or SEQ ID NO:30 or SEQ ID NO:32.

[0059] In some embodiments of this disclosure, the anti-LIV1 antibody or its antigen-binding fragment comprises a heavy chain as shown in SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39, and a light chain as shown in SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40.

[0060] In some embodiments of this disclosure, the anti-LIV1 antibody or its antigen-binding fragment comprises a heavy chain as shown in SEQ ID NO:33 and a light chain as shown in SEQ ID NO:34, or comprises a heavy chain as shown in SEQ ID NO:35 and a light chain as shown in SEQ ID NO:36, or comprises a heavy chain as shown in SEQ ID NO:37 and a light chain as shown in SEQ ID NO:38, or comprises a heavy chain as shown in SEQ ID NO:39 and a light chain as shown in SEQ ID NO:40.

[0061] In some embodiments of this disclosure, the Ab is selected from anti-Her2 antibodies or their antigen-binding fragments.

[0062] In some embodiments of this disclosure, the anti-Her2 antibody is trastuzumab.

[0063] In some embodiments of this disclosure, the Ab is selected from anti-LIV1 antibody or its antigen-binding fragment and anti-Her2 antibody or its antigen-binding fragment;

[0064] The anti-LIV1 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 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively. LCDR1, LCDR2, and LCDR3 as shown in NO:18; or the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively;

[0065] The anti-Her2 antibody or its antigen-binding fragment is trastuzumab.

[0066] In some embodiments of this disclosure, the Ab is selected from anti-LIV1 antibody or its antigen-binding fragment and anti-Her2 antibody or its antigen-binding fragment; wherein the anti-LIV1 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 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; or the heavy chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:9, respectively. HCDR1, HCDR2, and HCDR3 as shown in NO:15, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively; and the anti-Her2 antibody or its antigen-binding fragment is trastuzumab;

[0067] Y is Among them, the wavy line α represents being connected to Ab, and α represents being connected to X.

[0068] X is Among them, the wavy line α represents being connected to Y, and α represents being connected to Z;

[0069] Z is Among them, the wavy line α represents being connected to X, and α represents being connected to L1.

[0070] L1 is Furthermore, the -C(O)- terminal of L1 is connected to L2; where k is any integer from 0 to 8 (preferably any integer from 3 to 5, for example 4), j is any integer from 0 to 20 (preferably any integer from 5 to 20, more preferably any integer from 7 to 15, for example 7, 11 or 15), and p is 0, 1, 2, 3 or 4 (preferably 2, 3 or 4, for example 3);

[0071] L2 is a polypeptide fragment composed of 2-4 amino acids selected from the following: glycine, phenylalanine, valine, alanine, and citrulline, and the -NH-terminus of L2 is connected to L1.

[0072] L3 is α represents being connected to D;

[0073] D is Where R 1 C 1-6 Alkyl (preferably C) 1-4 Alkyl groups, such as methyl groups, R 2 It is a halogen (preferably F, Cl or Br, such as F);

[0074] t is 0 or 1 (e.g., 1);

[0075] m is any decimal or integer from 1 to 3 (e.g., 3);

[0076] n is any decimal or integer between 1 and 2 (e.g., 2).

[0077] Secondly, this disclosure provides an antibody-drug conjugate having a structure of formula (Ia).

[0078] Among them, Ab, Y, X, Z, L1, L2, m, and n are as defined above.

[0079] Thirdly, this disclosure provides the following specific antibody-drug conjugates or their pharmaceutically acceptable salts, and their stereoisomers:

[0080] Where t is selected from 0 or 1, and n is selected from 1 to 2 decimals or integers.

[0081] In some embodiments of this disclosure, n is 1 or 2, for example 2.

[0082] Fourthly, this disclosure provides a drug linker of formula (II), an isomer thereof, or a pharmaceutically acceptable salt thereof:

[0083] Z' is selected from α represents connection to L1; L1, L2, L3 and D are as defined in any of the present disclosures.

[0084] In some embodiments of this disclosure, L1 in formula (II) above is selected from chemical bonds, Furthermore, the -C(O)- terminal of L1 is connected to L2.

[0085] In some embodiments of this disclosure, L1 in equation (II) above is Furthermore, the -C(O)- terminal of L1 is connected to L2.

[0086] In some embodiments of this disclosure, L2 in formula (II) above is selected from -Val-Ala-, -Val-Cit-, -Gly-Gly-Phe-Gly-, and the -NH- end of L2 is connected to L1.

[0087] In some embodiments of this disclosure, L2 in formula (II) above is -L-Val-L-Ala-, -L-Val-L-Cit-, or -Gly-Gly-L-Phe-Gly-, wherein the -NH- end of L2 is connected to L1.

[0088] In some embodiments of this disclosure, L3 in formula (II) above is selected from... α represents being connected to D.

[0089] In some embodiments of this disclosure, D in formula (II) above is selected from...

[0090] Fifthly, this disclosure provides the following specific drug linkers, their isomers, or pharmaceutically acceptable salts thereof, selected from:

[0091] In a sixth aspect, this disclosure provides pharmaceutical compositions comprising the antibody-drug conjugates or pharmaceutically acceptable salts thereof provided in the first to third aspects of this disclosure, their stereoisomers, and pharmaceutically acceptable excipients. Preferably, the pharmaceutical composition further comprises one or more additional therapeutic agents.

[0092] In a seventh aspect, this disclosure provides the use of the antibody-drug conjugates or their pharmaceutically acceptable salts, stereoisomers thereof provided in the first to third aspects of this disclosure, or the pharmaceutical compositions provided in the sixth aspect of this disclosure, in the preparation of a medicament for treating a disease.

[0093] In some embodiments of this disclosure, in the above-described uses, preferably, the disease is a disease related to LIV1 expression or a disease related to Her2 expression.

[0094] In some embodiments of this disclosure, in the above-described uses, preferably, the disease is a disease related to LIV1 expression; more preferably, the disease related to LIV1 expression is a tumor; even more preferably, the tumor is a solid tumor, such as breast cancer, lung cancer, gastric cancer, head and neck squamous cell carcinoma, castration-resistant prostate cancer, and their metastatic forms. In some embodiments of this disclosure, in the above-described uses, preferably, the disease is a disease related to Her2 expression; more preferably, the disease related to Her2 expression is a tumor; even more preferably, the tumor is a solid tumor, such as breast cancer, lung cancer, gastric cancer, head and neck squamous cell carcinoma, castration-resistant prostate cancer, ovarian cancer, endometrial cancer, urothelial carcinoma, head and neck tumors, small cell lung cancer, nasopharyngeal carcinoma, non-small cell lung cancer, salivary gland tumors, colorectal cancer, esophageal cancer, bile duct cancer, gastroesophageal junction adenocarcinoma, and their metastatic forms.

[0095] In some embodiments of this disclosure, in the above-described uses, the tumor is breast cancer or gastric cancer. The breast cancer is, for example, MCF-7, MDA-MB-231, KPL-4, SKBR-3, or JIMT-1. The gastric cancer is, for example, NCI-N87.

[0096] Eighthly, this disclosure provides a method of treating a disease, comprising administering to a patient in need an effective amount of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a pharmaceutical composition provided in the sixth aspect of this disclosure.

[0097] In some embodiments of this disclosure, preferably, the disease described in the above method is a disease related to LIV1 expression or a disease related to Her2 expression. In some embodiments of this disclosure, preferably, the disease described in the above method is a disease related to LIV1 expression; more preferably, the disease related to LIV1 expression is a tumor; even more preferably, the tumor is a solid tumor, such as breast cancer, lung cancer, gastric cancer, head and neck squamous cell carcinoma, castration-resistant prostate cancer, and its metastatic forms.

[0098] In some embodiments of this disclosure, in the above method, preferably, the disease is a disease related to Her2 expression; more preferably, the disease related to Her2 expression is a tumor; even more preferably, the tumor is a solid tumor, such as breast cancer, lung cancer, gastric cancer, squamous cell carcinoma of the head and neck, castration-resistant prostate cancer, ovarian cancer, endometrial cancer, urothelial carcinoma, head and neck tumors, small cell lung cancer, nasopharyngeal carcinoma, non-small cell lung cancer, salivary gland tumors, colorectal cancer, esophageal cancer, bile duct cancer, adenocarcinoma of the gastroesophageal junction, and their metastatic forms.

[0099] In some embodiments of this disclosure, the tumor described in the above method is breast cancer or gastric cancer. The breast cancer is, for example, MCF-7, MDA-MB-231, KPL-4, SKBR-3, or JIMT-1. The gastric cancer is, for example, NCI-N87.

[0100] Ninthly, this disclosure provides antibody-drug conjugates or pharmaceutically acceptable salts thereof, stereoisomers thereof, or pharmaceutical compositions provided in the first to third aspects of this disclosure for the treatment of diseases.

[0101] In some embodiments of this disclosure, the disease is a disease associated with LIV1 expression or a disease associated with Her2 expression.

[0102] In some embodiments of this disclosure, preferably, the disease is a tumor associated with LIV1 expression; more preferably, the disease associated with LIV1 expression is a tumor; and even more preferably, the tumor is a solid tumor, such as breast cancer, lung cancer, gastric cancer, head and neck squamous cell carcinoma, castration-resistant prostate cancer, and their metastatic forms.

[0103] In some embodiments of this disclosure, preferably, the disease is a tumor associated with Her2 expression; more preferably, the disease associated with Her2 expression is a tumor; and even more preferably, the tumor is a solid tumor, such as breast cancer, lung cancer, gastric cancer, squamous cell carcinoma of the head and neck, castration-resistant prostate cancer, ovarian cancer, endometrial cancer, urothelial carcinoma, head and neck tumors, small cell lung cancer, nasopharyngeal carcinoma, non-small cell lung cancer, salivary gland tumors, colorectal cancer, esophageal cancer, bile duct cancer, adenocarcinoma of the gastroesophageal junction, and their metastatic forms.

[0104] In some embodiments of this disclosure, the tumor is breast cancer or gastric cancer. The breast cancer is, for example, MCF-7, MDA-MB-231, KPL-4, SKBR-3, or JIMT-1. The gastric cancer is, for example, NCI-N87.

[0105] Terms and Explanations

[0106] Unless otherwise stated, the terminology used in this disclosure has a general meaning in the art. 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 in this disclosure, they are intended to refer to the corresponding product or its active ingredient.

[0107] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if specifically and individually indicated to be incorporated by reference.

[0108] 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 in this disclosure 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 in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0109] 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.

[0110] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. BIOL. CHEM, 243, P3558 (1968).

[0111] As used in this disclosure, 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 have this structure. Each light chain consists of 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).

[0112] The term "antibody" in this disclosure may include complete antibodies (e.g., full-length monoclonal antibodies) and any antigen-binding fragment (i.e., antigen-binding portion) or their single chains, 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.).

[0113] Five main classes of antibodies are known in this art: IgA, IgD, IgE, IgG, and IgM, with their corresponding heavy chain constant domains designated 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 distinctly different types based on the amino acid sequence of their constant domains, termed κ and λ.

[0114] 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.

[0115] As used in this disclosure, the terms "anti-LIV1 antibody" or "LIV1-binding antibody" refer to an antibody that is capable of binding to LIV1 with sufficient affinity, or a fragment thereof.

[0116] As used in this disclosure, the terms "anti-Her2 antibody" or "Her2-binding antibody" refer to an antibody, or a fragment thereof, that is capable of binding to Her2 with sufficient affinity.

[0117] 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.

[0118] The term "Fc" is used in this disclosure 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.

[0119] 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.

[0120] The terms “full-length antibody,” “complete antibody,” and “intact antibody” may be used interchangeably in this disclosure to refer to an antibody whose structure is substantially similar to that of a natural antibody or which contains an FC region.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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, linear antibodies, single-chain antibodies (scFv), and bispecific or multispecific antibodies formed from antibody fragments, etc.

[0127] The term "Fab" or "Fab fragment" includes the heavy chain variable region and the light chain variable region, and also includes the light chain constant region and the first constant region CH1 of the heavy chain; it is a monovalent antibody fragment. The term "F(ab')2 fragment" contains two Fab fragments and a hinge region; it is a bivalent antibody fragment. 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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 in this disclosure, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0133] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably in this disclosure 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 operation such as conjugation with a labeled component. As used in this disclosure, 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 specified nucleic acid sequences.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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 linking 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 of this disclosure can be used to deliver cytotoxic agents or other payloads to target sites (e.g., tumorigenic cells and / or cells expressing LIV1). As used in this disclosure, the terms "drug" and "warhead" are used interchangeably and will refer to a bioactive or detectable molecule or compound, including anticancer agents. A "payload" may 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.

[0141] 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.

[0142] The term "effective dose" refers to a dosage of a pharmaceutical formulation of the antibody or its antigen-binding fragment 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.

[0143] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells in which exogenous nucleic acids are introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their progeny, regardless of the number of passages. Progeny may not be identical to parental cells in terms of nucleic acid content and may contain mutations. This disclosure includes mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells.

[0144] As used in this disclosure, the term "transfection" refers to the introduction of exogenous nucleic acids into eukaryotic cells. Transfection can be achieved by a variety of methods 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] As used in this disclosure, the terms “individual” or “subject” refer to any animal, such as a mammal or marsupial. Individuals in this disclosure 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.

[0151] As used in this disclosure, 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, the term "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).

[0152] As used in this disclosure, 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, and does not induce or inhibit the proliferation of normal cells.

[0153] As used in this disclosure, 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.

[0154] As used in this disclosure, the term "combination" refers to a treatment regimen that provides at least 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, which are used in combination when administered to a subject. 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.

[0155] As used in this disclosure, the term "linker" refers to a chemical structural fragment or bond that is linked to an antibody at one end and to a drug at the other end. It may also be linked to other linkers before being linked to a drug.

[0156] In this disclosure, the sulfur atom (S) at the α end of La in Formula I or any of its technical solutions, used to connect with Ab, actually originates from Ab. This sulfur atom S can be embodied in the La structural formula or omitted. For example, La is... With La It has the same meaning. The S atom at the corresponding position in the structures of other ADC compounds described in this disclosure is interpreted in the same way.

[0157] In this disclosure, H refers to the element hydrogen, which is a type of atom with one proton in its nucleus, including three isotopes: protium (P), deuterium (D), and tritium (T). H also represents one atom of the aforementioned element hydrogen.

[0158] In this disclosure, "independently selected" means that when multiple different variable groups and multiple substituents exist simultaneously, each variable group can be selected from the same or different ranges or options.

[0159] In this disclosure, "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0160] The term "alkyl" refers to a straight-chain or branched hydrocarbon group in which carbon atoms are linked by single bonds, both between carbon and hydrogen atoms. Alkyl groups are preferably C-type. 1-4 Or C 1-6 Alkyl group; C 1-4 "alkyl" refers to a straight-chain or branched alkyl group having 1-4 carbon atoms; C 1-4 Specific embodiments of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl, with methyl, ethyl, n-propyl, and isopropyl being preferred; "C 1-6 "Alkyl" refers to a straight-chain or branched alkyl group having 1-6 carbon atoms. C 1-6Specific embodiments of alkyl groups include, but are limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, and n-hexyl, with methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, and n-hexyl being preferred.

[0161] In this disclosure, "natural amino acids" refers to the following amino acids having the L-configuration: glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile I), proline (Pro), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), serine (Ser), threonine (Thr), cysteine ​​(Cys), methionine (Met), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), and histidine (His).

[0162] The term "non-natural amino acid" refers to amino acids other than natural amino acids, such as citrulline.

[0163] The term "optionally" means that the substituent may or may not be replaced by other substituents.

[0164] As used in this disclosure, the term "composition" means a product comprising specified amounts of each of the specified ingredients, and any product derived directly or indirectly from a combination of specified amounts of the specified ingredients. Those skilled in the art can vary the actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure so that the resulting amount of active compound is effectively targeted at a specific patient, composition, and route of administration to achieve the desired therapeutic response.

[0165] The term "pharmaceutically acceptable carrier" refers to a medium generally acceptable in the art for delivering bioactive pharmaceutical agents to animals, particularly mammals.

[0166] The term "excipient" generally refers to the carrier, diluent, and / or medium required to formulate an effective pharmaceutical composition.

[0167] The term "effective amount" refers to a compound of the present disclosure or a pharmaceutically acceptable salt thereof in sufficient quantity to provide a reasonable benefit / risk ratio for treating any medical condition and / or preventing the disorder.

[0168] The term "pharmaceutically acceptable salt" refers to a salt that, within the bounds of reliable medical judgment, is suitable for use in contact with human and lower animal tissues without causing excessive toxicity, irritation, allergic reactions, etc., and is commensurate with a reasonable benefit / risk ratio.

[0169] In this disclosure, a chemical bond in the substituent is labeled with When a hyphen appears in the substituent structure, it indicates the position where the substituent is connected to an adjacent group or structural segment. A hyphen "-" in the substituent structure indicates the connection point for the substituent; for example, -CH3 is connected via a carbon atom. The absolute configuration representing the center of a solid, i.e., the R or S configuration.

[0170] When a substituent's bond can be cross-linked to two atoms on a ring, this substituent can bond to any atom on that ring. For example, structural units. This indicates that the substituent R can be substituted at any position on the benzene ring.

[0171] The term "isomer" as used in this disclosure includes geometric isomers and stereoisomers, such as hindered trans isomers, cis-trans isomers, enantiomers, diastereomers, tautomers, and racemic mixtures and other mixtures thereof, 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 "hindered trans isomer" refers to a stereoisomer that can be separated due to hindered or very slow rotation of single bonds.

[0172] 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 a chiral column to achieve the separation of chiral compounds.

[0173] In this disclosure, the unit of solution concentration M represents mol / L, and nM represents nmol / L;

[0174] The chemical abbreviations used in this disclosure and the chemical names they refer to are as follows: Detailed Implementation

[0175] 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 reactions in this disclosure are carried out under continuous magnetic stirring in a dry nitrogen or argon atmosphere, using 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 using a rotary evaporator under reduced pressure, with a bath temperature not exceeding 60°C; (iii) the reaction process is monitored by thin-layer chromatography (TLC); and (iv) the final product has satisfactory 1H-NMR and / or mass spectrometry (MS) data.

[0176] Testing instruments:

[0177] The structures of the compounds disclosed herein were determined by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS). NMR chemical shifts (δ) are given in parts per million (ppm). NMR determinations were performed using a Bruker Neo 400M or Bruker Ascend 400 NMR instrument, with deuterated dimethyl sulfoxide (DMSO-d6) and deuterated water (D2O) as the solvents and tetramethylsilane (TMS) as the internal standard.

[0178] LC-MS determinations were performed using a Shimadzu 2030Plus-LCMS2020 mass spectrometer, an Agilent 1260-6125B single quadrupole mass spectrometer, or a Shimadzu LCMS-2020 mass spectrometer. HPLC determinations were performed using a Shimadzu LCMS-2020 or an Agilent 1260 high-performance liquid chromatograph.

[0179] Preparative high-performance liquid chromatography (HPLC) was performed using a Shimadzu FRC-40 equipped with LC-20AP and PDA-20A (column: Synergi Max-RP, 150×30mm, 4m) or a GILSON GX-281LC (column: Boston Prime C18 150*30mm*5μm; YMC-Actus Triart C18 150*30mm*5μm; YMC-Triart PFP 150*30mm*5μm; YMC-Triart Phenyl 150*30mm*5μm).

[0180] Example 1: Construction and expression of anti-LIV1 antibody

[0181] This disclosure discloses four groups of anti-LIV1 monoclonal antibodies obtained through hybridoma screening and humanization modification, named Ab1 to Ab4 respectively. The specific sequences are shown in Table 1. The division of the CDR of the antibody variable region is determined according to the Kabat definition.

[0182] Table 1. Amino acid sequences of each part of the variable region of the humanized antibody.

[0183] The gene fragments encoding the heavy and light chains of the aforementioned antibody molecules were cloned into the PTT5 expression vector to prepare transfection-grade expression plasmids, which were then transfected into Expi293 or ExpiCHO cells. Next, the transfected Expi293 or ExpiCHO cells were cultured in serum-free medium on a shaker at 37°C and 8% CO2 for 6–12 days. The supernatant was then collected, cell debris was removed by high-speed centrifugation, and affinity purification was performed using a Protein A column. The column was washed with PBS until the A column was reached. 280 The 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.

[0184] Example 2: Recombinant Expression and Purification of EndoSi Enzyme

[0185] The encoding gene of Endo Si from Streptococcus iniae was cloned into the pET22b vector (GenScript), and its amino acid sequence is SEQ ID NO:41, 34-928.

[0186] The plasmid containing the target gene was transformed into *E. coli* BL21(DE3) and plated on 2×YT agar plates containing 100 μg / mL ampicillin. The plates were incubated overnight at 37°C. Single colonies were picked and inoculated into 4 mL of 2×YT liquid medium containing 100 μg / mL ampicillin, and incubated overnight. 4 mL of the bacterial culture was then inoculated into 1 L of 2×YT broth medium containing 100 μg / mL ampicillin and incubated at 37°C until OD (Organic Growth Rate) was reached. 600 The culture was incubated until the concentration reached 0.8-1.0. Then, 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the culture and incubated at 20°C to induce protein overexpression. After 16 hours, the cells were harvested by centrifugation. The expression was then analyzed using B-PER. TM Bacterial Protein Extraction Reagent (Thermo) was used to lyse the cell pellet according to the manufacturer's instructions. The recombinant Endo Si protein was purified using the cOmplete His-Tag Purification Column (Roche) and the SDA030 protein purification system (Sepure). The protein was concentrated using an Amicon centrifuge filter (30 kDa, Millipore) and further processed through a HiLoad centrifuge. TM 26 / 600 Superdex TM Purification was performed using a 200 prep-grade column (Cytiva) via size exclusion. Fractions containing the Endo Si fusion protein were concentrated using an Amicon centrifugal filter (30 kDa, Millipore) and stored in storage buffer (20 mM PB, pH 7.5). Protein purity was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel imaging (Gel Doc EZ Imager, Bio-RAD) software. Protein concentration was quantified using a spectrophotometer (Nano-300).

[0187] Example 3: Synthesis of the disaccharide oxazoline compound G35

[0188] Reaction process:

[0189] Reaction steps:

[0190] Step 1: Weigh 26.2 g (100 mmol) of 2-[(azidoacetyl)amino]-2-deoxy-D-glucose and 64.1 g of UDP-Gal (disodium uridine 5′-bisphosphate galactose, 64.1 g, 105 mmol) into a 1000 mL single-necked reaction flask. Add 600 mL of deionized water and stir to dissolve. Add 4 g (42 mmol) of MgCl2 and 0.3 g of β-1,4-galactosyltransferase (NmLgtB-4) to catalyze the reaction. The system pH is 8.0, the reaction temperature is 37 °C, the stirring speed is 200 rpm, and the reaction is carried out overnight. After the reaction is complete, add an equal volume of ethanol to remove the enzyme. After centrifugation, concentrate the supernatant. Purify the concentrate by electrodialysis, concentrate through a membrane, and purify by silica gel column chromatography (dichloromethane:methanol = 10:1). Collect the fraction and concentrate to obtain compound G35-1 (8.2 g, yield 19%).

[0191] Step 2: At -15°C, TsCl (2.7 g, 14.16 mmol) was added to a 60 mL solution of pyridine containing compound G35-1 (2.0 g, 4.71 mmol) in a 100 mL three-necked flask, and the mixture was stirred for 13 hours. TLC (acetonitrile:water = 10:1) showed the reaction was complete. The mixture was diluted with ethyl acetate (200 mL), followed by 100 mL of saturated sodium bicarbonate solution. The mixture was separated, and the organic phase was washed with saturated brine (150 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 2.8 g of a yellow solid, compound G35-2, in 81% yield.

[0192] Step 3: Sodium azide (3.73 g, 57.38 mmol) was added to a 50 mL DMF solution containing compound G35-2 (2.8 g, 3.82 mmol) in a 100 mL three-necked flask, and the mixture was stirred at 60 °C for 40 hours. TLC (isopropanol:concentrated ammonia:water = 25:5:3) showed that the reaction was complete. The solution was concentrated under reduced pressure and prepared by C18 column chromatography (acetonitrile:water = 1:10-3:7) to give 1.8 g of crude white solid compound G35-3.

[0193] Step 4: At 0°C, potassium phosphate (K3PO4, 2.35 g, 11.07 mmol) and 2-chloro-1,3-dimethyl-1H-benzimidazole-3-onium chloride (CDMBI, 0.8 g, 3.68 mmol) were added to a 15 mL water solution (100 mL three-necked flask) containing compound G35-3 (0.35 g, 0.74 mmol), and the mixture was stirred overnight at 0°C. TLC (isopropanol:concentrated ammonia:water = 25:5:3) showed that the reaction was complete. Impurities were extracted with toluene (20 mL × 4), and the aqueous layer was directly prepared using C18 chromatography (acetonitrile:water = 1:10-1:3, preparative high-performance liquid chromatography (Wuhan Ruihe Chromatography Technology, LC 2100)) to give 166 mg of white solid compound G35, with a yield of 49%.

[0194] ESI-MS calc.for C 14 H 21 N 10 O8[M+H] + m / z=457.1, found m / z=457.1. 1 H NMR(400MHz,D2O)δ6.07(d,J=7.3Hz,1H),4.40–4.25(m,2H),4.20–4.11(m,1H),4 .10–3.92(m,1H),3.72(d,J=2.8Hz,1H),3.65(d,J=8.3Hz,2H),3.57–3.20(m,8H).

[0195] Example 4: Preparation of Compound 1

[0196] Step 1: In a 10 mL single-necked flask, add (2,5,8,11,14,17,20-heptaoxadecano-22-yloxy)acetic acid (1-1) (400.0 mg, 1.00 mmol) and HATU (380.0 mg, 1.00 mmol), and dissolve in DMF (5 mL). Add DIPEA (0.25 mL, 2.00 mmol) to the resulting solution and stir the mixture at room temperature for 15 minutes. Then add compound 1-2 (460.2 mg, 1.00 mmol, prepared according to WO 2019208820) to the mixture. Stir the reaction mixture at room temperature for 3 hours. Filter the reaction mixture and purify by preparative high-performance liquid chromatography to obtain compound 1-3.

[0197] LC-MS: 805.4 [M+H] + .

[0198] Step 2: Compound 1-3 (580.0 mg, 0.72 mmol) was added to a 50 mL single-necked flask and dissolved in TFA (2 mL) and dichloromethane (4 mL). The reaction solution was stirred at room temperature for 1 hour, then heated to 30 °C and stirred for 2 hours. The reaction solution was concentrated under reduced pressure, dissolved in 10 mL of toluene, and concentrated again under reduced pressure to obtain compound 1-4.

[0199] LC-MS: 749.4 [M+H] + .

[0200] Step 3: Compound 1-5 (75.4 mg, 0.10 mmol), DIPEA (0.05 mL, 0.3 mmol), and compound 1-4 (74.9 mg, 0.10 mmol) were added to a 100 mL single-necked flask and dissolved in DMF (2 mL). DMTMM (28.3 mg, 0.10 mmol) was added to the resulting solution in an ice bath, and the reaction mixture was stirred in an ice bath for 1 hour. The reaction mixture was filtered and purified by high-performance liquid chromatography to obtain compound 1-6.

[0201] LC-MS: 1485.7 [M+H] + .

[0202] Step 4: Add compound 1-6 (100.0 mg, 0.07 mmol) to a 10 mL single-necked flask and dissolve in DMF (2 mL). Add diethylamine (0.07 mL, 0.71 mmol) to the resulting solution. Stir the reaction mixture at room temperature for 1 hour. Concentrate the reaction mixture under reduced pressure, and slurry the residue with petroleum ether to obtain compound 1-7.

[0203] LC-MS: 1263.6 [M+H] + .

[0204] Step 5: Compounds 1-7 (80.0 mg, 0.06 mmol) and HATU (22.8 mg, 0.06 mmol) were added to a 10 mL single-necked flask and dissolved in DMF (2 mL). DIPEA (0.15 mL, 1.2 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. Then, {1-(9H-fluorene-9-yl)-3-oxonyl-4-aza-2,7,10,13-tetraoxapentadecan-15-yl]oxy}acetic acid (28.4 mg, 0.06 mmol) was added to the mixture. The reaction mixture was evaporated to dryness, and the silica gel column was eluted with 5% methanol-dichloromethane solution to obtain 1-8.

[0205] LC-MS: 1718.9 [M+H] + .

[0206] Step 6: Add compounds 1-8 (50.0 mg, 0.03 mmol) to a 10 mL single-necked flask and dissolve in DMF (1 mL). Add diethylamine (0.45 mL, 0.3 mmol) to the resulting solution. Stir the reaction mixture at room temperature for 1 hour. Concentrate the reaction mixture under reduced pressure, and slurry the residue with petroleum ether to obtain compounds 1-9.

[0207] LC-MS: 1496.8 [M+H] + .

[0208] Step 7: In a 100 mL single-necked flask, add DBCO-acid (213.12 mg, 0.70 mmol), HATU (398.11 mg, 1.05 mmol), and HOBT (141.48 mg, 1.05 mmol), and dissolve in DMF (5 mL). Add DIPEA (0.23 mL, 1.40 mmol) to the resulting solution. Stir the mixture at room temperature for 5 minutes, then add compounds 1-9 (1044.69 mg, 0.70 mmol). Stir the reaction mixture at room temperature for 3 hours. After the reaction is complete as monitored by LCMS, filter the reaction solution and purify by reverse-phase reaction to obtain a pale yellow solid, compound 1.

[0209] LC-MS: 1783.3 [M+H] + .

[0210] 1H NMR (400MHz, DMSO-d6) δ9.99 (s, 1H), 8.17 (d, J = 6.4Hz, 1H), 8.05 (d, J = 8.8Hz, 1H),7.88(d,J=8.8Hz,1H),7.80–7.73(m,2H),7.67–7.57(m,6H),7.51–7.42(m ,3H),7.38–7.28(m,6H),6.51(s,1H),5.45(s,2H),5.33–5.23(m,3H),5.08(s ,2H),5.02(d,J=14.4Hz,1H),4.41–4.35(m,2H),4.22–4.17(m,1H),3.91(s,2H ),3.83(s,2H),3.59–3.42(m,42H),3.30–3.26(m,2H),3.23(s,3H),3.14–3.0 3(m,5H),2.61–2.53(m,1H),2.37(s,3H),2.27–2.15(m,3H),2.02–1.95(m,2H) ,1.92–1.83(m,2H),1.79–1.72(m,1H),1.69–1.61(m,1H),1.58–1.50(m,1H),1 .42–1.36(m,2H),1.29(d,J=6.8Hz,3H),1.26–1.19(m,2H),0.90–0.81(m,9H).

[0211] Example 5: Preparation of Compound 2

[0212] Step 1: Compounds 1-9 (748.35 mg, 0.50 mmol) were dissolved in DMF (10 mL) in a 50 mL single-necked flask. PPTS (125.67 mg, 0.50 mmol), HOBT (33.78 mg, 0.25 mmol), and triethylamine (139.4 μL, 1.00 mmol) were added separately. The mixture was stirred at room temperature for 5 minutes, and then endo-BCN-O-PNB (189.20 mg, 0.60 mmol) was added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction was monitored by LCMS until complete. The reaction mixture was filtered and purified by reverse-phase chromatography (column: C18, 50*250 mm, 10 μm; gradient: 25-45% B; mobile phase A: 0.1% NH4Ac / H2O; mobile phase B: CH3CN; flow rate: 70 mL / min) to obtain a pale yellow solid, compound 2.

[0213] LC-MS: 1672.8 [M+H]+ .

[0214] Example 6: Preparation of Compound 3

[0215] Step 1: Compound 3-1 (559.0 mg, 1.0 mmol, prepared according to WO 2015155976) and (4-aminophenyl)methanol (123 mg, 1.0 mmol) were added to a 100 mL single-necked flask. The mixture was dissolved in dichloromethane (5 mL) and methanol (5 mL). EEDQ (296.7 mg, 1.2 mmol) was added dropwise to the solution, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 93:7) to obtain compound 3-2.

[0216] LC-MS: 664.3 [M+H] + .

[0217] Step 2: Compound 3-2 (0.66 g, 1.00 mmol) was dissolved in DMF (10 mL), and DIPEA (1.32 mL, 7.99 mmol) and di(p-nitrophenyl) carbonate (1.82 g, 5.99 mmol) were added. The mixture was stirred overnight at room temperature. The solution was purified by C18 column chromatography using acetonitrile:water as the eluent to give compound 3-3.

[0218] LC-MS: 851.4 [M+Na] + .

[0219] Step 3: Compound 3-3 (300 mg, 0.36 mmol) was dissolved in 10 mL of DMF. Ecinotecan mesylate (192.4 mg, 0.36 mmol) and HOBT (49.0 mg, 0.36 mmol) were added sequentially. After stirring briefly, N,N-DIPEA (0.12 mL, 0.72 mmol) was added, and the mixture was stirred at room temperature for 3 h. Aqueous products were added to precipitate the product. The precipitate was filtered, and the filter cake was lyophilized to obtain compound 3-4.

[0220] LC-MS: 1125.6 [M+H] + .

[0221] Step 4: Dissolve compound 3-4 (330 mg, 0.3 mmol) in DMF (8 mL), add diethylamine (0.3 mL, 3 mmol), and stir at room temperature for 30 min. Dry the reaction mixture by rotary evaporation, then slurry with petroleum ether to obtain compound 3-5.

[0222] LC-MS: 903.4 [M+H] + .

[0223] Step 5: Compound 3-5 (90.3 mg, 0.1 mmol) and HATU (57 mg, 0.15 mmol) were added to a 50 mL single-necked flask and dissolved in DMF (1 mL). DIPEA (25.8 mg, 0.2 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. Then, [(2,2-dimethyl-4-oxoylide-5-aza-3,8,11,14-tetraoxahexadecane-16-yl)oxy]acetic acid (52.6 mg, 0.15 mmol) was added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was evaporated to dryness and purified by preparative high-performance liquid chromatography to obtain compound 3-6.

[0224] LC-MS: 1236.5 [M+H] + .

[0225] Step 6: Add compound 3-6 (60 mg, 0.049 mmol) to a 50 mL single-necked flask, dissolve it in dichloromethane (1 mL), add trifluoroacetic acid (0.2 mL) to the solution, and stir the mixture at room temperature for 30 minutes. Rotate the reaction solution to dryness to obtain the crude product compound 3-7.

[0226] LC-MS: 1136.5 [M+H] + .

[0227] Step 7: In a 50 mL single-necked flask, add compound 3-7 (70 mg, 0.06 mmol) and DMTMM (26.5 mg, 0.09 mmol), and dissolve in DMF (1 mL). Add DIPEA (25.8 mg, 0.2 mmol) to the resulting solution and stir the mixture at room temperature for 15 minutes. Then add compound 1-4 (67 mg, 0.09 mmol) to the mixture. Stir the reaction mixture at room temperature for 3 hours. Rotate the reaction mixture to dryness and purify by preparative high-performance liquid chromatography to obtain compound 3-8.

[0228] LC-MS: 1867.9 [M+H] + .

[0229] Step 8: Add compound 3-8 (55.0 mg, 0.03 mmol) to a 50 mL single-necked flask and dissolve in DMF (1 mL). Add diethylamine (22.2 mg, 0.3 mmol) to the resulting solution. Stir the reaction mixture at room temperature for 1 hour. Concentrate the reaction mixture under reduced pressure, and slurry the residue with petroleum ether to obtain compound 3-9.

[0230] LC-MS: 1644.8 [M+H] + .

[0231] Step 9: In a 100 mL single-necked flask, add DBCO-acid (102.1 mg, 0.33 mmol), HATU (190.7 mg, 0.50 mmol), and HOBT (67.8 mg, 0.50 mmol), and dissolve in DMF (5 mL). Add DIPEA (0.12 mL, 0.69 mmol) to the resulting solution. Stir the mixture at room temperature for 5 minutes, then add compounds 3-9 (550 mg, 0.33 mmol). Stir the reaction mixture at room temperature for 3 hours. After the reaction is complete as monitored by LCMS, filter the reaction solution and purify it by reverse-phase preparation (column: C18, 50*250 mm, 10 μm; gradient: 25-45% B; mobile phase A: 0.1% NH4Ac / H2O; mobile phase B: CH3CN; flow rate: 70 mL / min) to obtain compound 3.

[0232] LC-MS: 1931.9 [M+H] + .

[0233] Example 7: Preparation of Compound 4

[0234] Step 1: Compound 3-9 (822.41 mg, 0.50 mmol) was dissolved in DMF (10 mL) in a 50 mL single-necked flask. PPTS (125.67 mg, 0.50 mmol), HOBT (33.78 mg, 0.25 mmol), and triethylamine (139.40 μL, 1.00 mmol) were added separately. The mixture was stirred at room temperature for 5 minutes, and then endo-BCN-O-PNB (189.20 mg, 0.60 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. The reaction was monitored by LCMS until complete. The reaction mixture was filtered and purified by reverse-phase chromatography (column: C18, 50*250 mm, 10 μm; gradient: 25-45% B; mobile phase A: 0.1% NH4Ac / H2O; mobile phase B: CH3CN; flow rate: 70 mL / min) to obtain compound 4.

[0235] LC-MS: 1820.9 [M+H] + .

[0236] Example 8: Preparation of Compound 5

[0237] Step 1: Compound 5-1 (70.0 mg, 0.09 mmol, CAS: 863971-53-3, purchased from Leyan), eczema mesylate (48.0 mg, 0.09 mmol), and HOBT (12.2 mg, 0.09 mmol) were added to a 50 mL single-necked flask and dissolved in DMF (1 mL). The resulting solution was stirred at room temperature for 30 minutes, and then DIPEA (0.03 mL, 0.18 mmol) was added. The reaction solution was stirred at room temperature for 3 hours, and the solution changed from turbid to clear. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 94:6) to obtain compound 5-2.

[0238] LC-MS: 1063.1 [M+H] + .

[0239] Step 2: Compound 5-2 (85.0 mg, 0.08 mmol) was added to a 25 mL single-necked flask and dissolved in DMF (2 mL). Diethylamine (0.07 mL, 0.71 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was slurried with petroleum ether to obtain compound 5-3.

[0240] LC-MS: 841.2 [M+H] + .

[0241] Step 3: Compound 5-3 (60 mg, 0.07 mmol), DIPEA (0.05 mL, 0.3 mmol), and compound 1-4 (74.9 mg, 0.10 mmol) were added to a 100 mL single-necked flask and dissolved in DMF (2 mL). The resulting solution was then treated with HOBT (2 mg, 0.01 mmol) and HATU (38 mg, 0.10 mmol) in an ice bath, and the reaction mixture was stirred in an ice bath for 1 hour. The reaction mixture was filtered and purified by high-performance liquid chromatography (HPLC) to obtain compound 5-4.

[0242] LC-MS: 1571.8 [M+H] + .

[0243] Step 4: Add compound 5-4 (86.0 mg, 0.055 mmol) to a 25 mL single-necked flask and dissolve it in DMF (2 mL). Add diethylamine (0.05 mL, 0.55 mmol) to the resulting solution. Stir the reaction mixture at room temperature for 1 hour. Concentrate the reaction mixture under reduced pressure, and slurry the residue with petroleum ether to obtain compound 5-5.

[0244] LC-MS: 1349.8 [M+H] + .

[0245] Step 5: Compound 5-5 (80.0 mg, 0.06 mmol) and HATU (22.8 mg, 0.06 mmol) were added to a 50 mL single-necked flask and dissolved in DMF (2 mL). DIPEA (0.15 mL, 1.2 mmol) was added to the resulting solution, followed by [(2,2-dimethyl-4-oxoylide-5-aza-3,8,11,14-tetraoxahexadecane-16-yl)oxy]acetic acid (21.0 mg, 0.06 mmol). The mixture was stirred at room temperature for 1 h. The reaction mixture was evaporated to dryness and purified by silica gel column chromatography (DCM:MeOH = 10:1) to obtain compound 5-6.

[0246] LC-MS: 1682.9 [M+H] + .

[0247] Step 6: Add compound 5-6 (50 mg, 0.03 mmol) to a 50 mL single-necked flask, dissolve it in dichloromethane (1 mL), add trifluoroacetic acid (0.2 mL) to the solution, and stir the mixture at room temperature for 30 minutes. Rotate the reaction solution to dryness to obtain the crude product compound 5-7. The crude product does not require purification and can be directly used for the next reaction.

[0248] LC-MS: 1582.9 [M+H] + .

[0249] Step 7: In a 100 mL single-necked flask, add DBCO-acid (152.67 mg, 0.50 mmol), HATU (285.18 mg, 0.75 mmol), and HOBT (101.34 mg, 0.75 mmol) and dissolve in DMF (5 mL). Add DIPEA (0.17 mL, 1.0 mmol) to the resulting solution. Stir the mixture at room temperature for 5 minutes, then add compounds 5-7 (791.40 mg, 0.50 mmol). Stir the reaction mixture at room temperature for 3 hours. Monitor the reaction by LCMS until complete. After filtration, the reaction solution is purified by reverse-phase chromatography (column: C18, 50*250 mm, 10 μm; gradient: 25-45% B; mobile phase A: 0.1% NH4Ac / H2O; mobile phase B: CH3CN; flow rate: 70 mL / min) to obtain compound 5.

[0250] LC-MS: 1870.1 [M+H] + .

[0251] Example 9: Preparation of Compound 6

[0252] Step 1: Compound 5-7 (791.39 mg, 0.50 mmol) was dissolved in DMF (10 mL) in a 50 mL single-necked flask. PPTS (125.67 mg, 0.50 mmol), HOBT (33.78 mg, 0.25 mmol), and triethylamine (139.40 μL, 1.00 mmol) were added separately. The mixture was stirred at room temperature for 5 minutes, and then endo-BCN-O-PNB (189.20 mg, 0.60 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. The reaction was monitored by LCMS until complete. The reaction mixture was filtered and purified by reverse-phase chromatography (column: C18, 50*250 mm, 10 μm; gradient: 25-45% B; mobile phase A: 0.1% NH4Ac / H2O; mobile phase B: CH3CN; flow rate: 70 mL / min) to obtain compound 6.

[0253] LC-MS: 1758.9 [M+H] + .

[0254] Example 10: Preparation of Compound 7

[0255] Step 1: In a 10 mL single-necked flask, add (2,5,8,11,14,17,20,23,26,29,32,35,38,41,44-pentadecaoxahexadecane-46-yloxy)acetic acid (750 mg, 1.00 mmol) and HATU (380 mg, 1.00 mmol), and dissolve in DMF (5 mL). Add DIPEA (0.25 mL, 2.00 mmol) to the resulting solution and stir the mixture at room temperature for 15 minutes. Then add compound 1-2 (460.2 mg, 1.00 mmol) to the mixture. Stir the reaction mixture at room temperature for 3 hours. Filter the reaction mixture and purify it by preparative high-performance liquid chromatography to obtain compound 7-1.

[0256] LC-MS: 1157.4 [M+H] + .

[0257] Step 2: Compound 7-1 (900.0 mg, 0.78 mmol) was added to a 50 mL single-necked flask and dissolved in TFA (3 mL) and dichloromethane (6 mL). The reaction solution was stirred at room temperature for 1 hour, then heated to 30 °C and stirred for 2 hours. The reaction solution was concentrated under reduced pressure, dissolved in 10 mL of toluene, and concentrated again under reduced pressure to obtain compound 7-2.

[0258] LC-MS: 1101.6 [M+H] + .

[0259] Step 3: Compound 7-2 (66.0 mg, 0.06 mmol) and HATU (22.8 mg, 0.06 mmol) were added to a 10 mL single-necked flask and dissolved in DMF (2 mL). DIPEA (0.15 mL, 1.2 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. Then, compound 5-3 (50 mg, 0.06 mmol) was added to the mixture. The reaction solution was evaporated to dryness, and the silica gel column was eluted with 5% methanol-dichloromethane solution to obtain compound 7-3.

[0260] LC-MS: 1925.0 [M+H] + .

[0261] Step 4: Compound 7-3 (65.0 mg, 0.03 mmol) was added to a 10 mL single-necked flask and dissolved in DMF (1 mL). Diethylamine (0.45 mL, 0.3 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was slurried with petroleum ether to obtain compound 7-4.

[0262] LC-MS: 1702.0 [M+H] + .

[0263] Step 5: In a 10 mL single-necked flask, add {[1-(9H-fluorene-9-yl)-3-oxonyl-4-aza-2,7,10,13-tetraoxapentadecan-15-yl]oxy}acetic acid (14.0 mg, 0.03 mmol) and HATU (11.4 mg, 0.03 mmol), and dissolve in DMF (2 mL). Add DIPEA (0.075 mL, 0.06 mmol) to the resulting solution and stir the mixture at room temperature for 15 minutes. Then add compound 7-4 (45 mg, 0.03 mmol) to the mixture. Rotate the reaction solution to dryness, and elute the silica gel column with 5% methanol-dichloromethane solution to obtain compound 7-5.

[0264] LC-MS: 1079.5 [M / 2+H] + .

[0265] Step 6: Add compound 7-5 (40.0 mg, 0.018 mmol) to a 10 mL single-necked flask and dissolve it in DMF (1 mL). Add diethylamine (0.45 mL, 0.3 mmol) to the resulting solution. Stir the reaction mixture at room temperature for 30 minutes. Concentrate the reaction mixture under reduced pressure, and slurry the residue with petroleum ether to obtain compound 7-6.

[0266] LC-MS: 1935.0 [M+H] + .

[0267] Step 7: In a 50 mL single-necked flask, add DBCO-acid (152.67 mg, 0.50 mmol), HATU (285.18 mg, 0.75 mmol), and HOBT (101.34 mg, 0.75 mmol), and dissolve in DMF (5 mL). Add DIPEA (0.17 mL, 1.0 mmol) to the resulting solution. Stir the mixture at room temperature for 5 minutes, then add compound 7-6 (967.6 mg, 0.50 mmol). Stir the reaction mixture at room temperature for 3 hours. After the reaction is complete as monitored by LCMS, filter the reaction solution and purify it by reverse-phase preparation (column: C18, 50*250 mm, 10 μm; gradient: 25-45% B; mobile phase A: 0.1% NH4Ac / H2O; mobile phase B: CH3CN; flow rate: 70 mL / min) to obtain compound 7.

[0268] LC-MS: 1111.6 [M / 2+H] + .

[0269] Example 11: Preparation of Compound 8

[0270] The overall synthesis method of compound 8 is the same as that of compound 2. Compound 8 was finally obtained by liquid phase preparation chromatography purification.

[0271] LC-MS: 1056.1 [M / 2+H] + .

[0272] Example 12: Preparation of Compound 9

[0273] The overall synthetic method of compound 9 was the same as that of compound 7. Compound 9 was finally obtained by preparative liquid chromatography (LC-MS) purification. LC-MS: 1023.5 [M / 2+H] + .

[0274] Example 13: Preparation of Compound 10

[0275] The overall synthesis method of compound 10 is the same as that of compound 2. The final product of compound 10 is obtained by liquid phase preparation chromatography purification.

[0276] LC-MS: 968.1 [M / 2+H] + .

[0277] Example 14: Preparation of Compound 11

[0278] The overall synthesis method of compound 11 is the same as that of compound 7. The final product of compound 11 is obtained by liquid phase preparation chromatography purification.

[0279] LC-MS: 980.5 [M / 2+H] + .

[0280] Example 15: Preparation of Compound 12

[0281] The overall synthesis method of compound 12 is the same as that of compound 2. Compound 12 was finally obtained by liquid phase preparation chromatography purification.

[0282] LC-MS: 925.0 [M / 2+H] + .

[0283] Example 16: Preparation of Compound 13

[0284] Step 1: Compound 13-1 (2.05 g, 10 mmol) and toluene (30 mL) were added to a 250 mL three-necked flask. Then, at room temperature, (prop-2-alkynyloxy)acetic acid-2-methylprop-2-yl ester (2.04 g, 12 mmol), cuprous iodide (38 mg, 0.2 mmol), triphenylphosphine (262 mg, 1 mmol), palladium dichloride diacetonitrile (77.7 mg, 0.3 mmol), and diisopropylamine (2.02 g, 20 mmol) were added. The mixture was purged with nitrogen three times and stirred overnight at room temperature. The reaction solution was filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate) to obtain compound 13-2.

[0285] LC-MS: 295.1 [M+H] + .

[0286] Step 2: Add compound 13-2 (2.0 g, 6.79 mmol) and dichloromethane (30 mL) to a 100 mL three-necked flask. Add m-chloroperoxybenzoic acid (5.86 g, 33.95 mmol) at room temperature, purge with nitrogen three times, and stir overnight at room temperature. Filter the reaction solution, concentrate the filtrate, and purify by column chromatography (eluent: petroleum ether / ethyl acetate) to obtain compound 13-3.

[0287] LC-MS: 327.1 [M+H] + .

[0288] Step 3: Compound 13-3 (1.5 g, 4.58 mmol) was added to a 100 mL single-necked flask and dissolved in trifluoroacetic acid (4 mL) and dichloromethane (20 mL). The reaction solution was stirred at room temperature for 1 hour, and then concentrated under reduced pressure. The crude product was purified by preparative high-performance liquid chromatography to obtain compound 13-4.

[0289] LC-MS271.0[M+H] + .

[0290] Step 4: Compound 1-9 (40.0 mg, 0.03 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (11.4 mg, 0.03 mmol) were added to a 10 mL single-necked flask and dissolved in N,N-dimethylformamide (1 mL). Diisopropylethylamine (0.03 mL, 0.06 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. Then, compound 13-4 (8 mg, 0.03 mmol) was added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative high-performance liquid chromatography to obtain compound 13.

[0291] LC-MS: 1748.8 [M+H] + .

[0292] 1H NMR (400MHz, DMSO-d6) δ9.99(s,1H),9.20(s,2H),8.17(d,J=6.6Hz,1H),8.05(d,J=8.5 Hz,1H),7.89(d,J=8.8Hz,1H),7.83(t,J=5.8Hz,1H),7.78(d,J=10.9Hz,1H),7.64(t,J= 7.0Hz,2H),7.59(d,J=8.4Hz,2H),7.36(d,J=8.5Hz,2H),7.31(s,1H),6.51(s,1H),5.45 (s,2H),5.28(s,3H),5.07(s,2H),4.59(s,2H),4.38(dd,J=13.6,6.7Hz,2H),4.23–4.15 (m,1H),4.04(s,2H),3.91(s,2H),3.84(s,2H),3.59–3.47(m,39H),3.45–3.40(m,7H), 3.28–3.24(m,2H),3.23(s,3H),3.14(s,1H),3.05(dd,J=13.5,7.0Hz,2H),2.38(s,3H), 2.19(s,2H),1.98(dd,J=13.7,6.9Hz,1H),1.87(tt,J=14.1,7.1Hz,2H),1.65(s,1H),1. 54(d,J=9.6Hz,1H),1.40(s,2H),1.29(d,J=7.1Hz,3H),1.23(s,2H),0.91–0.79(m,9H).

[0293] Coupling Example 17:

[0294] Example 17-1 ADC Analysis Method

[0295] 1. Determination of DAR value using hydrophobic chromatography-high-performance liquid chromatography (HIC-HPLC)

[0296] Sample preparation: The sample was diluted to 3.0 mg / mL with mobile phase B, centrifuged at 12000 rpm for 10 min, and the supernatant was used for HPLC analysis.

[0297] Chromatographic conditions:

[0298] Gradient elution procedure:

[0299] DAR value calculation formula:

[0300] DAR = Σ(weighted peak area) / 100, that is, DAR = (D0 peak area ratio × 0 + D1 peak area ratio × 1 + D2 peak area ratio × 2 + D3 peak area ratio × 3 + D4 peak area ratio × 4 + D5 peak area ratio × 5 + D6 peak area ratio × 6 + D7 peak area ratio × 7 + D8 peak area ratio × 8) / 100

[0301] 2. Determination of ADC purity by size exclusion chromatography-HPLC: Sample preparation: Sample concentration 1.0-5 mg / mL, filtered through a 0.22 μm filter membrane; Instrumentation: Agilent 1260 Infinity II Bio-Inert LC System; Column: TOSOH, TSKgel G3000SWxL, 5 μm, 7.8 mm × 300 mm; Mobile phase: 0.2 M PB, 5-15% isopropanol, pH 7.0; Flow rate: 0.5-1 mL / min; Detection wavelength: 280 nm & 360 nm; Column temperature: room temperature; Sample loading: 30 μg; SEC elution method: isocratic elution.

[0302] 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.

[0303] Example 17-2 Glycosite Coupling of ADC001

[0304] (1) Preparation of LIV-1-targeted glycoengineered antibody Ab1-G35

[0305] In a pH 7.4 1×PBS solution, anti-LIV-1 antibody Ab1, disaccharide oxazoline (compound G35, Wuhan Tangzhi Pharmaceutical Co., Ltd.), and wild-type glycoside endonuclease Endo Si (self-made) were prepared at concentrations of 10 mg / mL, 1.67 mM (25 times the antibody equivalent), and 0.6 mg / mL, respectively. The pH of the reaction system was adjusted to 7.4, the reaction temperature was 25℃, 800 rpm, and the reaction time was 3 h. Small molecule compounds and glycoside hydrolases were removed by protein A purification to obtain the corresponding G35-modified glycoengineered antibody Ab1-G35.

[0306] (2) Preparation of ADC001

[0307] In 1×PBS solution at pH 7.4, the prepared glycoengineered antibody Ab1-G35 and compound 1 were prepared at concentrations of 5 mg / mL and 0.33 mM, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated overnight at 25°C. The desired site-directed glycoengineering conjugate ADC001 was obtained by ultrafiltration. The measured HRMS value of the ADC001 heavy chain after deconvolution was 53200.46 Da.

[0308] (3) Preparation of ADC002~ADC007

[0309] The experimental procedures were as described in Examples 17-2 (1) and (2). The antibody was trastuzumab, which was purchased from Shanghai Langjing Biotechnology Co., Ltd.

[0310] The measured HRMS value after deconvolution for ADC002 is 157482.8 Da. The measured HRMS value after deconvolution for ADC003 is 156808.4 Da. The measured HRMS value after deconvolution for ADC004 is 158373.8 Da. The measured HRMS value after deconvolution for ADC005 is 157707.4 Da. The measured HRMS value after deconvolution for ADC006 is 158008.9 Da. The measured HRMS value after deconvolution for ADC007 is 157339.6 Da.

[0311] The ADC structure disclosed herein is as follows:

[0312] Where t is selected from 0 or 1.

[0313] Example 17-3 Coupling method of ADC022 and ADC013

[0314] Anti-LIV1 antibody Ab1 or trastuzumab was reduced in 20mM PBS (pH 6.0-7.4) with 6-20 equivalents of TCEP reducing agent at 4-40℃ for 0.5-6 hours. After reduction, add 5-30% DMSO (DMF or DMA can also be used as organic solvents), and add 6-30 equivalents of the loading-linker (e.g., compound 13) at 4-40℃, reacting for 0.5-24 hours. After the coupling reaction is complete, add 100mM N-acetyl-cysteine ​​solution at a quenching ratio of 20:1 (quencher: antibody), shake well, and quench at room temperature for 20 min to terminate the coupling reaction. Replace the ADC buffer with 20mM His-HAc buffer at pH 5.5 using desalting chromatography or ultrafiltration, adjusting the antibody-conjugate product concentration to approximately 3-10 mg / mL. Filter the ADC stock solution through a 0.22μm PES filter, aliquot according to experimental requirements, and store at -20℃ or -40℃. Measure the absorbance at 280nm and 370nm using UV chromatography to calculate the antibody concentration. Calculate the average DAR value using HIC-HPLC, and determine the ADC purity using SEC-HPLC.

[0315] Randomly coupled control ADC molecular structure:

[0316] The DAR value distribution data of the obtained products ADC022 and ADC013, as well as the sugar site-coupled products ADC001 to ADC007, are shown in Table 2.

[0317] Table 2 ADC Quality Control Data

[0318] Experimental data show that the homogeneity of ADC001 to ADC007 prepared by site-directed sugar coupling in this disclosure is superior to that of ADC022 and ADC013 prepared by random coupling.

[0319] Bioactivity experiment

[0320] The positive control molecule, hLIV22 antibody, has a sequence derived from WO2012078688A2 and was synthesized by Shanghai Baiying Biotechnology Co., Ltd.

[0321] The heavy chain amino acid sequence of the hLIV22 antibody is as follows:

[0322] The amino acid sequence of its antibody light chain is as follows:

[0323] Test Example 1: Antibody endocytosis experiment

[0324] Experimental objective: To evaluate the internalization capacity of anti-LIV1 antibody in overexpressing cell lines using the DT3C method.

[0325] Experimental Methods: CHOK1-hLIV1 cells in good growth condition were collected by centrifugation and seeded into 96-well white transparent plates at a specific cell density for overnight culture. The candidate antibody and positive control were mixed with DT3C at a 1:6 ratio and incubated at 37°C for 30 minutes to form a mAb-DT3C conjugate. The mAb-DT3C conjugate was serially diluted. The diluted mAb-DT3C conjugate was added to 96-well white transparent plates and incubated at 37°C with 5% CO2 for 6 days. After incubation, the 96-well plates were removed and equilibrated at room temperature for 30 minutes. A certain amount of [unspecified ingredient] was added to each well. Reagent was incubated at room temperature with shaking for 10 minutes, and the luminescence signal was detected using a microplate reader. The internalization activity data of the antibody on the CHOK1-hLIV1 cell line are shown in Table 3.

[0326] Table 3. Internalization ability of antibodies on CHOK1-hLIV1 cells

[0327] The experimental results show that the Ab1 disclosed in this invention has a stronger internalization ability in CHOK1-hLIV1 than the control antibody hLIV22.

[0328] Test Example 2: Antibody Binding Specificity

[0329] The binding activity of Ab1 with other LIV1 family members (ZIP10, ZIP4, ZIP5, ZIP12) was detected. Specifically, 100 μL / well of antigen (LIV1, ZIP10, ZIP4, ZIP5, ZIP12: 2 μg / mL) was coated and incubated overnight at 4°C. Blocking was performed at room temperature for 1 hour with 100 μL of blocking buffer (2% BSA in PBS). Washing was repeated three times with PBST using a BioTek automated plate washer. 100 μL of diluted antibody (starting concentration 200 nM, serially diluted 1:5) was added to each well. Incubation was performed at room temperature for 2 hours. Washing was repeated three times with PBST. 100 μL of diluted HRP anti-human secondary antibody was added to each well and incubated at room temperature for 1 hour. Washing was repeated six times with PBST. 100 μL of TMB substrate was added to each well and incubated at room temperature for 5 minutes, followed by 100 μL of stop solution to stop the reaction. The OD values ​​at a wavelength of 450 nm were read using an Envision microplate reader. The results are shown in Table 4.

[0330] Table 4. Binding specificity of antibody Ab1

[0331] The half-binding concentration (EC5) was calculated using software. 50 Ab1 showed no significant binding activity with other members of the ZIP family (ZIP10, ZIP4, ZIP5, ZIP12), indicating that the antibody obtained in this disclosure has good selectivity.

[0332] Test Example 3: In vivo efficacy of ADC in MCF-7 xenograft model

[0333] Experimental objective: To evaluate the in vivo antitumor activity of the ADC disclosed herein in a LIV1-positive breast cancer MCF-7 xenograft model.

[0334] Experimental methods: BALB / c Nude mice (purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.) were used as test animals to evaluate the efficacy of anti-LIV1-ADC after tail vein injection in a human breast cancer cell MCF-7 xenograft model.

[0335] MCF-7 is an estrogen-dependent breast cancer. Therefore, one week before cell inoculation, tumor-bearing mice were subcutaneously implanted with estrogen pads to maintain tumor growth. Mice were anesthetized with 3-4% isoflurane before implantation. The cells contained 1×10⁻⁶ estrogen. 7 100 μL of PBS suspension containing MCF-7 cells (source: ATCC) was mixed with an equal volume of Matrigel and subcutaneously injected into the right back of mice near the axilla. The injection volume was approximately 200 μL. Mice were anesthetized with 3-4% isoflurane before inoculation. When the tumor grew to an average size of approximately 150 mm... 3When the tumor volume and body weight were measured, the animals were randomly divided into groups of 7. The day of administration was defined as day 0.

[0336] The drug was administered via tail vein injection, once a week for a total of two administrations. Tumor volume and body weight were measured twice a week, and the data were recorded. Tumor inhibition rate (TGI) (%) = [1 - (T... i -T0) / (V i -V0)]×100,T i T0 and V represent the tumor volume of the experimental group on day i after drug administration and on the day of drug administration, respectively. i Vi and V0 represent the tumor volumes of the blank control group (Vehicle, PBS) on day i after group administration and on the day of group administration, respectively. Specific experimental results at the end of the experiment on day 28 after the start of administration are shown in Table 5.

[0337] Table 5. In vivo efficacy evaluation of the ADC compounds disclosed in this paper.

[0338] The experimental results showed that in the LIV1-positive MCF-7 xenograft model, the in vivo efficacy of ADC001 prepared by site-directed sugar conjugation of this disclosure was superior to that of ADC022 prepared by random conjugation.

[0339] Test Example 4: In vivo efficacy of ADC in the MDA-MB-231 xenograft model

[0340] Experimental objective: To evaluate the in vivo antitumor activity of the ADC disclosed herein in a LIV1-positive breast cancer MDA-MB-231 xenograft model.

[0341] Experimental methods: BALB / c Nude mice (purchased from Vital River Pharmaceuticals, Beijing) were used as test animals to evaluate the efficacy of anti-LIV1-ADC after tail vein injection in a human breast cancer cell MDA-MB-231 xenograft model.

[0342] Under aseptic conditions, a suspension of in vitro cultured MDA-MB-231 cells (source: Nanjing Kebai Biotechnology Co., Ltd.) was taken, centrifuged, and the cell concentration was adjusted to 10 × 10⁻⁶ cells using 0.9% NaCl injection. 7 Add an equal volume of Matrigel gel to a final cell concentration of 5 × 10⁶ cells / mL. 7 The dose was 0.1 mL / mouse, injected subcutaneously into the right axilla of each mouse. When the tumor grew to an average size of approximately 150 mm... 3 When the tumor volume and body weight were measured, the animals were randomly divided into groups of 7. The day of administration was defined as day 0.

[0343] The drug was administered via tail vein injection, once a week for a total of three administrations. Tumor volume and body weight were measured twice a week, and the data were recorded. Tumor inhibition rate (TGI) (%) = [1 - (T... i -T0) / (V i -V0)]×100,T i T0 and V represent the tumor volume of the experimental group on day i after drug administration and on the day of drug administration, respectively. i Vi and V0 represent the tumor volumes of the blank control group (Vehicle, PBS) on day i after drug administration and on the day of drug administration, respectively. Specific experimental results at the end of the experiment on day 24 after the start of drug administration are shown in Table 6.

[0344] Table 6. In vivo efficacy evaluation of the ADC compounds disclosed in this paper.

[0345] The experimental results showed that in the LIV1-positive MDA-MB-231 xenograft model, the in vivo efficacy of ADC001 prepared by site-directed sugar conjugation of this disclosure was superior to that of ADC022 prepared by random conjugation.

[0346] Test Example 5: Pharmacokinetics of ADCs in Cynomolgus Monkeys

[0347] Experimental objective: Using cynomolgus monkeys as the relevant species, this study investigates the pharmacokinetic characteristics of the ADC and control molecules disclosed herein after single-injection administration in cynomolgus monkeys.

[0348] Experimental Methods: Cynomolgus monkeys (Suzhou Xishan Zhongke Experimental Animal Co., Ltd.) were used. Two monkeys were used in the ADC001 group, and four monkeys were used in the ADC022 group (half male and half female). They were randomly assigned to each group and administered a single injection of a sample (prepared with an appropriate volume of physiological saline) at a dose of 30 MPa. Blood samples were collected before administration and at 35 minutes, 2.5 hours, 6.5 hours, 24.5 hours, 72.5 hours, 120.5 hours, 168.5 hours, 240.5 hours, 336.5 hours, 504.5 hours, and 672.5 hours after administration. The concentrations of total antibody and conjugated antibody (representing 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 ADC was determined using LC-HRMS. Pharmacokinetic parameters for different detection methods were calculated. The specific results of the pharmacokinetic characteristics are shown in Table 7, where ADC refers to the drug-conjugated form.

[0349] Table 7 Pharmacokinetic characteristics of ADCs in cynomolgus monkeys

[0350] Conclusion: In cynomolgus monkeys, at the same dose, the in vivo exposure of ADC001 disclosed herein was higher than that of the control ADC022, while the in vivo clearance rate was lower than that of the control ADC022.

[0351] Test Example 6: In vitro antitumor activity of ADC

[0352] Experimental objective: To detect the in vitro inhibitory activity of the disclosed ADC compound against NCI-N87 (human gastric cancer cells), KPL-4 (human breast cancer cells), or SKBR-3 (human breast adenocarcinoma cells).

[0353] Add 142.5 μL of a suspension of NCI-N87 (source: Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; 4000 cells / well), KPL-4 (source: Nanjing Kebai Biotechnology Co., Ltd.; 3000 cells / well), or SKBR-3 (source: Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; 3000 cells / well) tumor cells in logarithmic growth phase to cell culture plates. Incubate the cell culture plates at 37°C in a 5% CO2 cell culture incubator for 24 hours. Add 7.5 μL of sample (starting at 1000 nM, 5-fold dilution, 9 concentrations) to each well, gently mix, and incubate in the incubator. After 144 hours of incubation, add 50 μL of CellTiter-Glo TM (Promega, Catalog No.: G7572) Working solution, gently shake to lyse cells, and read the plate on a microplate reader. The cell proliferation inhibition rate is calculated as: Cell proliferation inhibition rate = (1 - Sample well / Control well) × 100%. Plot the sample concentration (Log value) on the x-axis and the cell proliferation inhibition rate on the y-axis, and perform four-parameter fitting to obtain the IC50 value for each sample. 50 Values. See Table 8 for specific results:

[0354] Table 8. In vitro antitumor activity assay of the disclosed ADC compounds.

[0355] Conclusion: The ADC drug disclosed in this paper has significant inhibitory activity against the proliferation of HER2-positive cells NCI-N87, KPL-4 and SKBR-3.

[0356] Test Example 7: Stability of ADCs in various plasmas

[0357] Experimental objective: To test the stability of the ADC drug disclosed herein in human plasma.

[0358] The ADC sample, healthy human plasma, and cynomolgus monkey plasma (Suzhou Fangda New Drug Development Co., Ltd.) were sterilized by filtration through a 0.22 μm filter. The ADC sample was added to the sterile plasma at a final concentration of 100 ug / ml and incubated in a cell culture incubator at 37°C. The day of incubation was marked as day 0. The sample was then removed on day 21 for free toxin analysis.

[0359] 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). Specific experimental results are shown in Table 9.

[0360] Table 9. Free toxin release rate of the disclosed ADC compounds on day 21.

[0361] Conclusion: The ADC compounds disclosed herein exhibit excellent in vitro plasma stability.

[0362] Test Example 8: In vivo efficacy of ADC in the NCI-N87 xenograft model

[0363] Using BALB / c Nude mice (purchased from Vital River Pharmaceuticals, Beijing) as test animals, the efficacy of anti-HER2-ADC administered via tail vein injection in nude mice with human gastric cancer cell NCI-N87 xenografts was evaluated.

[0364] Mice were subcutaneously inoculated with NCI-N87 cells in the right axilla (Source: ATCC) (5×10 6 Each individual (with 50% Matrigel) exhibited tumor growth for 9 days, reaching an average tumor volume of 150 mm. 3 Animals were randomly grouped according to tumor volume (D9), with 4 animals per group.

[0365] A single dose of 1 mpk was administered via tail vein injection. Tumor volume and body weight were measured twice weekly and recorded. Tumor inhibition rate (TGI) (%) = [1 - (T... i -T0) / (V i -V0)]×100,T i T0 and V represent the tumor volume on day i and day i respectively after drug administration in the experimental group. i Vi and V0 represent the tumor volumes of the blank control group (Vehicle, PBS) on day i and day i, respectively, after drug administration. Results for days 14 and 42 after drug administration are shown in Table 10.

[0366] Table 10. In vivo efficacy evaluation of the disclosed ADC compounds.

[0367] The results showed that the ADC drugs disclosed herein all exhibited excellent in vivo antitumor activity in the NCI-N87 xenograft model. Data from day 42 indicated that the antitumor activity of ADC003–ADC007, prepared by site-directed sugar conjugation according to this disclosure, was superior to that of ADC013, prepared by random conjugation.

[0368] Test Example 9: In vivo efficacy of ADC in the JIMT-1 xenograft model

[0369] Using BALB / c Nude mice (purchased from Vital River Pharmaceuticals, Beijing) as test animals, the efficacy of anti-HER2-ADC administered via tail vein injection in nude mice with human breast cancer cell JIMT-1 xenografts was evaluated.

[0370] JIMT-1 cells were subcutaneously injected into the right axilla of mice (Source: ATCC) (5×10 6 Each individual (with 50% Matrigel) exhibited tumor growth over 13 days, reaching an average tumor volume of 150 mm. 3 Animals were randomly grouped according to tumor volume (D13), with 5 animals per group.

[0371] The tumor was administered via tail vein injection at a dose of 1 or 3 mpk, twice weekly (one injection per week, for a total of two injections). Tumor volume and body weight were measured twice weekly and recorded. Tumor inhibition rate (TGI) (%) = [1 - (T...] i -T0) / (V i -V0)]×100,T i T0 and V represent the tumor volume on day i and day i respectively after drug administration in the experimental group. i Vi and V0 represent the tumor volumes of the blank control group (Vehicle, PBS) on day i and day i, respectively, after drug administration. The experimental results on day 28 after the start of drug administration are shown in Table 11.

[0372] Table 11. In vivo efficacy evaluation of the ADC compounds disclosed herein.

[0373] The results showed that the ADC drugs disclosed in this paper all exhibited excellent in vivo antitumor activity in the JIMT-1 xenograft model.

[0374] sequence list

Claims

1. The antibody-drug conjugate shown in formula (I), its isomers, or pharmaceutically acceptable salts thereof: Ab-[Y-X-(Z-L1-L2-L3-D) m ] n (I)； wherein The Ab is an antibody or antigen-binding fragment; Y is wherein the wavy line α represents being connected to Ab, and α represents being connected to X. X is a sugar or a sugar derivative; Z is the segment connecting X and L1; L1is a chemical bond, or L1is Furthermore, the -C(O)- terminal of L1 is connected to L2; Where k is selected from integers from 0 to 8, j is selected from integers from 0 to 20, and p is selected from 0, 1, 2, 3, or 4; L2 is selected from a peptide group composed of 2-10 amino acid residues; the amino acid is selected from natural amino acids or non-natural amino acids; L3 is the segment connecting L2 and D; D is a cytotoxic drug; t is selected from 0 and 1; m is selected from 1 to 3, and m is a decimal or an integer; n is selected from 1 to 2, and n is a decimal or an integer.

2. The antibody drug conjugate, isomer thereof or pharmaceutically acceptable salt thereof according to claim 1, characterized in that, It meets one or more of the following conditions: (1) X is a disaccharide derivative; (2) k is any integer from 3 to 5; (3) j is any integer from 5 to 20; (4) p is 2, 3 or 4; (5) L1is (6) L2 is a polypeptide fragment composed of 2-4 amino acids selected from the following: glycine, phenylalanine, valine, alanine, asparagine and citrulline, for example, a polypeptide fragment composed of 2-4 amino acids selected from the following: glycine, L-phenylalanine, L-valine, L-alanine, L-asparagine and L-citrulline, and the -NH-terminus of L2 is connected to L1; (7) D is a camptothecin derivative, for example wherein R 1 is C 1-6 alkyl, R 2 is halogen; (8) m is selected from 1 to 3 decimals or integers; preferably, m is selected from 2 to 3 decimals or integers; (9) n is selected from 1 to 2 decimals or integers; (10) The Ab is selected from anti-LIV1 antibody or its antigen-binding fragment, and anti-Her2 antibody or its antigen-binding fragment.

3. The antibody drug conjugate, isomer thereof or pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that, It meets one or more of the following conditions: (1) j is any integer from 7 to 15; (2) L1is a bond, Furthermore, the -C(O)- terminal of L1 is connected to L2; (3) L2 is a polypeptide fragment composed of 2-4 amino acids selected from the following: glycine, phenylalanine, valine, alanine and citrulline, for example glycine, L-phenylalanine, L-valine, L-alanine and L-citrulline, and the -NH-terminus of L2 is connected to L1. (4) D is wherein R 1 is C 1-4 alkyl, for example methyl; R 2 is F, CI or Br, for example F; (5) m is 2 or 3; (6) n is 1 or 2.

4. The antibody drug conjugate, isomer thereof or pharmaceutically acceptable salt thereof according to any one of claims 1-3, characterized in that, It meets one or more of the following conditions: (1) said X is a disaccharide derivative; preferably, X is wherein the wavy line α represents being connected to Y, and α represents being connected to Z; (2) The Z is selected from: wherein the wavy line α represents being connected to X, and α represents being connected to L1. (3) k is 4; (4) j is 7, 11 or 15; (5) p is 3; (6) L1is Furthermore, the -C(O)- terminal of L1 is connected to L2; (7) L2 is -Val-Ala-, -Val-Cit-, and -Gly-Gly-Phe-Gly- (SEQ ID NO:44), for example -L-Val-L-Ala-, -L-Val-L-Cit- or -Gly-Gly-L-Phe-Gly-, wherein the -NH- end of L2 is connected to L1; (8) L3 is α represents being connected to D; (9) D is (10) m is 3; (11) n is 2.

5. The antibody drug conjugate or a pharmaceutically acceptable salt thereof, stereoisomer thereof according to any one of claims 1-4, characterized in that, The Ab is selected from anti-LIV1 antibodies or their antigen-binding fragments; preferably, the anti-LIV1 antibody or its antigen-binding fragments comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively. LCDR1, LCDR2, and LCDR3 as shown in NO:16, SEQ ID NO:17, and SEQ ID NO:18; or the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively.

6. The antibody drug conjugate or a pharmaceutically acceptable salt thereof, stereoisomer thereof according to any one of claims 1-5, characterized in that, The anti-LIV1 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region each comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the following groups of heavy chain variable regions and light chain variable regions: (1) Heavy chain variable region SEQ ID NO:25 and light chain variable region SEQ ID NO:26; (2) Heavy chain variable region SEQ ID NO:27 and light chain variable region SEQ ID NO:28; (3) Heavy chain variable region SEQ ID NO:29 and light chain variable region SEQ ID NO:30; or (4) Heavy chain variable region SEQ ID NO:31 and light chain variable region SEQ ID NO:

32.

7. The antibody drug conjugate or a pharmaceutically acceptable salt thereof, stereoisomer thereof according to any one of claims 1-6, characterized in that, The anti-LIV1 antibody mentioned therein is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

8. The antibody drug conjugate or a pharmaceutically acceptable salt thereof, stereoisomer thereof according to any one of claims 1-7, characterized in that, The anti-LIV1 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:31 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:31, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:26 or SEQ ID NO:28 or SEQ ID NO:30 or SEQ ID NO:32 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:31, and / or the sequence shown in SEQ ID NO:26 or SEQ ID NO:28 or SEQ ID NO:30 or SEQ ID NO:

32. The sequence shown in NO:32 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity; Preferably, the anti-LIV1 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:31, and / or, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:26 or SEQ ID NO:28 or SEQ ID NO:30 or SEQ ID NO:32; More preferably, the anti-LIV1 antibody or its antigen-binding fragment comprises a heavy chain as shown in SEQ ID NO:33 or SEQ ID NO:35 or SEQ ID NO:37 or SEQ ID NO:39, and a light chain as shown in SEQ ID NO:34 or SEQ ID NO:36 or SEQ ID NO:38 or SEQ ID NO:40; More preferably, the anti-LIV1 antibody or its antigen-binding fragment comprises a heavy chain as shown in SEQ ID NO:33 and a light chain as shown in SEQ ID NO:34, or comprises a heavy chain as shown in SEQ ID NO:35 and a light chain as shown in SEQ ID NO:36, or comprises a heavy chain as shown in SEQ ID NO:37 and a light chain as shown in SEQ ID NO:38, or comprises a heavy chain as shown in SEQ ID NO:39 and a light chain as shown in SEQ ID NO:

40.

9. The antibody drug conjugate or a pharmaceutically acceptable salt thereof, stereoisomer thereof according to any one of claims 1-8, characterized in that, The Ab is selected from anti-Her2 antibody or its antigen-binding fragment; preferably, the anti-Her2 antibody is trastuzumab.

10. The antibody-drug conjugate or its pharmaceutically acceptable salt, or its stereoisomer, according to any one of claims 1-9, characterized in that, It has the following formula (Ia) structure: Wherein, Ab, Y, X, Z, L1, L2, m, and n are defined as in any one of claims 1 to 9.

11. The antibody-drug conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, or its stereoisomer, characterized in that, It is selected from: Where t is selected from 0 or 1, and n is selected from 1 to 2 decimals or integers.

12. The drug linker shown in formula (II), its isomer, or a pharmaceutically acceptable salt thereof: Z-L1-L2-L3-D (II); in, Z' is selected from Where α represents being connected to L1; L1, L2, L3 and D are defined as in any one of claims 1 to 11.

13. The drug linker, its isomer, or a pharmaceutically acceptable salt thereof according to claim 12, wherein, L1 is a chemical bond, Preferred Furthermore, the -C(O)- terminal of L1 is connected to L2; L2 is -Val-Ala-, -Val-Cit-, or -Gly-Gly-Phe-Gly-, for example -L-Val-L-Ala-, -L-Val-L-Cit-, or -Gly-Gly-L-Phe-Gly-, and the -NH- end of L2 is connected to L1. L3 is α represents being connected to D; D is Preferably, the drug linker, its isomer, or a pharmaceutically acceptable salt thereof is selected from:

14. A pharmaceutical composition comprising the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, a stereoisomer thereof, and a pharmaceutically acceptable excipient, preferably, the pharmaceutical composition further comprising one or more additional therapeutic agents.

15. The use of any one of the antibody-drug conjugates or pharmaceutically acceptable salts thereof, stereoisomers thereof, and the pharmaceutical composition of claim 14 in the preparation of a medicament for treating a disease, preferably, the disease being a disease associated with LIV1 expression and / or a disease associated with Her2 expression; More preferably, the disease is a disease related to LIV1 expression; more preferably, the disease related to LIV1 expression is a tumor; and even more preferably, the tumor is a solid tumor, such as breast cancer, lung cancer, gastric cancer, squamous cell carcinoma of the head and neck, castration-resistant prostate cancer, and its metastatic forms. Alternatively, the disease is a disease related to Her2 expression. More preferably, the disease related to Her2 expression is a tumor. More preferably, the tumor is a solid tumor, such as breast cancer, lung cancer, gastric cancer, squamous cell carcinoma of the head and neck, castration-resistant prostate cancer, ovarian cancer, endometrial cancer, urothelial carcinoma, head and neck tumors, small cell lung cancer, nasopharyngeal carcinoma, non-small cell lung cancer, salivary gland tumors, colorectal cancer, esophageal cancer, bile duct cancer, adenocarcinoma of the gastroesophageal junction, and their metastatic forms. More preferably, the tumor is breast cancer or gastric cancer; the breast cancer is, for example, MCF-7, MDA-MB-231, KPL-4, SKBR-3 or JIMT-1; the gastric cancer is, for example, NCI-N87.

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