Polycyclic drug conjugate, and preparation method therefor and use thereof

Abstract

A polycyclic drug conjugate, and a preparation method therefor and the use thereof. The polycyclic drug conjugate has the structure as shown in formula Ab-[M-L-E-D]x, the drug is selected from DNA topoisomerase inhibitors, and the prepared antibody-drug conjugate has excellent targeting and killing effects on solid tumors such as gastric cancer, breast cancer, lung cancer and urothelial carcinoma.

Description

Polycyclic drug conjugates and preparation methods and uses thereof

[0001] This application is based on the application with CN application number 202310538792.7 and application date May 12, 2023, the application with CN application number 202311413491.8 and application date October 27, 2023, and the application with CN application number 202410173901.4 and application date February 6, 2024, and claims the priority of the aforementioned applications. All contents of the said CN applications are hereby introduced into this application as a whole. Technical Field

[0002] The present application relates to the field of targeted therapy, and specifically to a polycyclic drug conjugate and its preparation method and use. Background Art

[0003] Antibody drug conjugates (ADCs) for tumor treatment typically consist of a monoclonal antibody, a bioactive molecule (primarily a tumor-killing cytotoxin), and a linker. The bioactive molecule is covalently coupled to the antibody via the linker. The antibody recognizes specific targets on the surface of tumor cells, guiding the ADC to the tumor microenvironment and the surface of cancer cells, where it is internalized. The bioactive molecule is then released inside the cancer cells and kills them by inhibiting their microtubules or damaging their DNA, minimizing damage to normal tissue cells.

[0004] The ErbB family of receptor tyrosine kinases is an important mediator of cell growth, differentiation, and survival. This family includes four members: epidermal growth factor receptor (EGFR or ErbB1), Her2 (ErbB2), Her3 (ErbB3), and Her4 (ErbB4). The anti-ErbB2 antibody trastuzumab (trade name Herceptin) is commonly used clinically to treat breast cancer with high ErbB2 expression, but the clinical response rate is low. In order to improve the therapeutic effect, conjugates of anti-ErbB2 antibodies with microtubule inhibitors such as maytansines (such as DM1) and auristatins (such as MMAE) or DNA topoisomerase I inhibitors (such as Dxd) (Trastuzumab emtansine, Disitamab vedotin, Trastuzumab deruxtecan) have been used in clinical treatment in recent years.

[0005] With the widespread clinical application of these ADCs for the treatment of Her2-expressing tumors, safety issues and drug resistance, including neurotoxicity, hematotoxicity, hepatotoxicity, and interstitial pneumonia, have gradually emerged (Pharmacology & Therapeutics 2019, 200, 110-125; Breast Cancer Research and Treatment 2020, 183, 23-39; JAMA Oncol. 2021, 7, 1873-1881; Drug Deliv. 2022, 29, 1335-1344; Cancers 2023, 15, 1130; Cancers 2023, 15, 1278).

[0006] Specifically, the linker portion of both Disitamab vedotin and Trastuzumab deruxtecan uses a maleimide linker (MC). Literature reports that MC linkers are prone to reverse Michael reactions and sulfhydryl exchange under physiological conditions, resulting in reduced efficacy and increased toxicity (Nat Biotechnol. 2012, 32, 184-189; Bioconjugate Chem. 2015, 26, 145-152). In terms of the hydrophilicity of the linker, Disitamab vedotin and Trastuzumab deruxtecan use the more hydrophobic valine-citrulline (Val-Cit) and glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), respectively. Literature reports that the hydrophilicity of the linker significantly affects the hydrophilicity of the ADC, thereby affecting the aggregation, pharmacokinetic properties, and toxicity of the ADC (Chemical Linkers in Antibody-Drug Conjugates (ADCs), Drug Discovery Series No. 81, Chapter 3.). Regarding the biologically active molecules that kill cancer cells, Trastuzumab emtansine uses the microtubule inhibitor DM1 as a cytotoxin, which results in a weak bystander effect when paired with a non-cleavable linker; Disitamab vedotin uses the auristatin toxin MMAE as a biological effector molecule, which is prone to problems such as neurotoxic accumulation after continuous repeated use; and both of the above ADC drugs are non-site randomly coupled, with a drug loading ratio (DAR) of approximately 4 and poor uniformity.

[0007] Summary of the Invention

[0008] The present invention aims to improve the above-mentioned problems in existing pharmaceutical technologies, specifically to provide a class of anti-Her2 antibody-drug conjugates that can be used to treat Her2-expressing tumors. The antibody-drug conjugates have good stability, homogeneity, hydrophilicity, efficacy, and safety.

[0009] The present application relates to an antibody drug conjugate, and exemplarily discloses an antibody drug conjugate having the general formula Ab-[MLED] with trastuzumab as the targeting moiety. x The antibody-drug conjugate of the structure shown in FIG. Results showed that the conjugate had excellent binding activity and proliferation inhibition against Her2-positive cells, and had a good targeted killing effect on Her2-positive tumors (e.g., breast cancer, lung cancer, or gastric cancer). Therefore, the present application provides an antibody-drug conjugate for treating Her2-expressing cancers, a pharmaceutical composition containing the antibody-drug conjugate, and their use in treating Her2-expressing cancers.

[0010] Antibody Drug Conjugates

[0011] In one aspect, the present application provides an antibody drug conjugate having the formula Ab-[MLED] x The structure shown, wherein:

[0012] Ab is an antibody or antigen-binding fragment thereof that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases:

[0013] M is a linker site with an antibody or antigen-binding fragment thereof;

[0014] L is a structural fragment connecting linkers M and E;

[0015] E is a structural fragment connecting L and D;

[0016] D is the cytotoxic drug fragment;

[0017] X is 1 to 10.

[0018] In the antibody-drug conjugate, the cytotoxic drug can be linked to the antibody or antigen-binding fragment thereof via the "MLE" fragment shown in this application.

[0019] In some embodiments, M is selected from the following substituted or unsubstituted structural fragments:

[0020] In some embodiments, M is selected from the following substituted or unsubstituted structural fragments:

[0021] In some embodiments, M is selected from the following substituted or unsubstituted structural fragments:

[0022] In some embodiments, M is selected from the following substituted or unsubstituted structural fragments:

[0023] In some embodiments, M is selected from the following substituted or unsubstituted structural fragments:

[0024] In some embodiments, M is selected from the following substituted or unsubstituted structural fragments:

[0025] In some embodiments, M is selected from the following substituted or unsubstituted structural fragments:

[0026] In some embodiments, M is selected from the following substituted or unsubstituted structural fragments:

[0027] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following: C 1-6Alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, 9-12 membered nitrogen-containing heterocyclic group, -N(R')-, -NH(R'), -N(R')2, carbonyl, -O-, natural amino acids or non-natural amino acids and their analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), Lys(R'), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Gl u, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly- Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG,SEQ ID NO:41), Gly-Gly-Val-Ala (GGVA, SEQ ID NO:42), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO:43), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO:44), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO:45)), Where R' represents hydrogen, C 1-6 Alkyl, -C 1-6 AlkyleneCO2H, -C 1-6 Alkylene SO3H, -SO3H, -PO3H2, -C 1-6 Alkylene-NHC 1-6 Alkyl, -C 1-6 Alkylene-N(C 1-6 Alkyl)2, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-heterocycle, -CH2NH-SO3H, -CH2N(C 1-6 Alkyl)-SO3H, -CH2NHC1-6 Alkylene -SO3H, -CH2N(C 1-6 Alkyl)C 1-6 Alkylene -SO3H, -CH2N(C 1-6 Alkylene -SO3H)2, -CH2N + (C 1-6 Alkylene -SO3H)3, -CH2N + (C 1-6 alkyl)2-C 1-6 Alkylene -SO3H, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene -SO3H)3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene -SO3H)3, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 alkyl)3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkyl)3, -CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 Alkyl)3, -CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 Alkyl)2-CH2CO2H, -CH2N(C 1-6 alkyl)-C 1-6 Alkylene -CO2H, -CH2N + (C 1-6 alkyl)2-C 1-6 Alkylene -CO2H, glucose, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl, -CH2N(C 1-6 alkyl)-C(=O)-(OCH2CH2) r -OC 1-6 Alkyl, -(CH2N(Me)-C(=O)) r -C 1-6Alkyl, or polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH2CH2O) r -C 1-6 alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), or NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue), wherein r is selected from an integer of 1-20; s is selected from an integer of 1-20.

[0028] In some embodiments, "-NOTA" refers to

[0029] In some embodiments, "-DOTA" refers to

[0030] In some embodiments, "-DOTAGA" refers to

[0031] In some embodiments, r is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 1-10, 1-8, 3-8, 1-6, 1-4, 1-2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0032] In some embodiments, s is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0033] In some embodiments, s is represented by n.

[0034] In some embodiments, L is selected from a divalent substituted or unsubstituted structural fragment consisting of one or more of the following groups: 1-6Alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, -N(R')-, carbonyl, -O-, natural amino acids or unnatural amino acids and their analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), Lys(R'), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Ci t, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), Where R' represents hydrogen, C 1-6 Alkyl, glucose, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl, or polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH2CH2O) r -C 1-6 alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), or NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue), wherein r is selected from an integer of 1 to 20; s is selected from an integer of 1 to 20;

[0035] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following: C 1-6 Alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, 9-12 membered nitrogen-containing heterocyclic group, -N(R')-, -NH(R'), -N(R')2, carbonyl, -O-, natural amino acids or non-natural amino acids and their analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val- Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val -Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), Where R' represents hydrogen, C 1-6 Alkyl, -C 1-6 AlkyleneCO2H, -C 1-6 Alkylene SO3H, -SO3H, -PO3H2, -C 1-6 Alkylene-NHC 1-6 Alkyl, -C 1-6 Alkylene-N(C 1-6 Alkyl)2, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-heterocycle, -CH2NH-SO3H, -CH2N(C 1-6 Alkyl)-SO3H, -CH2NHC 1-6 Alkylene -SO3H, -CH2N(C 1-6 Alkyl)C1-6 Alkylene -SO3H, -CH2N(C 1-6 Alkylene -SO3H)2, -CH2N + (C 1-6 Alkylene -SO3H)3, -CH2N + (C 1-6 alkyl)2-C 1-6 Alkylene -SO3H, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene -SO3H)3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene -SO3H)3, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 alkyl)3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkyl)3, -CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 Alkyl)3, -CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 Alkyl)2-CH2CO2H, -CH2N(C 1-6 alkyl)-C 1-6 Alkylene -CO2H, -CH2N + (C 1-6 alkyl)2-C 1-6 Alkylene -CO2H, glucose, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl, -CH2N(C 1-6 alkyl)-C(=O)-(OCH2CH2) r -OC 1-6 Alkyl, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl, or polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH2CH2O) r -C1-6 alkyl), wherein r is selected from an integer of 1-20; s is selected from an integer of 1-20;

[0036] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following: C 1-6 Alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, -N(R')-, carbonyl, -O-, natural amino acids or non-natural amino acids and their analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Va l-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Gl u-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), Where R' represents hydrogen, C 1-6 Alkyl, glucose, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl, or polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH2CH2O) r -C 1-6 alkyl), wherein r is selected from an integer of 1-20; s is selected from an integer of 1-20.

[0037] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following: C 1-6 Alkylene, carbonyl, 9-12 membered nitrogen-containing heterocyclic group, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gl y-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys- Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, wherein s is selected from an integer of 1 to 20;

[0038] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following: Val-Ala, Val-Cit, Ala-Ala-Ala, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Gly-Gly-Gly-Gly, wherein s is selected from an integer of 1 to 20;

[0039] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following:

[0040] In some embodiments, the substructure of L is selected from the following fragments:

[0041] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following:

[0042] In some embodiments, L is selected from the following substituted or unsubstituted structural fragments:

[0043] In some embodiments, L is selected from a divalent substituted or unsubstituted structural fragment consisting of one or more of the following groups:

[0044] s is selected from an integer from 1 to 20. In some embodiments, L is selected from a divalent substituted or unsubstituted structural fragment consisting of one or more of the following groups:

[0045] s is an integer selected from 1-20.

[0046] In some embodiments, L is selected from a divalent substituted or unsubstituted structural fragment consisting of one or more of the following groups:

[0047] s is selected from an integer of 1-20, preferably s is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 5-10, 8-10, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0048] In some embodiments, L is selected from a divalent substituted or unsubstituted structural fragment consisting of one or more of the following groups:

[0049] s is selected from an integer of 1-20, preferably s is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 5-10, 8-10, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0050] In some embodiments, L is selected from One of the The divalent substituted or unsubstituted structural fragment is composed of s, an integer selected from 1-20, preferably an integer selected from 1-15, such as an integer of 1-12, 3-12, 5-10, 8-10, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0051] In some embodiments, E is a single bond, a substituted or unsubstituted -NH-CH2-, or a substituted or unsubstituted structural fragment selected from the following:

[0052] In some embodiments, E is a single bond, substituted or unsubstituted -NH-CH2- or

[0053] In some embodiments, E is substituted or unsubstituted -NH-CH2- or

[0054] In some embodiments, E is a single bond or substituted or unsubstituted -NH-CH2-.

[0055] In some embodiments, Selected from the following substituted or unsubstituted structures:

[0056] s is an integer selected from 1-20.

[0057] In some embodiments, Selected from the following substituted or unsubstituted structures:

[0058] n is selected from integers of 1-20.

[0059] In some embodiments, Selected from the following substituted or unsubstituted structural fragments:

[0060] n is selected from integers of 1-20.

[0061] [Corrected 30.05.2025 in accordance with Rule 26] In some embodiments, Selected from the following substituted or unsubstituted structural fragments:

[0062] [Corrected 30.05.2025 in accordance with Rule 26] n is an integer selected from 1-20.

[0063] [Corrected 30.05.2025 in accordance with Rule 26] In some embodiments, Selected from the following substituted or unsubstituted structural fragments:

[0064] [Corrected 30.05.2025 according to Rule 26] n is an integer selected from 1-20, preferably n is an integer selected from 1-15, for example an integer from 1-12, 3-12, 5-10, 8-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10.

[0065] [Corrected 30.05.2025 in accordance with Rule 26] In some embodiments, Selected from the following substituted or unsubstituted structural fragments:

[0066] [Corrected 30.05.2025 in accordance with Rule 26] In some embodiments, Selected from the following substituted or unsubstituted structural fragments:

[0067] [Corrected 30.05.2025 according to Rule 26] n is an integer selected from 1-20, preferably n is an integer selected from 1-15, for example an integer from 1-12, 3-12, 5-10, 8-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10.

[0068] [Corrected 30.05.2025 in accordance with Rule 26] In some embodiments, Selected from the following substituted or unsubstituted structural fragments:

[0069] [Corrected 30.05.2025 in accordance with Rule 26] In some embodiments, Selected from the following substituted or unsubstituted structural fragments:

[0070] [Corrected 30.05.2025 according to Rule 26] n is an integer selected from 1-20, preferably n is an integer selected from 1-15, for example an integer from 1-12, 3-12, 5-10, 8-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10.

[0071] [Corrected 30.05.2025 in accordance with Rule 26] In some embodiments, Selected from the following substituted or unsubstituted structural fragments:

[0072] In some embodiments, the cytotoxic drug is selected from anti-tubulin agents, DNA intercalators, DNA topoisomerase inhibitors, RNA polymerase inhibitors and gene transcription inhibitors. In some embodiments, the anti-tubulin agent is an auristatin compound, a maytansine compound or an eribulin compound. In some embodiments, the DNA intercalator is a pyrrolobenzodiazepine (PBD) compound, trabectedin or rubitidine. In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., doxorubicin, doxorubicin, PNU-159682 and its analogs, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide). In some embodiments, the RNA polymerase inhibitor is α-amanitin. In some embodiments, the gene transcription inhibitor is triptolide and pharmaceutically acceptable salts, esters, and analogs thereof.

[0073] In some embodiments, the cytotoxic drug is selected from PNU-159682 analogs.

[0074] The cytotoxic drugs disclosed in this application generally contain multiple functional groups, such as hydroxyl (-OH), carboxyl (-COOH), primary amino (-NH2), secondary amine (-NR a H), tertiary amine group (-NR b R c ), where R a 、R b 、R c Here, only non-hydrogen substituents on N, or sulfhydryl (-SH) groups are represented, and these functional groups can react with appropriate functional groups in the rest of the conjugate to achieve attachment.

[0075] In some embodiments, the cytotoxic drug is linked to E in the antibody-drug conjugate via a -OH, primary amino, secondary amine, or tertiary amine group, or -SH group. In some embodiments, D is a monovalent structure obtained by losing one H from a -OH, -NH2, or secondary amine group on the cytotoxic drug.

[0076] In some embodiments, the cytotoxic drug has the structure shown in Formula (I):

[0077] in, Indicates a single bond or no chemical bond;

[0078] R1, R2, R3, R4, R6, and R7 are each independently selected from hydrogen, -CN, halogen, -OH, -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl; the alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl groups are optionally substituted by one or more selected from -CN, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 6-10 substituted by aryl and 5-10 membered heteroaryl substituents;

[0079] R5 is -(C=O)-D2-D3-X6, wherein D2 does not exist or is -O-, -S-, or -NR X -, where R X Selected from hydrogen or C 1-6 Alkyl, the C 1-6 The alkyl group is optionally substituted with one or more radicals selected from halogen, -OH, -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -CN substituent substituted; D3 is C 1-6 Alkylene, C 2-6 Alkenylene or C 2-6 Alkyne; the alkylene, alkenylene and alkynylene are optionally substituted by one or more selected from -CN, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 6-10 substituted by aryl and 5-10 membered heteroaryl substituents; X6 is selected from hydrogen, halogen, -OH, C 1-6 Alkoxy, -SH, -SC 1-6 Alkyl, -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl) 2, the C 1-6 The alkyl group is optionally substituted with one or more radicals selected from halogen, -OH, -NH2, -NH(C 1-6 Alkyl), -N(C1-6 Alkyl)2, -CN, C 1-6 Substitution of alkoxy groups;

[0080] Y1 and Y2 are -C(R8)2-, wherein R8 are independently selected from hydrogen, -CN, halogen, -OH, -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, or two R8 attached to the same carbon atom together form =O;

[0081] X1, X2 and X5 are each independently selected from substituted or unsubstituted C 1-6 Alkylene, -O-, -NR Y -or-S-; where R Y Each occurrence is independently selected from hydrogen or optionally substituted by one or more selected from halogen, -OH, -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -CN substituted C 1-6 alkyl;

[0082] when When it represents a single bond, X4 is selected from substituted or unsubstituted C 1-6 Alkylene, -O-, -NR Y -or-S-; where R Y Each occurrence is independently selected from hydrogen or optionally substituted by one or more selected from halogen, -OH, -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -CN substituted C 1-6 alkyl;

[0083] when When there is no chemical bond, X4 is selected from substituted or unsubstituted C 1-6 Alkyl, -OR Y 、-N(R Y )2 or -S-; wherein R Y Each occurrence is independently selected from hydrogen or optionally substituted by one or more selected from halogen, -OH, -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -CN substituted C 1-6 alkyl;

[0084] X3 selected from CR Z or N, where R Z is selected from hydrogen or optionally substituted by one or more selected from halogen, -OH, -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -CN substituted C 1-6 alkyl.

[0085] In some embodiments, R1, R2, R3, R4, R6, and R7 are each independently selected from hydrogen, -CN, halogen, -OH, -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, C 1-6 Alkyl and C 1-6 Alkoxy; the alkyl, alkoxy is optionally one or more selected from -CN, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 6-10 substituted by aryl and 5-10 membered heteroaryl substituents;

[0086] In some embodiments, R1, R2, R3, R4, R6, and R7 are each independently selected from hydrogen, -OH, and C 1-6 Alkoxy; the alkoxy is optionally substituted by one or more selected from -CN, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 6-10 substituted by aryl and 5-10 membered heteroaryl substituents;

[0087] In some embodiments, R1, R2, R3, R4, R6 and R7 are each independently selected from hydrogen, -OH, C 1-6 Alkyl and C 1-6 Alkoxy.

[0088] In some embodiments, R1, R2, R3, R4, R6, and R7 are each independently selected from hydrogen, -OH, and C 1-6 Alkoxy.

[0089] In some embodiments, R2, R3, and R4 are all -OH.

[0090] In some embodiments, R1 and R7 are each independently selected from C 1-6Alkoxy, such as methoxy, ethoxy, n-propoxy or isopropoxy. In some embodiments, R1 and R7 are both methoxy.

[0091] In some embodiments, R6 is selected from C 1-6 Alkyl, such as methyl, ethyl, n-propyl or isopropyl. In some embodiments, R6 is methyl.

[0092] In some embodiments, R1 and R7 are both methoxy; R2, R3, and R4 are all -OH; and R6 is methyl.

[0093] In some embodiments, R5 is -(C=O)-D2-D3-X6, wherein D2 is -O-, -S-, -NR X -, where R X Selected from hydrogen or C 1-6 Alkyl; D3 is C 1-6 Alkylene; the alkylene is optionally substituted by one or more selected from -CN, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 6-10 substituted by aryl and 5-10 membered heteroaryl substituents; X6 is selected from -OH, C 1-6 Alkoxy, -SH, -SC 1-6 Alkyl, -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl) 2, the C 1-6 The alkyl group is optionally substituted with one or more radicals selected from halogen, -OH, -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -CN, C 1-6 Substitution of alkoxy groups;

[0094] In some embodiments, R5 is -(C=O)-D2-D3-X6, wherein D2 is -O-, -S-, -NR X -, where R X Selected from hydrogen or C 1-6 Alkyl; D3 is C 1-6 Alkylene; X6 is selected from -OH, C 1-6 Alkoxy, -SH, -SC 1-6 Alkyl, -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2.

[0095] In some embodiments, R5 is -(C=O)-D2-D3-X6, wherein D2 is -O-, -S- or -NR X -, where R X Selected from hydrogen, methyl and ethyl; D3 is selected from methylene, ethylene, -(CH2)3, -CH2-CH(CH3)- and -CH2-C(CH3)2-; X6 is selected from -OH, methoxy, ethoxy, -SH, -S-CH3, -NH2, -NH(CH3) and -N(CH3)2.

[0096] In some embodiments, R5 is -(C=O)-D2-D3-X6, wherein D2 is selected from -O-, -S-, -NH- and -N(CH3)-; D3 is selected from methylene, ethylene, -(CH2)3, -CH2-CH(CH3)- and -CH2-C(CH3)2-; and X6 is selected from -OH, methoxy, ethoxy, -SH, -S-CH3, -NH2, -NH(CH3) and -N(CH3)2.

[0097] In some embodiments, R5 is -(C=O)-D2-D3-X6, wherein D2 is -O-, -S-, -NR X -, where R X Selected from hydrogen or C 1-3 Alkyl; D3 is C 1-6 Alkylene; X6 is selected from -OH, -SH, -NH2.

[0098] In some embodiments, R5 is selected from:

[0099] In some embodiments, Y1 and Y2 are -C(R8)2-, wherein R8 are independently selected from hydrogen, -CN, halogen, -OH, -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Alkoxy, or two R8 attached to the same carbon atom together form =O;

[0100] Preferably, Y1 and Y2 are -(C=O)-.

[0101] In some embodiments, X1, X2 and X5 are each independently selected from substituted or unsubstituted C 1-6 Alkylene, -O- or -NR Y ; where R Y Each occurrence is independently selected from hydrogen or optionally substituted by one or more selected from halogen, -OH, -NH2, -NH(C 1-6 Alkyl), -N(C 1-6Alkyl)2, -CN substituted C 1-6 alkyl;

[0102] Preferably, X1, X2, X4 and X5 are all -O-.

[0103] In some embodiments, when When it represents a single bond, X4 is selected from substituted or unsubstituted C 1-6 Alkylene, -O-; Preferably, X4 is selected from -O-;

[0104] In some embodiments, when When there is no chemical bond, X4 is selected from substituted or unsubstituted C 1-6 Alkyl, -OR Y ; where R Y Each occurrence is independently selected from hydrogen or optionally substituted by one or more selected from halogen, -OH, -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -CN substituted C 1-6 Alkyl; preferably, X4 is selected from -OH.

[0105] In some embodiments, X3 is selected from CR Z or N, where R Z is selected from hydrogen or C optionally substituted by one or more substituents selected from halogen, -OH, -NH2, -CN 1-6 alkyl;

[0106] In some embodiments, X3 is N.

[0107] In some embodiments, the cytotoxic drug has the structure shown in Formula (III)-1 or (III)-2:

[0108] Among them, X1~X5, R1~R7, Y1, and Y2 are as described above.

[0109] In some embodiments, the cytotoxic drug has the structure shown in Formula (IV)-1 or (IV)-2:

[0110] Wherein, R5 is as described above.

[0111] All technical features disclosed in this specification, except for mutually exclusive technical features, can be combined in any manner.

[0112] The present invention encompasses compounds resulting from any combination of the various embodiments.

[0113] In some embodiments, the cytotoxic drug is selected from the following compounds or isotopically labeled compounds thereof:

[0114] In some embodiments, the cytotoxic drug is selected from the following compounds or isotopically labeled compounds thereof:

[0115] In some embodiments, the cytotoxic drug is selected from the following compounds or isotopically labeled compounds thereof:

[0116] In some embodiments, the cytotoxic drug fragment has the structure shown in the following formula (I'):

[0117] Wherein: X1-X5, Y1, Y2, D2, D3, R1-R4, R6, R7 are as described above; X6' is selected from -O-, -S-, -NH-, or -N(C 1-6 alkyl)-.

[0118] In some embodiments, the cytotoxic drug fragment has the following structure:

[0119] Those skilled in the art will appreciate that the antibody-drug conjugates described herein can be prepared modularly. For example, a free-form "drug-linker" (which can be understood as M'-LED, where M' is the structural form of M before covalently linking to the antibody or its antigen-binding fragment) is first obtained, and then covalently linked to the antibody or its antigen-binding fragment to obtain the antibody-drug conjugate described herein. Accordingly, M' in the free-form "drug-linker" is linked to one or more sulfhydryl (-SH) or amino (-NH2) groups on the antibody or its antigen-binding fragment by a substitution reaction (e.g., removal of structures such as -SO2Me or pentafluorophenol thereon) or by an addition reaction.

[0120] In one aspect, the present application provides a compound or a pharmaceutically acceptable salt thereof having a structure shown in formula DEL-M', wherein:

[0121] M' is -M-Lg, wherein Lg is a leaving group of the nucleophilic substitution reaction and M is a structural fragment that binds to the targeting moiety;

[0122] L is the structural fragment connecting M and E;

[0123] E is a structural fragment connecting L and D;

[0124] D is a cytotoxic drug fragment.

[0125] In some embodiments, Lg is selected from halogen (eg, F, Cl, Br, I), halogenated C 1-6 Alkyl, C 1-6 Alkylsulfonyl, halo C 1-6 Alkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 Alkyl sulfoxide, halogenated phenoxy, hydroxyl (-OH), thiol (-SH), amino (-NH2), nitro, azido, cyano, alkenyl, alkynyl and alkynyl-containing structural fragments, the halogenated C 1-6 Alkyl, C 1-6 Alkylsulfonyl, halo C 1-6 Alkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 The alkylsulfoxide group, halophenoxy group, alkenyl group, alkynyl group and alkynyl group-containing structural fragments are optionally substituted with one or more suitable substituents.

[0126] In some embodiments, Lg is selected from halogen (eg, F, Cl, Br, I), halogenated C 1-6 Alkyl, C 1-6 Alkylsulfonyl, halo C 1-6 Alkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 Alkylsulfoxide, halogenated phenoxy, hydroxyl (-OH), thiol (-SH), amino (-NH2), nitro, azide, cyano, alkenyl, alkynyl and structural fragments containing alkynyl.

[0127] In some embodiments, Lg is selected from halogen, substituted or unsubstituted C 1-6 Alkylsulfonyl (C 1-6 alkyl-SO2-), halogenated phenoxy, hydroxyl (-OH), mercapto (-SH) or amino (-NH2).

[0128] In some embodiments, Lg is selected from halogen, substituted or unsubstituted methylsulfonyl, halophenoxy, hydroxyl (-OH), thiol (-SH), or amino (-NH2).

[0129] In some embodiments, Lg is selected from C 1-6Alkylsulfonyl or halogenated phenoxy.

[0130] In some embodiments, Lg is selected from methylsulfonyl or pentafluorophenoxy.

[0131] In some embodiments, in the compound having the structure shown in DEL-M' or a pharmaceutically acceptable salt thereof,

[0132] M' is -M-Lg,

[0133] Lg is selected from halogen, substituted or unsubstituted C 1-6 Alkylsulfonyl (C 1-6 alkyl-SO2-), halogenated phenoxy, hydroxyl (-OH), mercapto (-SH) or amino (-NH2), preferably Lg is selected from methylsulfonyl or pentafluorophenoxy;

[0134] M is selected from the following substituted or unsubstituted structural fragments:

[0135] L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following:

[0136] s is selected from an integer of 1-20, preferably s is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 5-10, 8-10, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

[0137] E is a single bond or a substituted or unsubstituted -NH-CH2-, preferably a single bond or -NH-CH2-;

[0138] D is a cytotoxic drug fragment, wherein the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0139] In some embodiments, in the compound having the structure shown in DEL-M' or a pharmaceutically acceptable salt thereof,

[0140] M' is -M-Lg,

[0141] Lg is selected from C 1-6 Alkylsulfonyl, preferably methylsulfonyl;

[0142] M is the following substituted or unsubstituted structural fragment:

[0143] Preferably, L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following:

[0144] s is selected from an integer of 1-20, preferably s is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 5-10, 8-10, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

[0145] Preferably, L is selected from One of the A divalent substituted or unsubstituted structural fragment composed of s, s is selected from an integer of 1-20, preferably s is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 5-10, 8-10, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

[0146] E is a single bond or a substituted or unsubstituted -NH-CH2-, preferably a single bond or -NH-CH2-;

[0147] D is a cytotoxic drug fragment, wherein the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0148] Preferred are compounds 6-7 or isotope-labeled compounds thereof.

[0149] In some embodiments, the free form of the "drug-linker" is selected from K-1 to K-18 shown below:

[0150] In some embodiments, the free form of the "drug-linker" is selected from the group consisting of K'-17 to K'-18 shown below:

[0151] wherein n is selected from an integer of 1-20, preferably n is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 5-10, 8-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10.

[0152] In some embodiments, the drug-linker is selected from K-1 to K-18, K'-17 to K'-18, wherein n is selected from an integer of 1-20, preferably n is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 5-10, 8-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10.

[0153] In some embodiments, the drug-linker is selected from K-1 to K-9.

[0154] In some embodiments, the present application provides compounds shown in K-1 to K-18, K'-17 to K'-18 or pharmaceutically acceptable salts thereof, wherein n is selected from an integer of 1-20, preferably n is selected from an integer of 1-15, for example, an integer of 1-12, 3-12, 5-10, 8-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10.

[0155] In some embodiments, the present application provides compounds shown as K-1 to K-18 or pharmaceutically acceptable salts thereof.

[0156] In some embodiments, the present application provides compounds shown as K-1 to K-9 or pharmaceutically acceptable salts thereof.

[0157] In some embodiments, the present application provides compounds shown in K-1 to K-16, K'-17 to K'-18 or pharmaceutically acceptable salts thereof, wherein n is selected from an integer of 1-20, preferably n is selected from an integer of 1-15, for example, an integer of 1-12, 3-12, 5-10, 8-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10.

[0158] In some embodiments, the aforementioned compounds or pharmaceutically acceptable salts thereof may be optionally substituted with one or more suitable substituents.

[0159] In some embodiments, Ab is an antibody or an antigen-binding fragment thereof. In some embodiments, Ab is an antibody or an antigen-binding fragment thereof that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases.

[0160] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0161] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL):

[0162] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5 or a variant thereof, CDR-H2 of SEQ ID NO: 6 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0163] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20 or a variant thereof, CDR-H2 of SEQ ID NO: 21 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0164] wherein the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0165] or,

[0166] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0167] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof, CDR-H2 of SEQ ID NO: 19 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0168] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33 or a variant thereof, CDR-H2 of SEQ ID NO: 34 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0169] wherein the variant described in any one of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0170] or,

[0171] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0172] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0173] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof, CDR-H2 of SEQ ID NO: 27 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0174] wherein the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0175] or,

[0176] (4) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0177] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13 or a variant thereof, CDR-H2 of SEQ ID NO: 14 or a variant thereof, and CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof, CDR-L2 of SEQ ID NO: 17 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0178] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 28 or a variant thereof, CDR-H2 of SEQ ID NO: 29 or a variant thereof, and CDR-H3 of SEQ ID NO: 30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 31 or a variant thereof, CDR-L2 of SEQ ID NO: 32 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0179] Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0180] In some embodiments, the antibody or antigen-binding fragment thereof comprises

[0181] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0182] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5 or a variant thereof, CDR-H2 of SEQ ID NO: 6 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0183] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20 or a variant thereof, CDR-H2 of SEQ ID NO: 21 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0184] wherein the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0185] or,

[0186] (2) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0187] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof, CDR-H2 of SEQ ID NO: 19 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0188] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33 or a variant thereof, CDR-H2 of SEQ ID NO: 34 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0189] wherein the variant described in any one of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0190] or,

[0191] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0192] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0193] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof, CDR-H2 of SEQ ID NO: 27 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0194] wherein the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0195] or,

[0196] (4) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0197] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13 or a variant thereof, CDR-H2 of SEQ ID NO: 14 or a variant thereof, and CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof, CDR-L2 of SEQ ID NO: 17 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0198] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 28 or a variant thereof, CDR-H2 of SEQ ID NO: 29 or a variant thereof, and CDR-H3 of SEQ ID NO: 30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 31 or a variant thereof, CDR-L2 of SEQ ID NO: 32 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0199] Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0200] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0201] (1) The following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0202] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6, and CDR-H3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0203] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20, CDR-H2 of SEQ ID NO: 21, and CDR-H3 of SEQ ID NO: 22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0204] or,

[0205] (2) The following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0206] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18, CDR-H2 of SEQ ID NO: 19, and CDR-H3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0207] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33, CDR-H2 of SEQ ID NO: 34, and CDR-H3 of SEQ ID NO: 22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0208] or,

[0209] (3) the following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0210] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11, CDR-H2 of SEQ ID NO: 12, and CDR-H3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0211] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26, CDR-H2 of SEQ ID NO: 27, and CDR-H3 of SEQ ID NO: 22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0212] or,

[0213] (4) The following heavy chain variable region (VH) and light chain variable region (VL), where the CDRs are defined according to the IMGT numbering system:

[0214] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13, CDR-H2 of SEQ ID NO: 14, and CDR-H3 of SEQ ID NO: 15; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16, CDR-L2 of SEQ ID NO: 17, and CDR-L3 of SEQ ID NO: 10; or

[0215] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:28, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:30; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:31, CDR-L2 of SEQ ID NO:32, and CDR-L3 of SEQ ID NO:25.

[0216] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0217] (a) VH or a variant thereof shown in SEQ ID NO: 1, and / or VL or a variant thereof shown in SEQ ID NO: 2; or

[0218] (b) VH or a variant thereof shown in SEQ ID NO: 3, and / or VL or a variant thereof shown in SEQ ID NO: 4;

[0219] wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0220] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0221] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0222] (b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4.

[0223] In some embodiments, the antibody or antigen-binding fragment thereof further comprises:

[0224] (a) a heavy chain constant region (CH) of a human immunoglobulin, or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived; and

[0225] (b) a light chain constant region (CL) of a human immunoglobulin, or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived.

[0226] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region.

[0227] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, wherein the variant has up to 20 conservative amino acid substitutions compared to SEQ ID NO: 35 (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4 or 5 conservative substitutions of amino acids).

[0228] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, wherein the variant has up to 20 conservative amino acid substitutions compared to SEQ ID NO: 36 (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4 or 5 conservative substitutions of amino acids).

[0229] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 and a light chain constant region (CL) as shown in SEQ ID NO: 36.

[0230] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0231] (1) a heavy chain comprising the VH sequence of SEQ ID NO: 1 and the heavy chain constant region (CH) of SEQ ID NO: 35, and a light chain comprising the VL sequence of SEQ ID NO: 2 and the light chain constant region (CL) of SEQ ID NO: 36; or

[0232] (2) A heavy chain comprising the VH sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.

[0233] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0234] (1) a heavy chain comprising the sequence shown in SEQ ID NO: 37, and a light chain comprising the sequence shown in SEQ ID NO: 38;

[0235] (2) A heavy chain comprising the sequence shown in SEQ ID NO: 39, and a light chain comprising the sequence shown in SEQ ID NO: 40.

[0236] In certain embodiments of the antibodies or antigen-binding fragments disclosed herein, the heavy chain constant domain may comprise a C-terminal lysine or lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide. In some embodiments of the antibodies or antigen-binding fragments thereof, the N-terminal amino acid of the antibodies or antigen-binding fragments thereof may be cyclized to pyroglutamic acid.

[0237] As known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0238] In certain embodiments, provided herein are compositions comprising the antibodies or antigen-binding fragments disclosed herein, wherein each antibody or antigen-binding fragment may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid, the N-terminal amino acid cyclized to pyroglutamic acid, or the N-terminal amino acid cyclized to pyroglutamate.

[0239] In certain embodiments, the antibodies or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to an antigen and may include post-translational modifications thereof (e.g., cleavage of a C-terminal lysine in a heavy chain, conversion of an N-terminal glutamine or glutamic acid in a heavy or light chain to pyroglutamic acid or pyroglutamate), which may occur upon recombinant expression in a host cell (e.g., a CHO cell) or during purification / storage.

[0240] In certain embodiments, the N-terminal glutamine of the VH of SEQ ID NO: 1 or 3 or its variants or the heavy chain of SEQ ID NO: 37 or 39 or its variants undergoes cyclization to form pyroglutamate or pyroglutamate salt.

[0241] In certain embodiments, the heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof or the heavy chain of the sequence as shown in SEQ ID NO: 37 or 39 or a variant thereof lacks a C-terminal lysine.

[0242] In some embodiments, the antibody or antigen-binding fragment thereof is selected from Trastuzumab or Pertuzumab, the amino acid sequence of Trastuzumab has an accession number in the IMGT database (IMGT / mAb-DB ID): 97, and the amino acid sequence of Pertuzumab has an accession number in the IMGT database (IMGT / mAb-DB ID): 80.

[0243] In some embodiments, M is linked to a sulfhydryl (—SH) or amino (—NH 2 ) group on Ab.

[0244] In some embodiments, M is linked to a sulfhydryl (—SH) group on Ab.

[0245] In some embodiments, in the presence of Ab-[MLED] x In the antibody drug conjugate of the structure shown, MLED is formed by the compound represented by DEL-M', preferably by removing Lg from DEL-M', wherein the compound represented by DEL-M' is as defined above, and Ab is as defined above.

[0246] In some embodiments, in the presence of Ab-[MLED] x In the antibody-drug conjugate of the structure shown, MLED is formed by the compound represented by K-1 to K-18 or K'-17 to K'-18 (e.g., K-1 to K-18), preferably by removing the -SO2Me or pentafluorophenoxy group of the compound, and Ab is as defined above.

[0247] In some embodiments, in the presence of Ab-[MLED] x In the antibody drug conjugate of the structure shown, MLED is formed by removing -SO2Me from the compound represented by K'-17 to K'-18 (eg, K-17 to K-18), and Ab is as defined above.

[0248] In some embodiments, in the presence of Ab-[MLED] x In the antibody drug conjugate of the structure shown,

[0249] M is the following substituted or unsubstituted structural fragment:

[0250] Preferably, L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following:

[0251] s is selected from an integer of 1-20, preferably s is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 5-10, 8-10, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

[0252] Preferably, L is selected from One of the A divalent substituted or unsubstituted structural fragment composed of s, s is selected from an integer of 1-20, preferably s is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 5-10, 8-10, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

[0253] E is a single bond or a substituted or unsubstituted -NH-CH2-, preferably a single bond or -NH-CH2-;

[0254] D is a cytotoxic drug fragment, wherein the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0255] Preferably, it is compound 6-7 or an isotope-labeled compound thereof;

[0256] Ab is an antibody or its antigen-binding fragment that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases.

[0257] The antibody or antigen-binding fragment thereof is as defined above,

[0258] Preferably, the antibody or antigen-binding fragment thereof comprises:

[0259] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL):

[0260] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5 or a variant thereof, CDR-H2 of SEQ ID NO: 6 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0261] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20 or a variant thereof, CDR-H2 of SEQ ID NO: 21 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0262] wherein the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0263] or,

[0264] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0265] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof, CDR-H2 of SEQ ID NO: 19 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0266] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33 or a variant thereof, CDR-H2 of SEQ ID NO: 34 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0267] wherein the variant described in any one of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0268] or,

[0269] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0270] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0271] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof, CDR-H2 of SEQ ID NO: 27 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0272] wherein the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0273] or,

[0274] (4) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0275] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13 or a variant thereof, CDR-H2 of SEQ ID NO: 14 or a variant thereof, and CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof, CDR-L2 of SEQ ID NO: 17 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0276] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 28 or a variant thereof, CDR-H2 of SEQ ID NO: 29 or a variant thereof, and CDR-H3 of SEQ ID NO: 30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 31 or a variant thereof, CDR-L2 of SEQ ID NO: 32 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0277] wherein the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0278] Preferably, the antibody or antigen-binding fragment thereof comprises:

[0279] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0280] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5 or a variant thereof, CDR-H2 of SEQ ID NO: 6 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0281] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20 or a variant thereof, CDR-H2 of SEQ ID NO: 21 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0282] wherein the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0283] or,

[0284] (2) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0285] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof, CDR-H2 of SEQ ID NO: 19 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0286] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33 or a variant thereof, CDR-H2 of SEQ ID NO: 34 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0287] wherein the variant described in any one of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0288] or,

[0289] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0290] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0291] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof, CDR-H2 of SEQ ID NO: 27 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0292] wherein the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0293] or,

[0294] (4) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0295] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13 or a variant thereof, CDR-H2 of SEQ ID NO: 14 or a variant thereof, and CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof, CDR-L2 of SEQ ID NO: 17 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0296] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 28 or a variant thereof, CDR-H2 of SEQ ID NO: 29 or a variant thereof, and CDR-H3 of SEQ ID NO: 30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 31 or a variant thereof, CDR-L2 of SEQ ID NO: 32 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0297] wherein the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0298] Preferably, the antibody or antigen-binding fragment thereof comprises:

[0299] (1) The following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0300] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6, and CDR-H3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0301] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20, CDR-H2 of SEQ ID NO: 21, and CDR-H3 of SEQ ID NO: 22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0302] or,

[0303] (2) The following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0304] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18, CDR-H2 of SEQ ID NO: 19, and CDR-H3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0305] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33, CDR-H2 of SEQ ID NO: 34, and CDR-H3 of SEQ ID NO: 22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0306] or,

[0307] (3) the following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0308] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11, CDR-H2 of SEQ ID NO: 12, and CDR-H3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0309] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26, CDR-H2 of SEQ ID NO: 27, and CDR-H3 of SEQ ID NO: 22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0310] or,

[0311] (4) The following heavy chain variable region (VH) and light chain variable region (VL), where the CDRs are defined according to the IMGT numbering system:

[0312] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13, CDR-H2 of SEQ ID NO: 14, and CDR-H3 of SEQ ID NO: 15; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16, CDR-L2 of SEQ ID NO: 17, and CDR-L3 of SEQ ID NO: 10; or

[0313] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 28, CDR-H2 of SEQ ID NO: 29, and CDR-H3 of SEQ ID NO: 30; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 31, CDR-L2 of SEQ ID NO: 32, and CDR-L3 of SEQ ID NO: 25;

[0314] Preferably, the antibody or antigen-binding fragment thereof comprises:

[0315] (a) VH or a variant thereof shown in SEQ ID NO: 1, and / or VL or a variant thereof shown in SEQ ID NO: 2; or

[0316] (b) VH or a variant thereof shown in SEQ ID NO: 3, and / or VL or a variant thereof shown in SEQ ID NO: 4;

[0317] wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0318] Preferably, the antibody or antigen-binding fragment thereof comprises:

[0319] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0320] (b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4;

[0321] Preferably, the antibody or antigen-binding fragment thereof is selected from Trastuzumab or Pertuzumab or an antigen-binding fragment thereof.

[0322] In some embodiments, the antibody drug conjugate is selected from ADC K-1 to ADC K-18 shown below:

[0323] Wherein, HA in each antibody-drug conjugate represents an antibody or antigen-binding fragment thereof that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family receptor tyrosine kinase;

[0324] in, Or it indicates the specific connection mode between the sulfhydryl group in the antibody or antigen-binding fragment thereof and the M fragment;

[0325] Indicates the specific connection method between the amino group in the antibody or its antigen-binding fragment and the M fragment.

[0326] In some embodiments, the antibody drug conjugate is selected from the group consisting of ADC K'-17 to ADC K'-18 shown below:

[0327] Wherein, HA in each antibody-drug conjugate represents an antibody or antigen-binding fragment thereof that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases; n is selected from an integer of 1-20, preferably an integer of 1-15, such as an integer of 1-12, 3-12, 5-10, or 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, preferably 5, 8, or 10;

[0328] in, Or it represents the specific connection method between the sulfhydryl group in the antibody or its antigen-binding fragment and the M fragment.

[0329] In some embodiments, the antibody or antigen-binding fragment thereof in each antibody drug conjugate is as defined above.

[0330] In some embodiments, the HA comprises:

[0331] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL):

[0332] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5 or a variant thereof, CDR-H2 of SEQ ID NO: 6 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0333] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20 or a variant thereof, CDR-H2 of SEQ ID NO: 21 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0334] wherein the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0335] or,

[0336] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0337] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof, CDR-H2 of SEQ ID NO: 19 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0338] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33 or a variant thereof, CDR-H2 of SEQ ID NO: 34 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0339] wherein the variant described in any one of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0340] or,

[0341] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0342] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0343] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof, CDR-H2 of SEQ ID NO: 27 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0344] wherein the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0345] or,

[0346] (4) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0347] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13 or a variant thereof, CDR-H2 of SEQ ID NO: 14 or a variant thereof, and CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof, CDR-L2 of SEQ ID NO: 17 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0348] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 28 or a variant thereof, CDR-H2 of SEQ ID NO: 29 or a variant thereof, and CDR-H3 of SEQ ID NO: 30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 31 or a variant thereof, CDR-L2 of SEQ ID NO: 32 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0349] Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0350] In some embodiments, the HA comprises

[0351] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0352] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5 or a variant thereof, CDR-H2 of SEQ ID NO: 6 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0353] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20 or a variant thereof, CDR-H2 of SEQ ID NO: 21 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0354] wherein the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0355] or,

[0356] (2) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0357] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof, CDR-H2 of SEQ ID NO: 19 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0358] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33 or a variant thereof, CDR-H2 of SEQ ID NO: 34 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0359] wherein the variant described in any one of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0360] or,

[0361] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0362] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0363] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof, CDR-H2 of SEQ ID NO: 27 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0364] wherein the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0365] or,

[0366] (4) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0367] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13 or a variant thereof, CDR-H2 of SEQ ID NO: 14 or a variant thereof, and CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof, CDR-L2 of SEQ ID NO: 17 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0368] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 28 or a variant thereof, CDR-H2 of SEQ ID NO: 29 or a variant thereof, and CDR-H3 of SEQ ID NO: 30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 31 or a variant thereof, CDR-L2 of SEQ ID NO: 32 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0369] Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0370] In some embodiments, the HA comprises:

[0371] (1) The following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0372] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6, and CDR-H3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0373] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20, CDR-H2 of SEQ ID NO: 21, and CDR-H3 of SEQ ID NO: 22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0374] or,

[0375] (2) The following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0376] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18, CDR-H2 of SEQ ID NO: 19, and CDR-H3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0377] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33, CDR-H2 of SEQ ID NO: 34, and CDR-H3 of SEQ ID NO: 22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0378] or,

[0379] (3) the following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0380] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11, CDR-H2 of SEQ ID NO: 12, and CDR-H3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0381] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26, CDR-H2 of SEQ ID NO: 27, and CDR-H3 of SEQ ID NO: 22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0382] or,

[0383] (4) The following heavy chain variable region (VH) and light chain variable region (VL), where the CDRs are defined according to the IMGT numbering system:

[0384] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13, CDR-H2 of SEQ ID NO: 14, and CDR-H3 of SEQ ID NO: 15; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16, CDR-L2 of SEQ ID NO: 17, and CDR-L3 of SEQ ID NO: 10; or

[0385] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:28, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:30; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:31, CDR-L2 of SEQ ID NO:32, and CDR-L3 of SEQ ID NO:25.

[0386] In some embodiments, the HA comprises:

[0387] (a) VH or a variant thereof shown in SEQ ID NO: 1, and / or VL or a variant thereof shown in SEQ ID NO: 2; or

[0388] (b) VH or a variant thereof shown in SEQ ID NO: 3, and / or VL or a variant thereof shown in SEQ ID NO: 4;

[0389] wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0390] In some embodiments, the HA comprises:

[0391] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0392] (b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4.

[0393] In some embodiments, the HA further comprises:

[0394] (a) a heavy chain constant region (CH) of a human immunoglobulin, or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived; and

[0395] (b) a light chain constant region (CL) of a human immunoglobulin, or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived.

[0396] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region.

[0397] In some embodiments, the HA comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 35, or a variant thereof, which has up to 20 conservative amino acid substitutions compared to SEQ ID NO: 35 (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; for example, 1, 2, 3, 4 or 5 conservative amino acid substitutions).

[0398] In some embodiments, the HA comprises a light chain constant region (CL) as set forth in SEQ ID NO: 36, or a variant thereof, which has up to 20 conservative amino acid substitutions compared to SEQ ID NO: 36 (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; for example, 1, 2, 3, 4 or 5 conservative amino acid substitutions).

[0399] In some embodiments, the HA comprises a heavy chain constant region (CH) as set forth in SEQ ID NO:35 and a light chain constant region (CL) as set forth in SEQ ID NO:36.

[0400] In some embodiments, the HA comprises:

[0401] (1) a heavy chain comprising the VH sequence of SEQ ID NO: 1 and the heavy chain constant region (CH) of SEQ ID NO: 35, and a light chain comprising the VL sequence of SEQ ID NO: 2 and the light chain constant region (CL) of SEQ ID NO: 36; or

[0402] (2) A heavy chain comprising the VH sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.

[0403] In some embodiments, the HA comprises:

[0404] (1) a heavy chain comprising the sequence shown in SEQ ID NO: 37, and a light chain comprising the sequence shown in SEQ ID NO: 38; or

[0405] (2) A heavy chain comprising the sequence shown in SEQ ID NO: 39, and a light chain comprising the sequence shown in SEQ ID NO: 40.

[0406] In certain embodiments of the antibodies or antigen-binding fragments disclosed herein, the heavy chain constant domain may comprise a C-terminal lysine or lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide. In some embodiments of the antibodies or antigen-binding fragments thereof, the N-terminal amino acid of the antibodies or antigen-binding fragments thereof may be cyclized to pyroglutamic acid.

[0407] As known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0408] In certain embodiments, provided herein are compositions comprising the antibodies or antigen-binding fragments disclosed herein, wherein each antibody or antigen-binding fragment may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid, the N-terminal amino acid cyclized to pyroglutamic acid, or the N-terminal amino acid cyclized to pyroglutamate.

[0409] In certain embodiments, the antibodies or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to an antigen and may include post-translational modifications thereof (e.g., cleavage of a C-terminal lysine in a heavy chain, conversion of an N-terminal glutamine or glutamic acid in a heavy or light chain to pyroglutamic acid or pyroglutamate), which may occur upon recombinant expression in a host cell (e.g., a CHO cell) or during purification / storage.

[0410] In certain embodiments, the N-terminal glutamine of the VH of SEQ ID NO: 1 or 3 or its variants or the heavy chain of SEQ ID NO: 37 or 39 or its variants undergoes cyclization to form pyroglutamate or pyroglutamate salt.

[0411] In certain embodiments, the heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof or the heavy chain of the sequence as shown in SEQ ID NO: 37 or 39 or a variant thereof lacks a C-terminal lysine.

[0412] In some embodiments, HA is selected from Trastuzumab or Pertuzumab, the amino acid sequence of Trastuzumab has an IMGT database accession number (IMGT / mAb-DB ID): 97, and the amino acid sequence of Pertuzumab has an IMGT database accession number (IMGT / mAb-DB ID): 80.

[0413] In some embodiments, HA in each antibody drug conjugate represents trastuzumab, pertuzumab, or an antigen-binding fragment thereof.

[0414] In some embodiments, HA in each antibody drug conjugate represents the following antibody or antigen-binding fragment:

[0415] (1) a heavy chain comprising the VH sequence of SEQ ID NO: 1 and the heavy chain constant region (CH) of SEQ ID NO: 35, and a light chain comprising the VL sequence of SEQ ID NO: 2 and the light chain constant region (CL) of SEQ ID NO: 36; or

[0416] (2) A heavy chain comprising the VH sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.

[0417] In certain embodiments of the antibodies or antigen-binding fragments disclosed herein, the heavy chain constant domain may comprise a C-terminal lysine or lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide. In some embodiments of the antibodies or antigen-binding fragments thereof, the N-terminal amino acid of the antibodies or antigen-binding fragments thereof may be cyclized to pyroglutamic acid.

[0418] As known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0419] In certain embodiments, provided herein are compositions comprising the antibodies or antigen-binding fragments disclosed herein, wherein each antibody or antigen-binding fragment may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid, the N-terminal amino acid cyclized to pyroglutamic acid, or the N-terminal amino acid cyclized to pyroglutamate.

[0420] In certain embodiments, the antibodies or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to an antigen and may include post-translational modifications thereof (e.g., cleavage of a C-terminal lysine in a heavy chain, conversion of an N-terminal glutamine or glutamic acid in a heavy or light chain to pyroglutamic acid or pyroglutamate), which may occur upon recombinant expression in a host cell (e.g., a CHO cell) or during purification / storage.

[0421] In certain embodiments, the N-terminal glutamine of the VH of SEQ ID NO: 1 or 3 or its variants or the heavy chain of SEQ ID NO: 37 or 39 or its variants undergoes cyclization to form pyroglutamate or pyroglutamate salt.

[0422] In certain embodiments, the heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof or the heavy chain of the sequence as shown in SEQ ID NO: 37 or 39 or a variant thereof lacks a C-terminal lysine.

[0423] In some embodiments, x in the conjugate represented by Ab-[MLED]x is 1-10, e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, or 4-10.

[0424] In some embodiments, x in the conjugate represented by Ab-[MLED]x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0425] In some embodiments, x in the conjugate represented by Ab-[MLED]x is 1, 2, 3, or 4.

[0426] In some embodiments, x in the conjugate represented by Ab-[MLED]x is 3, 4, or 5.

[0427] In some embodiments, x in the conjugate represented by Ab-[MLED]x is 2 or 4.

[0428] In some embodiments, the conjugate of the present invention is an antibody drug conjugate (ADC).

[0429] In some embodiments, the conjugates described herein are optionally substituted with one or more suitable substituents.

[0430] The present application further provides a formula Ab-[L'-D] x The antibody-drug conjugate shown, wherein:

[0431] Ab is an antibody or an antigen-binding fragment thereof;

[0432] D is a cytotoxic drug fragment;

[0433] L' is a linker connecting Ab and D;

[0434] x is an integer from 1 to 10;

[0435] The antibody or antigen-binding fragment thereof is coupled to a drug-linker selected from the group consisting of:

[0436] In certain embodiments, the antibody comprises the heavy chain amino acid sequence of SEQ ID NO:37, and the light chain amino acid sequence of SEQ ID NO:38.

[0437] In certain embodiments, the antibody is Trastuzumab.

[0438] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-4 (e.g., 1, 2, 3, or 4) of the following structures:

[0439] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0440] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-4 (e.g., 1, 2, 3, or 4) of the following structures:

[0441] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0442] Composition

[0443] In another aspect, the present application provides a composition of an antibody drug conjugate (ADC) as described herein. Such a composition may comprise a plurality of ADCs as described herein, wherein each ADC comprises a drug-linker as described herein, wherein x is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In other words, each antibody molecule in the composition can be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 drug-linkers. Thus, the composition is characterized in that the "drug-antibody" ratio (DAR) is in the range of about 1 to about 10. Methods for determining DAR are well known to those skilled in the art, including methods using reverse phase chromatography or HPLC-MS.

[0444] For example, in any embodiment, the ADC compositions described herein have a DAR of about 1 to about 10, or any subrange therebetween, e.g., about about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to 10, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7, about 2 to 8, about 2 to 9, about 1 to 10. or about 9 to 10.

[0445] In certain embodiments, the DAR of the ADC compositions described herein is about 1 to 8, e.g., about 1.0 to 1.5, about 1.5 to 2.0, about 2.0 to 2.5, about 2.5 to 3.0, about 3.0 to 3.5, about 3.5 to 4.0, about 4.0 to 4.5, about about 4.5 to 5.0, about 5.0 to 5.5, about 5.5 to 6.0, about 5.5 to 6.5, about 5.5 to 7.0, about 5.5 to 7.5, about 5.5 to 8.0, about 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5, about 6.0 to 8.5, about 6.5 to 7.0, about 6.5 to 7.5, about 6.5 to 8.0, about 6.5 to 8.5, about 7.0 to 7.5, about 7.0 to 8.0.

[0446] In certain embodiments, the DAR of the ADC compositions described herein is about 1 to 5, e.g., about 1.0 to 1.5, about 1.5 to 2.0, about 2.0 to 2.5, about 2.5 to 3.0, about 3.0 to 3.5, about 3.5 to 4.0, about 3.5 to 4.5, about 4.0 to 4.5, about 4.5 to 5.0.

[0447] In certain embodiments, the DAR of the ADC compositions described herein is about 1 to 3, e.g., about 1.0 to 1.5, about 1.0 to 2.0, about 1.0 to 2.5, about 1.0 to 3.0, about 1.5 to 2.0, about 1.5 to 2.5, about 1.5 to 3.0, about 2.0 to 2.5, about 2.0 to 3.0, about 2.5 to 3.0.

[0448] In certain embodiments, the DAR of the ADC compositions described herein is from about 1.0 to 6.0, e.g., from about 1.0 to 5.5, from about 1.0 to 5.0, from about 1.5 to 6.0, from about 1.5 to about 5.5, from about 1.5 to 5.0, 2.0 to 5.5, from about 2.0 to about 5.0, e.g., 1.0, about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.1, about 1.11, about 1.12, about 1.13, about 1.14, about 1.15, about 1.16, about 1.17, about 1.18, about 1.19, about 1.2, about 1.21, about 1.22, about 1.23, about 1.24, about 1.25 .25, about 1.26, about 1.27, about 1.28, about 1.29, about 1.3, about 1.31, about 1.32, about 1.33, about 1.34, about 1.35, about 1.36, about 1.37, about 1.38, about 1.39, about 1.4, about 1.41, about 1.42, about 1.43, about 1.44, about 1.45, about 1. 46, about 1.47, about 1.48, about 1.49, about 1.5, about 1.51, about 1.52, about 1.53, about 1.54, about 1.55, about 1.56, about 1.57, about 1.58, about 1.59, about 1.6, about 1.61, about 1.62, about 1.63, about 1.64, about 1.65, about 1.66, about 1.6 7, about 1.68, about 1.69, about 1.7, about 1.71, about 1.72, about 1.73, about 1.74, about 1.75, about 1.76, about 1.77, about 1.78, about 1.79, about 1.8, about 1.81, about 1.82, about 1.83, about 1.84, about 1.85, about 1.86, about 1.87, about 1.88 , about 1.89, about 1.9, about 1.91, about 1.92, about 1.93, about 1.94, about 1.95, about 1.96, about 1.97, about 1.98, about 1.99, about 2.0, about 2.01, about 2.02, about 2.03, about 2.04, about 2.05, about 2.06, about 2.07, about 2.08, about 2.09, about 2.1, about 2.11, about 2.12, about 2.13, about 2.14, about 2.15, about 2.16, about 2.17, about 2.18, about 2.19, about 2.2, about 2.21, about 2.22, about 2.23, about 2.24, about 2.25, about 2.26, about 2.27, about 2.28, about 2.29, about 2.3, about 2 .31, about 2.32, about 2.33, about 2.34, about 2.35, about 2.36, about 2.37, about 2.38, about 2.39, about 2.4, about 2.41, about 2.42, about 2.43, about 2.44, about 2.45, about 2.46, about 2.47, about 2.48, about 2.49, about 2.5, about 2.51, about 2.52, about 2.53, about 2.54, about 2.55, about 2.56, about 2.57, about 2.58, about 2.59, about 2.6, about 2.61, about 2.62, about 2.63, about 2.64, about 2.65, about 2.66, about 2.67, about 2.68, about 2.69, about 2.7, about 2.71, about 2.72, about 2. 73, about 2.74, about 2.75, about 2.76, about 2.77, about 2.78, about 2.79, about 2.8, about 2.81, about 2.82, about 2.83, about 2.84, about 2.85, about 2.86, about 2.87, about 2.88, about 2.89, about 2.9, about 2.91, about 2.92, about 2.93, about 2.9 4, about 2.95, about 2.96, about 2.97, about 2.98, about 2.99, about 3.0, about 3.01, about 3.02, about 3.03, about 3.04, about 3.05, about 3.06, about 3.07, about 3.08, about 3.09, about 3.1, about 3.11, about 3.12, about 3.13, about 3.14, about 3.1 5, about 3.16, about 3.17, about 3.18, about 3.19, about 3.2, about 3.21, about 3.22, about 3.23, about 3.24, about 3.25, about 3.26, about 3.27, about 3.28, about 3.29, about 3.3, about 3.31, about 3.32, about 3.33, about 3.34, about 3.35, about 3.36 , about 3.37, about 3.38, about 3.39, about 3.4, about 3.41, about 3.42, about 3.43, about 3.44, about 3.45, about 3.46, about 3.47, about 3.48, about 3.49, about 3.5, about 3.51, about 3.52, about 3.53, about 3.54, about 3.55, about 3.56, about 3.57 , about 3.58, about 3.59, about 3.6, about 3.61, about 3.62, about 3.63, about 3.64, about 3.65, about 3.66, about 3.67, about 3.68, about 3.69, about 3.7, about 3.71, about 3.72, about 3.73, about 3.74, about 3.75, about 3.76, about 3.77, about 3.78, about 3.79, about 3.8, about 3.81, about 3.82, about 3.83, about 3.84, about 3.85, about 3.86, about 3.87, about 3.88, about 3.89, about 3.9, about 3.91, about 3.92, about 3.93, about 3.94, about 3.95, about 3.96, about 3.97, about 3.98, about 3.99, about 4.0, about 4.01, about 4.02, about 4.03, about 4.04, about 4.05, about 4.06, about 4.07, about 4.08, about 4.09, about 4.1, about 4.11, about 4.12, about 4.13, about 4.14, about 4.15, about 4.16, about 4.17, about 4.18, about 4.19, about 4.2, about 4.21, about 4.22, about 4.23, about 4.24, about 4.25, about 4.26, about 4.27, about 4.28, about 4.29, about 4.3, about 4.31, about 4.32, about 4.33, about 4.34, about 4.35, about 4.36, about 4.37, about 4.38, about 4.39, about 4.4, about 4.41, about 4.42, about 4.43, about 4.44, about 4.45, about 4.46, about 4.47, about 4.48, about 4.49, about 4.5, about 4.51, about 4.52, about 4.53, about 4.54, about 4.55, about 4.56, about 4.57, about 4.58, about 4.59, about 4.6 4.61, about 4.62, about 4.63, about 4.64, about 4.65, about 4.66, about 4.67, about 4.68, about 4.69, about 4.7, about 4.71, about 4.72, about 4.73, about 4.74, about 4.75, about 4.76, about 4.77, about 4.78, about 4.79, about 4.8, about 4.81, about 4.82, about 4.83, about 4.84, about 4.85, about 4.86, about 4.87, about 4.88, about 4.89, about 4.9, about 4.91, about 4.92, about 4.93, about 4.94, about 4.95, about 4.96, about 4.97, about 4.98, about 4.99, about 5.0.

[0449] In certain embodiments, the DAR of the ADC compositions described herein is about 4.28 or about 4.44.

[0450] In certain embodiments, the DAR of the ADC compositions described herein is 4.28 or 4.44.

[0451] In certain embodiments, the composition is composed of Ab-[L'-D] x In certain embodiments, the composition comprises an antibody drug conjugate. In certain embodiments, the DAR of the composition is 2-6, such as 3-5, such as about 4. In certain embodiments, the DAR of the composition is 4.28. In certain embodiments, the DAR of the composition is 4.44. In certain embodiments, the DAR of the composition is 4.28, and the antibody drug conjugates with x being 4 account for 50-90% (e.g., 60%-90%, such as 65%-75%, and another example 71.74%). In certain embodiments, the DAR of the composition is 4.44, and the antibody drug conjugates with x being 4 account for 50-90% (e.g., 50%-70%, such as 55%-65%, and another example 58.67%).

[0452] intermediates

[0453] In some embodiments, the present application provides an intermediate compound having the structure shown below, or a salt, stereoisomer, tautomer, or isotope-labeled compound thereof:

[0454] In some embodiments, the present application provides an intermediate compound having the structure shown below, or a salt, stereoisomer, tautomer, or isotope-labeled compound thereof:

[0455] in

[0456] PG1 and PG2 are each independently H or an amino protecting group, wherein the amino protecting group is an alkoxycarbonyl amino protecting group, such as benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), methyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methyl (or ethyl)oxycarbonyl; an acyl amino protecting group, such as phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa) , o-(p-)nitrobenzenesulfonyl (Ns), pivaloyl, benzoyl, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, benzyloxycarbonyl, p-toluenesulfonyl, p-nitrobenzenesulfonyl, trifluoroacetyl, methoxycarbonyl, or ethoxycarbonyl; alkyl amino protecting groups, such as trityl (Trt), 2,4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), benzyl (Bn);

[0457] PG3 is each independently H or a carboxyl protecting group, such as C 1-6 Alkyl, allyl, benzyl, 2,4-dimethoxybenzyl, p-methoxybenzyl, methoxyethoxymethyl, pentafluorophenyl, 4-p-methylbenzyloxybenzyl;

[0458] PG4 is each independently H or a hydroxyl protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), methyl, tert-butyl, allyl, benzyl, methoxymethyl (MOM), ethoxyethyl, 2-tetrahydropyranyl (THP), formyl, acetyl, benzoyl, p-nitrobenzoyl, methylsulfonyl, trifluoromethanesulfonyl, nitrobenzenesulfonyl or p-toluenesulfonyl.

[0459] In another aspect, the present application provides use of the aforementioned intermediate compound or its salt, stereoisomer, tautomer or isotope-labeled compound in preparing the compound of the present invention or a pharmaceutically acceptable salt thereof.

[0460] Synthesis Method The present application provides a method for preparing the following compound K'-17:

[0461] The method comprises reacting compound Int-2' and compound K-17-7 to obtain compound K'-17:

[0462] In some embodiments, the reaction is carried out in the presence of DIPEA and / or HATU reagent.

[0463] In some embodiments, the present application provides a method for preparing compound K-17-7, which comprises removing the protecting group PG1 from compound K-17-6' to obtain compound K-17-7:

[0464] In some embodiments, the reaction is carried out in the presence of an organic basic reagent.

[0465] In some embodiments, the organic basic reagent is piperidine.

[0466] In some embodiments, the present application provides a method for preparing compound K-17-6', comprising reacting compound K-17-5' with (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylic acid to obtain compound K-17-6':

[0467] In some embodiments, the reaction is carried out in the presence of a DIPEA reagent.

[0468] In some embodiments, the reaction is performed in the presence of a PyBOP reagent.

[0469] In some embodiments, the reaction is carried out under nitrogen protection.

[0470] In some embodiments, the present application provides a method for preparing compound K-17-5', comprising reacting compound K-17-4' with 1-amino-2-methylpropane-2-thiol to obtain compound K-17-5':

[0471] In some embodiments, the reaction is carried out in the presence of a basic reagent.

[0472] In some embodiments, the alkaline agent is sodium bicarbonate.

[0473] In some embodiments, the present application provides a method for preparing compound K-17-4', ​​which comprises reacting compound K-17-3' with N-hydroxysuccinimide to obtain compound K-17-4':

[0474] In some embodiments, the reaction is carried out in the presence of an EDCI reagent.

[0475] In some embodiments, the present application provides a method for preparing compound K-17-3', which comprises reacting compound K-17-2' with L-alanine to obtain compound K-17-3':

[0476] In some embodiments, the reaction is carried out in the presence of a basic reagent.

[0477] In some embodiments, the alkaline agent is sodium bicarbonate.

[0478] In some embodiments, the reaction includes a post-processing step.

[0479] In some embodiments, the post-treatment step comprises the steps of pH adjustment, extraction, and washing.

[0480] In some embodiments, the pH of the reaction system is adjusted to 3-6, preferably 4-5, in the pH adjustment step.

[0481] In some embodiments, the present application provides a method for preparing compound K-17-2', comprising reacting compound K-17-1' with N-hydroxysuccinimide to obtain compound K-17-2':

[0482] In some embodiments, the reaction is carried out in the presence of an EDCI reagent.

[0483] The present application provides a method for preparing the following compound K'-18:

[0484] The method comprises reacting compound Int-1' and compound K-17-7 to obtain compound K'-18:

[0485] In some embodiments, the reaction is carried out in the presence of DIPEA and / or HATU reagent.

[0486] In some embodiments, the reaction is carried out in the presence of DIPEA and / or HATU reagent.

[0487] Pharmaceutical composition

[0488] In another aspect, the present application provides a pharmaceutical composition comprising any of the above-mentioned antibody-drug conjugates, any of the above-mentioned compounds, or any of the above-mentioned drug-linkers, and one or more pharmaceutical excipients.

[0489] The antibody drug conjugates, compounds or drug-linkers described herein can be formulated in a unit injectable form together with a pharmaceutically acceptable parenteral vehicle for parenteral use, such as bolus injection, intravenous injection, intratumoral injection, etc. Optionally, the antibody drug conjugate having the desired purity is mixed with a pharmaceutically acceptable diluent, carrier, excipient or stabilizer in the form of a lyophilized agent or solution (Remington's Pharmaceutical Sciences (1980) 16 th The antibody drug conjugates described herein or pharmaceutical compositions containing the same can be administered by any route appropriate to the subject to be treated.

[0490] application

[0491] The antibody drug conjugates, drug-linkers, compositions, or pharmaceutical compositions thereof described herein can be used to treat a variety of diseases or conditions, such as Her2-expressing cancers, including solid tumors or hematological malignancies, such as urothelial carcinoma, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, particularly lung adenocarcinoma), or lymphoma.

[0492] Therefore, the present application provides use of any of the above-mentioned antibody-drug conjugates, drug-linkers, compositions, or pharmaceutical compositions containing the same in the preparation of drugs for treating Her2-expressing cancers.

[0493] At the same time, the present application provides any of the above-mentioned antibody-drug conjugates, drug-linkers, compositions, or pharmaceutical compositions containing the same, and their use in drugs for treating Her2-expressing cancers.

[0494] At the same time, the present application also provides a method for treating Her2-expressing cancer, which comprises the step of administering an effective amount of any of the above-mentioned antibody-drug conjugates, drug-linkers, compositions, or pharmaceutical compositions containing the same to a subject in need thereof.

[0495] In some embodiments, the antibody drug conjugate, drug-linker, combination thereof, or pharmaceutical composition is sufficient (e.g., in a subject):

[0496] (1) Inhibit the proliferation of cells (such as tumor cells);

[0497] (2) inhibit tumor growth;

[0498] (3) induce and / or increase antibody-dependent cellular cytotoxicity activity;

[0499] (4) inhibiting HER2-mediated signal transduction;

[0500] (5) preventing and / or treating HER2-mediated diseases / disorders; or

[0501] (6) Any combination of (1) to (5) above.

[0502] In some embodiments, the cancer is selected from a solid tumor or a hematological malignancy; for example, selected from gastric cancer, breast cancer, lung cancer (eg, non-small cell lung cancer, particularly lung adenocarcinoma), and urothelial carcinoma.

[0503] All technical features disclosed in this specification, such as the definitions of various groups, except for mutually exclusive technical features, and all embodiments can be combined in any manner to obtain different general formula ranges or specific solutions. These ranges and solutions are all within the scope of the present invention.

[0504] definition

[0505] Unless otherwise defined below, the meanings of all technical and scientific terms used herein are intended to be the same as those generally understood by those skilled in the art. Reference to the techniques used herein is intended to refer to techniques generally understood in the art, including variations of those techniques that are obvious to those skilled in the art or replacements with equivalent techniques. Furthermore, laboratory procedures such as genomics, nucleic acid chemistry, and molecular biology used herein are conventional procedures widely used in the corresponding fields. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention.

[0506] The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, with heavy chains also containing a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain, CL. The constant domains are not directly involved in the binding of antibodies to antigens, but exhibit a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding site, respectively. The allocation of amino acids to each region or domain can follow various numbering systems known in the art. The term "antibody" also includes embodiments in which the heavy chain constant region comprises a C-terminal lysine, or lacks a C-terminal lysine or a C-terminal glycine-lysine dipeptide. The term also includes embodiments in which the N-terminal amino acid of the antibody variable region has been cyclized to pyroglutamate. Thus, in a composition comprising the antibodies disclosed herein, each antibody therein may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or comprise an N-terminal glutamine or glutamic acid or have the N-terminal amino acid cyclized to pyroglutamate.

[0507] The term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable regions of the heavy and light chains each contain three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, as defined in the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). For a given antibody, a person skilled in the art will easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (eg, see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003).

[0508] In the present invention, the CDRs contained in an antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art, such as those defined by the Kabat, Chothia, IMGT, or AbM numbering systems. In certain embodiments, the CDRs contained in an antibody or antigen-binding fragment thereof are defined by the Chothia numbering system.

[0509] The following general rules (published at www.bioinf.org.uk: Professor Andrew CR Martin's research group) can be used to define CDRs in antibody sequences, which include amino acids that specifically interact with amino acids that make up the epitope to which the antibody binds. In rare cases, these generally constant features do not appear; however, Cys residues are the most conserved feature.

[0510] V H The entire amino acid sequence of a V is generally numbered according to Kabat, and the three CDRs within the variable region may be defined according to any of the above numbering systems. H The amino acid positions in the sequence may be numbered sequentially starting from amino acid position 1 to the end of the sequence, or according to Kabat numbering. H and V L The amino acid positions in are defined according to sequential numbering.

[0511] Amino acid positions in the heavy chain constant region can be numbered sequentially starting from amino acid position 1 and continuing to the end of the sequence, or numbered according to Eu. The amino acid sequence of the IgG1 heavy chain constant region has 330 amino acids, numbered sequentially from 1 to 330. The corresponding sequence numbered according to Eu begins at position 118 and ends at position 447. Unless otherwise indicated, amino acid positions in the heavy and light chains described herein are defined according to sequential numbering.

[0512] The term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0513] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0514] The term "antigen-binding fragment" of an antibody refers to polypeptides that are fragments of an antibody, such as polypeptides that are fragments of a full-length antibody, which retain the ability to specifically bind to the same antigen bound by the full-length antibody and / or compete with the full-length antibody for specific binding to the antigen, and are also referred to as "antigen-binding portions." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fd, Fv, scFv, di-scFv, (scFv)2, disulfide-stabilized Fv proteins ("dsFv"), single domain antibodies (sdAbs, nanobodies), and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0515] The term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means a fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, consisting of one complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

[0516] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0517] The term "Fc" refers to an antibody fragment formed by disulfide bonds between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but is not involved in antigen binding.

[0518] The term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS (SEQ ID NO: 46) amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO: 47) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, a VH-VH-COOH domain comprising NH2-VH-VH-COOH, NH 2- VL-VL-COOH scFv.

[0519] The term "single-domain antibody (sdAb)" has the meaning generally understood by those skilled in the art, and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as the full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also called nanobodies.

[0520] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0521] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0522] Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

[0523] The term "murine antibody" refers to antibodies obtained by fusing B cells from immunized mice with myeloma cells, screening for murine hybrid fusion cells that can both proliferate indefinitely and secrete antibodies, followed by screening, antibody preparation, and antibody purification; or refers to antibodies secreted by plasma cells formed by the differentiation and proliferation of B cells in mice after antigen invasion.

[0524] The term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc. The donor antibody can be a mouse, rat, rabbit or non-human primate (e.g., cynomolgus monkey) antibody with the expected properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response).

[0525] The term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 total positions match). Typically, two sequences are compared when aligned for maximum identity. Such alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0526] The term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which amino acid residues are substituted with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). 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, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0527] The twenty conventional amino acids referred to herein are denoted according to conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0528] The term "linker" refers to a structural fragment that connects a cytotoxic drug to an antibody or antigen-binding fragment. For example, a fragment of the formula Ab-[MLED] x -MLE- structure fragment in.

[0529] The term "drug-linker" refers to the structure of the cytotoxic drug and linker described herein prior to linkage to the antibody or antigen-binding fragment thereof. For example, a "drug-linker" refers to M'-LED, where M' represents the structure of M prior to covalent linkage to the antibody or antigen-binding fragment thereof. The covalent linkage of the drug-linker to the antibody or antigen-binding fragment thereof yields the antibody-drug conjugate described herein.

[0530] The "drug-linker" also includes all pharmaceutically acceptable isotope-labeled compounds thereof, which are identical to the "drug-linker" compounds of the present invention except that one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature. Examples of isotopes suitable for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2 H. 3 H, deuterium D, tritium T); carbon isotopes (such as 11 C. 13 C and 14 C); isotopes of chlorine (e.g. 37 Cl); isotopes of fluorine (e.g. 18 F); isotopes of iodine (such as 123 I and 125 I); isotopes of nitrogen (e.g. 13 N and 15 N); oxygen isotopes (e.g. 15 O. 17 O and 18 O); and sulfur isotopes (e.g. 35 S).

[0531] The terms "comprises," "comprising," "having," "containing," or "involving," and other variations thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0532] The term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched hydrocarbon group, for example, "C 1-20 Alkyl", "C 1-10 Alkyl", "C 1- 6 alkyl", "C 1-4 Alkyl", "C 1-3alkyl”, etc., specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0533] The term "isotopically labeled compound" means a compound that is identical in structure to a compound of the present invention except that one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature. Examples of suitable isotopes for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2 H. 3 H, deuterium D, tritium T); carbon isotopes (such as 11 C. 13 C and 14 C); isotopes of chlorine (e.g. 37 Cl); isotopes of fluorine (e.g. 18 F); isotopes of iodine (such as 123 I and 125 I); isotopes of nitrogen (e.g. 13 N and 15 N); oxygen isotopes (e.g. 15 O. 17 O and 18 O); and sulfur isotopes (e.g. 35 S).

[0534] As used herein, the term "suitable substituent" refers to modifications that can be made to a compound by one skilled in the art according to the needs of the compound substituent. "Suitable substituents" include oxo (=O), halogen, cyano, NR 8 R 9 , carboxyl, thiol, hydroxyl, ester (e.g. -C 1-6 Alkyl-C(=O)-OC 1-6 Alkyl), C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkyl-OC 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 3-10 membered heterocyclic group, 5-10 membered heteroaryl, C 6-10Aryl, benzyl, hydroxy substituted benzyl, indolylmethylene and C 1-6 Haloalkoxy, R 8 、R 9 Each independently selected from H, C 1-6 Alkyl, C 3-6 Cycloalkyl, 3-10 membered heterocyclic group, 5-10 membered heteroaryl, C 6-10 Aryl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 Haloalkoxy, halogen, hydroxy, carboxyl and ester groups (e.g. -C 1-6 Alkyl-C(=O)-OC 1- 6 alkyl).

[0535] The term "substituted" refers to the replacement of one or more (e.g., 1, 2, 3, 4, or 5) hydrogens on the specified compound or structural fragment by a substituent, provided that the normal valence of the specified atom in the current situation is not exceeded and the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form stable compounds. In some embodiments, the substituents are each independently composed of one or more of the following structures: -O-, -S-, -NR'-, halogen, -CN, -OH, -NH2, -NO2, -CN, =O, C1-C6 (alkylene), C1-C6 haloalkylene, C1-C6 alkoxy, C2-C6 (alkenyl), C2-C6 (alkynyl), C3-C8 (cycloalkyl), 3-8 membered (heterocyclyl), C6-C 10 (Ethyl)aryl and 5-10 membered (Ethyl)heteroaryl, etc. In some embodiments, the substituents are each independently composed of one or more of the following structures: NR 8 R 9 , -O-, -S-, -NR'-, halogen, -CN, -OH, -SH, -NH2, -NO2, -C(O)-, -CN, =O, C1-C6 (alkylene), C1-C6 haloalkylene, C1-C6 alkoxy, C2-C6 (alkenyl), C2-C6 (alkynyl), C3-C8 (cycloalkyl), 3-10 membered (heterocyclyl), C6-C 10 (Ethyl)aryl and 5-10 membered (Ethyl)heteroaryl, etc., wherein R 8 、R 9 and R' are as defined above. For example, the substituent may be a suitable substituent as described above.

[0536] If a functional group or structural moiety is described as "substituted or unsubstituted," the functional group or structural moiety may be (1) unsubstituted or (2) substituted.

[0537] As used herein, the term "one or more" means 1 or more than 1, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, where reasonable.

[0538] Unless otherwise indicated, as used herein, the point of attachment of a substituent may be from any suitable position of the substituent.

[0539] Whether explicitly stated or not, the numerical values ​​in this application are modified by the term “about.” The term “about” means within ±20%, ±10%, preferably ±5%, and more preferably ±2% of the numerical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0540] Figure 1 shows the tumor inhibition effect of anti-human HER2 antibody-drug conjugate ADC on the NCI-N87 cell subcutaneous tumor-bearing mouse model.

[0541] FIG2 shows the changes in body weight of mice in each group in the human gastric cancer cell NCI-N87 CDX model.

[0542] Figure 3 shows the efficacy of anti-human HER2 antibody-drug conjugate ADC in the JIMT-1 cell subcutaneous tumor-bearing mouse model.

[0543] Figure 4 Changes in body weight of mice in each group in the human breast cancer cell JIMT-1CDX model. DETAILED DESCRIPTION

[0544] The present invention will be further described below by describing specific embodiments, but this is not intended to limit the present invention. Those skilled in the art can make various modifications or improvements based on the teachings of the present invention without departing from the basic idea and scope of the present invention.

[0545] The information of the sequences involved in the present invention is described in the following table:

[0546] The abbreviations used in this document have the following meanings:

[0547] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0548] Nuclear magnetic resonance (1H NMR) measurements were performed using a Bruker 400 MHz NMR spectrometer; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0549] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the examples are shown below.

[0550] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: hertz, DMSO-d6: deuterated dimethyl sulfoxide. δ values ​​are expressed in ppm.

[0551] Mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0552] Intermediate Example 1: Preparation of 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatriacontane-31-carboxylic acid (INT-1):

[0553] Step 1: Preparation of 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatriacontane-31-carboxylic acid (INT-1-2)

[0554] Use standard solid phase synthesis methods:

[0555] 1) Resin Preparation: 2-CTC resin (3.00 mmol, 3.70 g, 0.81 mmol / g), N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methylglycine (3.00 mol, 933 mg, 3.00 equiv), and DIPEA (4.00 equiv) were added to dichloromethane (10.0 mL) and reacted under a nitrogen atmosphere for 2 hours. MeOH (1.0 mL) was then added to the resin under a nitrogen bubbling atmosphere over 30 minutes, and the resin was filtered to obtain the obtained product.

[0556] 2) Coupling: A solution of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methylglycine (5.60 g, 6.00 equiv) and HATU (6.58 g, 5.70 equiv) in DMF (10.0 mL) was added to the resin under nitrogen bubbling. DIPEA (6.00 equiv) was added dropwise, and nitrogen was bubbled at 20°C for 30 minutes. The resin was washed with DMF (30.0 mL x 5) before proceeding to the next step.

[0557] 3) Deprotection: A 20% piperidine DMF solution (30.0 mL) was added to the resin and bubbled with nitrogen at 20° C. for 30 minutes. The resin was then washed with DMF (30.0 mL) × 5.

[0558] 4) Repeat steps 2 and 3 using the amino acids in Table 1: numbers 2-10 in Table 1.

[0559] 5) The resin was then washed with DMF (30.0 mL x 5) and MeOH (30.0 mL x 5), and then dried under vacuum.

[0560] Table 1:

[0561] Peptide cleavage and purification:

[0562] 1) Add cleavage solution (TFA / DCM, 1 / 100, v / v, 200.0 mL) to the flask containing the side-chain protected peptide at room temperature and stir for 3 minutes twice.

[0563] 2) After filtration, the filtrates were combined and concentrated.

[0564] 3) The crude product was purified by HPLC and freeze-dried to give the title compound (1117.1 mg, TFA salt).

[0565] Its structural characterization data are as follows:

[0566] ESI-MS (m / z): 1010.4 (M+H) + .

[0567] The purification method is as follows:

[0568] Step 2: Preparation of 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatriacontane-31-carboxylic acid (INT-1)

[0569] 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatriacontane-31-carboxylic acid (665 mg, 615.04 μmol) was added to water (7.5 mL) and acetonitrile (15 mL), and sodium periodate (1.32 g, 6.15 mmol) and ruthenium trichloride hydrate (51.03 mg, 246.02 μmol) were added, and the mixture was reacted at 25°C for 1 hour. The reaction solution was directly purified by reverse phase purification (acetonitrile / water (0.05% formic acid) = 0-25%) and freeze-dried to obtain the title compound (515 mg, 449.69 μmol).

[0570] Its structural characterization data are as follows:

[0571] ESI-MS (m / z): 1074.4 (M+H) + .

[0572] Intermediate Example 2: Preparation of 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid (INT-2)

[0573] Step 1: Preparation of 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid methyl ester (INT-2-2)

[0574] The raw materials, methyl 3,5-dibromobenzoate (720 mg, 2.45 mmol), 2-methylthiopyrimidine-5-boronic acid (874 mg, 5.14 mmol), XPhosPd G3 (207 mg, 245 μmol), and K3PO4 (1.56 g, 7.35 mmol) were added to dioxane (12 mL) and water (4 mL). The reaction system was stirred at 90°C under a nitrogen atmosphere for 3 hours. The reaction was monitored by LC-MS, filtered through celite, and water and ethyl acetate were added to the filtrate. The mixture was extracted and concentrated to give a crude product, which was purified by column chromatography (EA / PE = 0-25%) to give 710 mg of methyl 3,5-di(2-(methylthio)pyrimidin-5-yl)benzoate.

[0575] Its structural characterization data are as follows:

[0576] ESI-MS (m / z): 385.1 [M+H] + .

[0577] Step 2: Preparation of 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid (INT-2-3)

[0578] Methyl 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoate (650 mg, 1.69 mol) and lithium hydroxide (121 mg, 5.07 mmol) were dissolved in THF (2 mL), MeOH (2 mL), and H₂O (2 mL). The reaction was stirred at 25°C for 2 hours and monitored by LC-MS. The pH of the system was adjusted to approximately 2 with 1N HCl, resulting in the precipitation of a large amount of solid. The filter cake was collected by filtration and dried to yield 560 mg of 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid.

[0579] Its structural characterization data are as follows:

[0580] ESI-MS (m / z): 371.1 [M+H] + .

[0581] Step 3: Preparation of 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid tert-butyl ester (INT-2-4)

[0582] 3,5-Bis(2-(methylthio)pyrimidin-5-yl)benzoic acid (3.00 g, 8.10 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptane-27-ate (4.03 g, 8.10 mmol) were added to DMF (40 mL), and HOBt (3.28 g, 24.3 mmol), EDCI (4.66 g, 24.3 mmol) and DIPEA (4.19 g, 32.4 mmol, 5.64 mL) were added in sequence, and the reaction system was stirred at 60 ° C for 2 hours. Water (100 mL) and ethyl acetate (60 mL x 3) were added to the reaction solution, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give tert-butyl 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oate (4.20 g, 4.14 mmol), which was used in the next step without purification.

[0583] Step 4: Preparation of 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid (INT-2-5)

[0584] Dissolve tert-butyl 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosadecane-29-oate (3.60 g, 4.24 mmol) in dichloromethane (30 mL). Add TFA (15.3 g, 134 mmol, 10 mL). Stir the reaction system at 25°C for 6 hours. Add water (60 mL) and extract with ethyl acetate (40 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by preparative high performance liquid chromatography and freeze-dried to give 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid (2.93 g, 3.63 mmol).

[0585] Its structural characterization data are as follows:

[0586] ESI-MS (m / z): 794.3 [M+H] + .

[0587] The purification method is as follows:

[0588] Chromatographic column: Phenomenex luna C18 (250mm*70mm*10μm)

[0589] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0590] Step 5: Preparation of 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid (INT-2)

[0591] 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosanetan-29-oic acid (148 mg, 0.186 mmol) was added to acetonitrile (15 mL) and water (7.5 mL), and sodium periodate (398.71 mg, 1.86 mmol) and ruthenium trichloride were added. The hydrate (15.47 mg, 74.56 μmol) was added to the reaction system, stirred at 25 ° C for 30 minutes, and the reaction system was extracted with water and ethyl acetate and concentrated to give 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid (155 mg).

[0592] Its structural characterization data are as follows:

[0593] ESI-MS (m / z): 858.3 [M+H] + .

[0594] Intermediate Example 3: Preparation of 2-((((4-((allyloxy)carbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (INT-3)

[0595] Step 1: Preparation of allyl 1-(N-((2-(tert-butoxy)-2-oxoethoxy)carbonyl)aminosulfonyl)piperidine-4-carboxylate (INT-3-2)

[0596] Chlorosulfonyl isocyanate (2.14 g, 15.13 mmol, FR) was added to dichloromethane (50 mL) and cooled in an ice-water bath for 10 minutes. Tert-butyl 2-hydroxyacetate (2 g, 15.13 mmol, FR) was then added to the reaction system and stirred in the ice-water bath for 2 hours. Allyl piperidine-4-carboxylate hydrochloride (3.74 g, 18.16 mmol) and triethylamine (4.59 g, 45.40 mmol) were added to the reaction system. The ice-water bath was removed, and the mixture was allowed to return to room temperature and stirred for 3 hours. Water (100 mL) was added to the reaction system to quench the reaction. The mixture was extracted with ethyl acetate three times (50 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered and concentrated to obtain the crude product. The product was purified on a silica gel column (DCM / PE = 0-100%) to yield the title compound (1.87 g, 4.19 mmol).

[0597] Its structural characterization data are as follows:

[0598] 1 H NMR (400MHz, DMSO) δ11.64(s,1H),6.06-5.79(m,1H),5.35-5.17(m,2H),4.61-4.48(m,4H),4.03(q,J=7.1Hz,1H),3.5 9(dt,J=12.4,3.3Hz,2H),3.04-2.88(m,2H),1.93(dd,J=13.5,3.2Hz,2H),1.59(dd,J=13.4,3.6Hz,2H),1.43(s,9H).

[0599] Step 2: Preparation of 2-((((4-((allyloxy)carbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (INT-3)

[0600] Allyl 1-(N-((2-(tert-Butoxy)-2-oxoethoxy)carbonyl)aminosulfonyl)piperidine-4-carboxylate (1.75 g, 4.31 mmol, FR) was added to a mixed solvent of trifluoroacetic acid (5 mL) and dichloromethane (10 mL) and reacted at 25°C for 2 hours. The reaction system was concentrated to dryness, diluted with ethyl acetate, and the pH was adjusted to approximately 8 with aqueous sodium bicarbonate. Impurities were removed by extraction, and the pH was adjusted to approximately 3 with 3N dilute hydrochloric acid. The mixture was extracted with ethyl acetate three times (30 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the title compound (1.4 g, 4.00 mmol).

[0601] Its structural characterization data are as follows:

[0602] ESI-MS (m / z): 351.1 [M+H] + .

[0603] Preparation Example 1: (2S,4S)-2,5,12-trihydroxy-N-(2-hydroxyethyl)-7-methoxy-4-((((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamide (6-3)

[0604] Step 1: Preparation of (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylic acid (6-3-2)

[0605] (8S,10S)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-10-((((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydronaphthacene-5,12-dione (157 mg, 244.69 μmol) was added to methanol (3 mL) and water (2 mL), and sodium periodate (78.51 mg, 367.04 μmol) was added. After the addition was complete, the mixture was stirred at room temperature in the dark. After the reaction was completed, the mixture was concentrated under reduced pressure to remove the solvent, and the solvent was removed by lyophilization to obtain the title compound (142 mg, 210.42 μmol), which was used directly in the next step without purification.

[0606] Its structural characterization data are as follows:

[0607] MSm / z(ESI):628.2[M+H] +

[0608] Step 2: Preparation of (2S,4S)-2,5,12-trihydroxy-N-(2-hydroxyethyl)-7-methoxy-4-((((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxamide (6-3)

[0609] To DMF (2 mL) was added (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylic acid (50 mg, 79.67 μmol), 4-dimethylaminopyridine (38.93 mg, 318.68 μmol), EDCI (30.55 mg) and 2-aminoethanol (14 mg, 229.20 μmol). After the addition was complete, the temperature was raised to 35°C / 45°C under nitrogen protection for reaction. After the reaction, the title compound (2.04 mg, 2.89 μmol) was obtained by direct purification using preparative HPLC and freeze-drying.

[0610] Its structural characterization data are as follows:

[0611] MSm / z(ESI):671.4[M+H] +

[0612] The separation and purification method is as follows

[0613] Column: Waters XBridge Prep C18 OBD (5μm*19mm*150mm)

[0614] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate)

[0615] Preparation Example 2: (2-Hydroxyethyl (2S, 4S) -2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylate (6-6)

[0616] To DMF (2 mL) was added (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylic acid (50 mg, 79.67 μmol), potassium carbonate (28.06 mg, 203.06 μmol), and sodium iodide (12.17 mg, 81.22 μmol). The reaction was stirred at room temperature for 20 min. Finally, 2-bromoethanol (20.30 mg, 162.45 μmol) was added, and the temperature was raised to 65°C. After the reaction, the title compound (5.95 mg, 8.42 μmol) was obtained by direct purification using preparative HPLC and freeze-drying.

[0617] Its structural characterization data are as follows:

[0618] MSm / z(ESI):672.2[M+H] +

[0619] The separation and purification method is as follows

[0620] Column: Waters XBridge Prep C18 OBD (5μm*19mm*150mm)

[0621] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate)

[0622] Preparation Example 3: S-(1-amino-2-methylpropan-2-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carbonothioate (6-7)

[0623] Step 1: Preparation of (9H-fluoren-9-yl)methyl (2-mercapto-2-methylpropyl)carbamate (6-7-2)

[0624] 1-Amino-2-methylpropane-2-thiol hydrochloride (0.5 g, 3.53 mmol) was dissolved in THF (10 mL) and water (2 mL). Sodium bicarbonate (593.01 mg, 7.06 mmol) was added, followed by 9-fluorenylmethyl-N-succinimidyl carbonate (1.13 g, 3.53 mmol). The mixture was stirred at room temperature for 2 hours. After completion of the reaction, 10 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate three times (10 mL x 3). The mixture was washed with brine (10 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude title compound (1.12 g, 3.43 mmol), which was used directly in the next reaction without purification.

[0625] Its structural characterization data are as follows:

[0626] MS m / z(ESI):350.1[M+Na] +

[0627] Step 2: Preparation of S-(1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-methylpropan-2-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carbonothioate (6-7-3)

[0628] (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylic acid (86m g, 137.03 μmol) and DIPEA (35.42 mg, 274.06 μmol) and (9H-fluoren-9-yl)methyl (2-mercapto-2-methylpropyl) carbamate (67.30 mg, 205.55 μmol) were dissolved in DMF (4 mL). PyBOP (85.57 mg, 164.44 μmol) was added under nitrogen and stirred at room temperature for 0.5 h. Water (5.0 mL) was added to the reaction solution, which was extracted three times with ethyl acetate (5 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (3 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-90%) and concentrated again under reduced pressure to give the title compound (41 mg, 43.76 μmol).

[0629] Its structural characterization data are as follows:

[0630] MS m / z(ESI):937.3[M+H] +

[0631] Step 3: Preparation of S-(1-amino-2-methylpropan-2-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-thiocarbonate (6-7)

[0632] S-(1-(9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-methylpropan-2-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carbonothioate (30 mg, 32.02 umol) was dissolved in DMF (1.5 mL). A solution of piperidine (8.18 mg, 96.05 μmol) in DMF (1.5 mL) was added dropwise under nitrogen. After the addition, the system turned purple-red and stirred at room temperature for 40 minutes. The reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (4.12 mg, 5.48 μmol).

[0633] Its structural characterization data are as follows:

[0634] MS m / z(ESI):715.3[M+H] +

[0635] 1H NMR (400MHz, DMSO) δ14.07(s,1H),13.25(s,1H),7.96-7.93(m,2H),7.69-7.66(m,1H),5.45(s,1H),5.25(t,J=4.4Hz,1H),5.02(s,1H),4.58(d,J =2.0Hz,1H),4.23(d,J=2.0Hz,1H),4.21-4.13(m,1H),4.00(s,3H),3.93 (dd,J=6.0,1.6Hz,1H),3.66(t,J=8.8Hz,1H),3.51(dd,J=7.6,3.6Hz,1H) ,3.39(dd,J=13.2,7.2Hz,2H),3.31(s,3H),3.19(dd,J=13.2,6.0Hz,1H),3.03(dd,J=45.2,18.4Hz,2H),2.73-2.58(m,2H),2.29(dd,J=14.8,5.6 Hz,1H),2.20(d,J=13.6Hz,1H),2.00(dd,J=14.4,6.8Hz,1H),1.69(s,2H ),1.45(s,1H),1.28(d,J=5.6Hz,4H),1.23(s,2H),1.21(d,J=6.4Hz,3H).

[0636] The separation and purification method is as follows:

[0637] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0638] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate)

[0639] Preparation Example 4: Preparation of S-(1-hydroxy-2-methylpropan-2-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-thiocarbonate (6-8)

[0640] (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylic acid (60 mg, 95.60 μmol), DIPEA (24.71 mg, 191.21 μmol) and 2-mercapto-2-methylpropan-1-ol (30.46 mg, 286.81 μmol) were dissolved in DMF (3 mL), PyBOP (59.70 mg, 114.72 μmol) was added, and the mixture was stirred at room temperature for 0.5 h. The reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (5.74 mg, 7.22 μmol).

[0641] Its structural characterization data are as follows:

[0642] MS m / z(ESI):716.3[M+H] +

[0643] 1 H NMR (400MHz, CDCl3) δ13.88(s,1H),13.29(s,1H),8.03(d,J=7.2Hz,1H),7.78(t,J=8.0Hz,1H),7.39(d,J=8.4Hz,1H),5.50(s,1 H),5.29(s,1H),5.05(s,1H),4.73(s,1H),4.52(s,1H),4.10(d,J=12.8Hz,5H),3.95(s,2H),3.80(q,J=11.6Hz,2H),3.60(d,J= 11.2Hz,1H),3.46(d,J=4.8Hz,3H),3.44-3.36(m,1H),3.29(d,J=18.8Hz,1H),3.09(d,J=18.8Hz,1H),2.88(s,1H),2.77(s,1H) ,2.59(d,J=14.8Hz,1H),2.12(dd,J=14.8,3.6Hz,1H),2.02(s,1H),1.82-1.75(m,2H),1.48(d,J=3.6Hz,5H),1.45-1.37(m,4H).

[0644] The separation and purification method is as follows:

[0645] Chromatographic column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0646] Mobile phase A: acetonitrile; mobile phase B: water

[0647] Preparation Example 5: Preparation of (2S,4S)-2,5,12-trihydroxy-4-(((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methoxymorpholinyl)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-N-(2-hydroxyethyl)-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamide (6-9)

[0648] Step 1: Preparation of (2S,4S)-2,5,12-trihydroxy-4-(((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methoxymorpholinyl)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylic acid (6-9-2)

[0649] (8S,10S)-6,8,11-trihydroxy-10-(((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methoxymorpholinyl)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-8-(2-hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetraphenyl-5,12-dione (100 mg, 155.37 μmol) was added to a mixture of methanol (4 mL) and water (2 mL). Sodium periodate (60 mg) was added, and the mixture was allowed to react at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure to remove methanol and freeze-dried to obtain the crude title compound (80 mg, 120.71 μmol), which was used directly in the next step without purification.

[0650] Its structural characterization data are as follows:

[0651] MS m / z(ESI):630.2[M+1] +

[0652] Step 2: Preparation of (2S,4S)-2,5,12-trihydroxy-4-(((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methoxymorpholinyl)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-N-(2-hydroxyethyl)-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamide (6-9)

[0653] (2S,4S)-2,5,12-trihydroxy-4-(((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methoxymorpholinyl)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylic acid (13 mg, 23.82 μmol) was dissolved in DMF (2 mL). 2-Aminoethanol (3 mg), DIPEA (15 mg), and HATU (15 mg) were added and reacted at room temperature for 1 hour. The reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (7.25 mg, 10.67 μmol).

[0654] Its structural characterization data are as follows:

[0655] MS m / z(ESI):673.2[M+1] +

[0656] 1 H NMR (400MHz, DMSO) δ14.08(s,1H),13.26(s,1H),7.94(dd,J=11.7,5.3Hz,3H),7.71-7.64(m,1H),5.38(s,1H),5.30(s,1H ),4.98(s,1H),4.74(t,J=5.4Hz,1H),4.38(s,1H),4.09(s,1H),4.04-3.98(m,3H),3.79-3.71(m,1H),3.58(s,1H),3.49- 3.39(m,3H),3.26(s,3H),3.24-3.17(m,2H),2.99(dd,J=40.3,18.5Hz,2H),2.63(d,J=10.6Hz,1H),2.44(s,2H),2.25(dd ,J=14.3,5.3Hz,2H),2.11(d,J=13.7Hz,1H),1.83(dd,J=12.6,9.2Hz,1H),1.57(d,J=10.0Hz,1H),1.11(d,J=6.5Hz,3H).

[0657] The preparation method is as follows:

[0658] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0659] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0660] Preparation Example 6: Preparation of (2S,4S)-2,5,12-trihydroxy-N-(2-hydroxyethyl)-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-N-methyl-6,11-dioxo-1,2,4,6,11-hexahydrotetracene-2-carboxamide (6-10)

[0661] (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylic acid (30 mg, 47.80 μmol) was dissolved in DMF (1 mL) and 2-(methylamino)ethane-1-ol (10 mg, 133.14 μmol) was added. The system turned black. DIPEA (12.36 mg, 95.60 μmol) and PyBOP (29.85 mg, 57.36 μmol) were added and the reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (7.17 mg, 9.90 μmol).

[0662] Its structural characterization data are as follows:

[0663] MS m / z(ESI):685.2[M+1] +

[0664] 1H NMR(400MHz,DMSO)δ14.06(s,1H),13.31(s,1H),7.95-7.88(m,2H),7.68-7.62(m,1H),5.71-5.69(m,1H),5.26-5.23(m,1H), 5.04-4.99(m,1H),4.84-4.80(m,1H),4.62-4.50(m,2H),4.23(d,J=2.0Hz,1H),4.10-4.02(m,1H),3.99(s,3H),3.93(d,J=6. 0Hz,1H),3.82-3.72(m,1H),3.70-3.60(m,2H),3.50(dd,J=7.6,3.6Hz,1H),3.40-3.36(m,4H),3.31(d,J=4.4Hz,6H),2.88(d ,J=17.6Hz,1H),2.79(s,1H),2.72-2.61(m,1H),2.45-2.35(m,2H),2.26-2.16(m,1H),1.69-1.64(m,1H),1.25-1.20(m,2H).

[0665] The preparation method is as follows:

[0666] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0667] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate)

[0668] Preparation Example 7: Preparation of (2S,4S)-N-(2-aminoethyl)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-N-methyl-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamide (6-11)

[0669] Step 1: Preparation of tert-butyl (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)(methyl)carbamate (6-11-2)

[0670] Dissolve tert-butyl (2-aminoethyl)(methyl)carbamate (0.5 g, 2.87 mmol) and 9-fluorenylmethyl-N-succinimidyl carbonate (967.99 mg, 2.87 mmol) in THF (5 mL). Add water (5 mL) and then sodium bicarbonate (241.07 mg, 2.87 mmol). Stir at room temperature for 2 hours. After completion, the reaction solution was diluted with 20 mL of ethyl acetate and washed twice with 10 mL of brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude title compound (1.2 g, 2.18 mmol), which was used directly in the next step without purification.

[0671] Its structural characterization data are as follows:

[0672] MS m / z(ESI):297.1[M+1-Boc] +

[0673] Step 2: Preparation of (9H-fluoren-9-yl)methyl (2-(methylamino)ethyl)carbamate hydrochloride (6-11-3)

[0674] To tert-butyl (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)(methyl)carbamate (1.2 g, 3.03 mmol) was added a 4M hydrogen chloride / dioxane solution (10 mL). The mixture was stirred at room temperature for 1 hour. After completion, the reaction solution was concentrated under reduced pressure. To the residue was added 20 mL of water, and the pH was adjusted to 4-5 with 4N HCl. The mixture was extracted with 10 mL of ethyl acetate twice. The organic phase was discarded, and the aqueous phases were combined and freeze-dried to afford the title compound (670 mg, 1.96 mmol) without further purification.

[0675] Its structural characterization data are as follows:

[0676] MS m / z(ESI):297.3[M+1] +

[0677] Step 3: Preparation of (9H-fluoren-9-yl)methyl (2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-N-methyl-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamido)ethyl)carbamate (6-11-4)

[0678] (9H-Fluoren-9-yl)methyl(2-(methylamino)ethyl)carbamate hydrochloride, (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylic acid (50 mg, 79.67 μmol) were dissolved in DMF (1 mL), and DIPEA (30.89 mg, 239.01 μmol) and PyBOP (49.75 mg, 95.60 μmol) were added, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was diluted with 5 mL of water and extracted with 5 mL of ethyl acetate x 3. The combined organic phases were washed with 5 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 0-100%) and concentrated again under reduced pressure to obtain the title compound (18 mg, 19.87 μmol).

[0679] Its structural characterization data are as follows:

[0680] MS m / z(ESI):906.3[M+1] +

[0681] Step 4: Preparation of (2S,4S)-N-(2-aminoethyl)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-N-methyl-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxamide (6-11)

[0682] (9H-Fluoren-9-yl)methyl (2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-N-methyl-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamido)ethyl)carbamate (18 mg, 19.87 μmol) was dissolved in DMF (2 mL), and piperidine (16.92 mg, 198.69 μmol) was added dropwise, and the mixture was stirred at room temperature for 5 minutes. After the reaction was completed, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (2.61 mg, 3.74 μmol).

[0683] Its structural characterization data are as follows:

[0684] MS m / z(ESI):684.3[M+1] +

[0685] 1 H NMR (400MHz, DMSO) δ7.95-7.85(m,2H),7.68-7.60(m,1H),5.40-5.35(m,1H),5.27-5.20(m,1H),5.05-4.95( m,1H),4.58(s,1H),4.22(s,1H),4.17-4.03(m,1H),4.01-3.95(s,3H),3.92(d,J=4.0Hz,2H),3.66(t,J=12. 0Hz,2H),3.30(s,6H),3.06-2.97(m,2H),2.97-2.8(m,2H),2.80-2.73(m,1H),2.73-2.55(m,5H),2.34-2.25 (m,3H),2.25-2.20(m,1H),2.20-2.11(m,1H),2.08-1.94(m,1H),1.72-1.61(m,2H),1.25-1.19(d,J=8.0,2H)

[0686] The preparation method is as follows:

[0687] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0688] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate)

[0689] Preparation Example 8: Preparation of 1-hydroxypropan-2-yl (2S, 4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S, 3R, 4aS, 9S, 9aR, 10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4', 3': 4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylate (6-12)

[0690] (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylic acid (30.0 mg, 47.8 μmol) and 2-bromopropanol (99.7 mg, 717.03 μmol) were dissolved in DMF (2 mL), potassium carbonate (99.10 mg, 717.03 μmol) was added, and the temperature was raised to 50°C for 6 hours. The reaction solution was poured into dichloromethane (15 ml) and water (10 ml), the organic phase was separated, washed with saturated aqueous sodium chloride solution (2 ml), and concentrated to give a crude product, which was purified by preparative HPLC and freeze-dried to give the title compound (3.50 mg, 4.95 μmol).

[0691] Its structural characterization data are as follows:

[0692] MS m / z(ESI):686.2[M+H] +

[0693] 1 H NMR (400MHz, DMSO-d6): δ14.06(s,1H),13.28(s,1H),7.93(m,2H),7.70-7.64(m,1H),5.24 (m,1H),4.96(m,1H),4.82-4.76(m,1H),4.59(d,J=2.0Hz,1H),4.23(d,J=1.6Hz,1H),4.19 -4.15(m,1H),4.12(dd,J=10.4,5.2Hz,1H),3.99(s,2H),3.95-3.85(m,2H),3.7 9-3.72(m,1H),3.69-3.63(m,1H),3.51-3.48(m,1H),3.33(s,3H),3.30(s,2H), 3.17(d,J=5.2Hz,3H),3.11-3.05(m,1H),3.00-3.95(m,1H),2.65-2.61(m,1H), 2.27-2.24(m,1H),1.67-1.62(m,2H),1.22(d,J=6.4Hz,3H),1.11-1.02(m,3H).

[0694] The preparation method is as follows:

[0695] Chromatographic column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0696] Mobile phase A: acetonitrile; mobile phase B: 0.05% formic acid in water

[0697] Example 1: Preparation of perfluorophenyl 1-(N-((9S,12S,15S)-9,12,15-trimethyl-1,8,11,14,17-pentaoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-yl)-5,19-dioxa-2,7,10,13,16-pentaazaeicosanoyl)sulfamoyl)piperidine-4-carboxylate (K-9)

[0698] Step 1: Preparation of (S)-(1-(9H-fluoren-9-yl)-5-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-yl)carbamic acid benzyl ester (K-9-2)

[0699] To dichloromethane (20 mL) was added methyl (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)acetate (1.53 g, 7.85 mmol), benzyl (2-hydroxyethyl)carbamate (3.60 g, 9.41 mmol), and pyridinium p-toluenesulfonate (985.73 mg, 3.92 mmol). The mixture was heated to 40°C and reacted for 20 h. The reaction solution was purified on a silica gel column (ethyl acetate / petroleum ether = 0% to 80%) and concentrated again to obtain the title compound (1.08 g, 1.98 mmol).

[0700] Its structural characterization data are as follows:

[0701] MS m / z(ESI):518.2[M+H] +

[0702] Step 2: Preparation of (S)-(2-((2-aminopropionamido)methoxy)ethyl)carbamic acid benzyl ester (K-9-3)

[0703] To DMF (8 mL) were added benzyl (S)-(1-(9H-fluoren-9-yl)-5-methyl-3,6-dioxo-2,9-dioxa-4,7-diazoundec-11-yl)carbamate (632 mg, 1.22 mmol) and diethylamine (267 mg, 3.66 mmol). The mixture was stirred at 25°C for 2 h. After completion of the reaction, the residual diethylamine and solvent were removed under reduced pressure to obtain the crude title compound (360 mg), which was used directly in the next step without purification.

[0704] Step 3: Preparation of (9H-fluoren-9-yl)methylbenzyl ((7S,10S,13S)-7,10-dimethyl-6,9,12-trioxo-3-oxa-5,8,11-triazatetradecane-1,13-diyl) dicarbamate (K-9-4)

[0705] To DMF (8 mL) were added crude benzyl (S)-(2-((2-aminopropionamido)methoxy)ethyl)carbamate (360 mg), (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine (466.14 mg, 1.22 mmol), DIPEA (787.69 mg, 6.09 mmol), and DMTMM condensing agent (718 mg, 2.44 mmol). The reaction was stirred at 25°C for 2 h. After completion of the reaction, water (40 mL) was added to the reaction solution to precipitate a solid, which was filtered and dried to obtain the crude title compound (800 mg), which was used directly in the next step without purification.

[0706] Step 4: Preparation of ((7S,10S,13S)-13-amino-7,10-dimethyl-6,9,12-trioxo-3-oxa-5,8,11-triazatetradecyl)carbamic acid benzyl ester (K-9-5)

[0707] Crude (9H-fluoren-9-yl)methylbenzyl ((7S,10S,13S)-7,10-dimethyl-6,9,12-trioxo-3-oxa-5,8,11-triazatetradecane-1,13-diyl) dicarbamate (800 mg) and diethylamine (204 mg, 2.80 mmol) were added to DMF (8 mL) and stirred at 25°C for 2 h. The reaction mixture was purified by reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 0% to 30%) and lyophilized to obtain the title compound (394 mg, 855.56 μmol).

[0708] Its structural characterization is as follows:

[0709] MS m / z(ESI):438.2[M+H] +

[0710] Step 5: Preparation of 1-(N-((11S,14S,17S)-11,14,17-trimethyl-3,10,13,16,19-pentaoxo-1-phenyl-2,7,21-trioxa-4,9,12,15,18-pentaazadocosan-22-yl)sulfamoyl)piperidine-4-carboxylic acid allyl ester (K-9-6)

[0711] To DMF (1 mL) was added benzyl ((7S,10S,13S)-13-amino-7,10-dimethyl-6,9,12-trioxo-3-oxa-5,8,11-triazatetradecyl)carbamate (150 mg, 342.87 μmol), 2-((((4-((allyloxy)carbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (132.84 mg, 341.25 μmol), DIPEA (120.28 mg, 930.69 μmol), and HATU (153.35 mg, 403.30 μmol). The reaction was stirred at 25°C for 3 h. The reaction solution was purified by reverse-phase column chromatography (acetonitrile / 0.05% aqueous formic acid = 0% to 80%) and lyophilized to obtain the title compound (98 mg, 120.94 μmol).

[0712] Its structural characterization is as follows:

[0713] MS m / z(ESI):770.3[M+H] +

[0714] Step 6: Preparation of 1-(N-((11S,14S,17S)-11,14,17-trimethyl-3,10,13,16,19-pentaoxo-1-phenyl-2,7,21-trioxa-4,9,12,15,18-pentaazadocosan-22-yl)sulfamoyl)piperidine-4-carboxylic acid (K-9-7)

[0715] To DMF (1 mL) was added allyl 1-(N-((11S,14S,17S)-11,14,17-trimethyl-3,10,13,16,19-pentaoxo-1-phenyl-2,7,21-trioxa-4,9,12,15,18-pentaazadocosan-22-yl)sulfamoyl)piperidine-4-carboxylate (45 mg, 58.46 μmol). Under nitrogen, tetrakistriphenylphosphine palladium (33.77 mg, 29.23 μmol) and tetrahydropyrrole (12.47 mg, 175.37 μmol) were added. After addition, the mixture was stirred at 25°C for 3 h. The reaction solution was purified by reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 0% to 60%) and lyophilized to obtain the title compound (35 mg, 45.56 μmol).

[0716] Its structural characterization is as follows:

[0717] MS m / z(ESI):730.3[M+H] +

[0718] Step 7: Preparation of 1-(N-((6S,9S,12S)-18-amino-6,9,12-trimethyl-4,7,10,13-tetraoxo-2,16-dioxa-5,8,11,14-tetraazaoctadecanoyl)sulfamoyl)piperidine-4-carboxylic acid (K-9-8)

[0719] To MeOH (8 mL) was added 1-(N-((11S,14S,17S)-11,14,17-trimethyl-3,10,13,16,19-pentaoxo-1-phenyl-2,7,21-trioxa-4,9,12,15,18-pentaazadocosan-22-yl)sulfamoyl)piperidine-4-carboxylic acid (35 mg, 45.56 μmol) and 10% Pd / C (80 mg). The mixture was stirred under hydrogen balloon pressure at 25°C for 6 h. After completion of the reaction, the mixture was filtered and the solvent removed under reduced pressure to obtain the crude title compound (27 mg), which was used directly in the next step without purification.

[0720] Its structural characterization is as follows:

[0721] MS m / z(ESI):596.2[M+H] +

[0722] Step 8: Preparation of 1-(N-((9S,12S,15S)-9,12,15-trimethyl-1,8,11,14,17-pentaoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-yl)-5,19-dioxa-2,7,10,13,16-pentaazaeicosane-20-yl)sulfamoyl)piperidine-4-carboxylic acid (K-9-9)

[0723] To DMF (1 mL) was added 2,5-dioxopyrrolidin-1-yl (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylate (13.69 mg, 18.8 After addition of crude 1-(N-((6S,9S,12S)-18-amino-6,9,12-trimethyl-4,7,10,13-tetraoxo-2,16-dioxa-5,8,11,14-tetraazaoctadecanoyl)sulfamoyl)piperidine-4-carboxylic acid (9 mg), and DIPEA (6 mg, 46.43 μmol), the mixture was stirred at 25°C for 0.5 h. The reaction solution was purified by preparative HPLC and lyophilized to obtain the title compound (5 mg, 3.73 μmol).

[0724] Its structural characterization is as follows:

[0725] MS m / z(ESI):1206.3[M+H] +

[0726] The preparation method is as follows:

[0727] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0728] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate)

[0729] Step 9: Preparation of perfluorophenyl 1-(N-((9S,12S,15S)-9,12,15-trimethyl-1,8,11,14,17-pentaoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-yl)-5,19-dioxa-2,7,10,13,16-pentaazaeicosanoyl)sulfamoyl)piperidine-4-carboxylate (K-9)

[0730] To DMF (1 mL) was added 1-(N-((9S,12S,15S)-9,12,15-trimethyl-1,8,11,14,17-pentaoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxadiazol-1-yl)-1-nitropropane To the mixture were added (5 mg, 3.73 μmol), pentafluorophenol (2 mg, 11.12 μmol), and EDCI (1.07 mg, 5.56 μmol), and the mixture was stirred at 25°C for 3 h. The reaction mixture was purified by HPLC and lyophilized to afford the title compound (1.5 mg, 1.1 μmol).

[0731] Its structural characterization is as follows:

[0732] MS m / z(ESI):1372.3[M+H] +

[0733] The preparation method is as follows:

[0734] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0735] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium formate aqueous solution)

[0736] Example 2: Preparation of perfluorophenyl 1-(N-((7S,10S,13S)-2,7,10,13-tetramethyl-1,6,9,12,15-pentaoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-yl)-17-oxa-2,5,8,11,14-pentaazaoctadecane-18-yl)sulfamoyl)piperidine-4-carboxylate (K-11)

[0737] Step 1: Preparation of tert-butyl ((5S,8S,11S)-1-(9H-fluoren-9-yl)-5,8,11-trimethyl-3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-yl)(methyl)carbamate (K-11-2)

[0738] (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanyl-L-alanine (1 g, 2.21 mmol) and tert-butyl (2-aminoethyl)(methyl)carbamate (384.22 mg, 2.21 mmol) were dissolved in DMF (10 mL). HATU (1.01 g, 2.65 mmol) and DIPEA (569.99 mg, 4.41 mmol) were added and stirred at room temperature for 1 hour. After completion of the reaction, 150 mL of water was added to the reaction solution, causing a large amount of solid to precipitate. After stirring for 10 minutes, the mixture was filtered, the filter cake was washed with appropriate amount of water, and the solid was collected and dehydrated in a lyophilizer to obtain the crude title compound (900 mg, 1.48 mmol).

[0739] Its structural characterization data are as follows:

[0740] MS m / z(ESI):610.3[M+H] +

[0741] Step 2: Preparation of (9H-fluoren-9-yl)methyl ((7S,10S,13S)-7,10-dimethyl-6,9,12-trioxo-2,5,8,11-tetraazatetradec-13-yl)carbamate (K-11-3)

[0742] To tert-butyl ((5S,8S,11S)-1-(9H-fluoren-9-yl)-5,8,11-trimethyl-3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-yl)(methyl)carbamate (350 mg, 574.04 μmol) was added 1,4-dioxane (1 mL), followed by a 4 M hydrogen chloride / dioxane solution (3 mL). The mixture was stirred at room temperature for 1 hour, during which time solids gradually precipitated. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to afford the crude hydrochloride salt of the title compound (310 mg, 567.71 μmol).

[0743] Its structural characterization data are as follows:

[0744] MS m / z(ESI):510.3[M+H] +

[0745] Step 3: Preparation of (9H-fluoren-9-yl)methyl((7S,10S,13S)-2,7,10-trimethyl-1,6,9,12-tetraoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-yl)-2,5,8,11-tetraazatetradec-13-yl)carbamate (K-11-4)

[0746] (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylic acid (367.32 mg, 585.29 μmol) and ( 9H-fluoren-9-yl)methyl ((7S,10S,13S)-7,10-dimethyl-6,9,12-trioxo-2,5,8,11-tetraazatetradec-13-yl)carbamate (0.376 g, 585.29 μmol) was dissolved in DMF (6 mL). DIPEA (226.93 mg, 1.76 mmol) and HATU (267.05 mg, 702.34 μmol) were added and stirred at room temperature for 2 hours. After completion of the reaction, 20 mL of water was added to the reaction solution, and the mixture was extracted with 10 mL of ethyl acetate x 5. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude title compound (755 mg, 337.30 μmol).

[0747] Its structural characterization data are as follows:

[0748] MS m / z(ESI):1120.4[M+H] +

[0749] Step 4: Preparation of (2S,4S)-N-(2-((S)-2-((S)-2-((S)-2-aminopropionamido)propionamido)propionamido)ethyl)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-N-methyl-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamide (K-11-5)

[0750] (9H-fluoren-9-yl)methyl((7S,10S,13S)-2,7,10-trimethyl-1,6,9,12-tetraoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3 (-c) [1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-yl)-2,5,8,11-tetraazatetradec-13-yl)carbamate (755 mg, 674.61 μmol) was dissolved in DMF (15 mL), and piperidine (287.20 mg, 3.37 mmol) was added dropwise. The mixture was stirred at room temperature for 0.5 hours. After completion of the reaction, the reaction solution was purified by reverse-phase silica gel column chromatography (0.05% aqueous ammonium bicarbonate-acetonitrile = 0-80%) and lyophilized to obtain the crude product. The crude product was further purified by preparative high-performance liquid chromatography and lyophilized to obtain the title compound (80 mg, 89.19 μmol).

[0751] Its structural characterization data are as follows:

[0752] MS m / z(ESI):897.3[M+H] +

[0753] The preparation method is as follows:

[0754] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0755] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate)

[0756] Step 5: Preparation of 1-(N-((7S,10S,13S)-2,7,10,13-tetramethyl-1,6,9,12,15-pentaoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-yl)-17-oxa-2,5,8,11,14-pentaazaoctadecane-18-yl)sulfamoyl)piperidine-4-carboxylic acid allyl ester (K-11-6)

[0757] 2-((((4-((allyloxy)carbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (21.09 mg, 60.21 μmol) was dissolved in DMF (2 mL), HATU (22.89 mg, 60.21 μmol) and DIPEA (16.21 mg, 125.43 μmol) were added, and the reaction was stirred at room temperature for 0.5 hour. Then, (2S,4S)-N-(2-((S)-2-((S)-2-((S)-2-aminopropionamido)propionamido)propionamido)ethyl)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-N-methyl-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamide (45 mg, 50.17 umol) was added and the reaction was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was extracted with water and dichloromethane, the organic phase was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residual solvent was removed by lyophilization to obtain the title compound as a crude product (60 mg, 48.81 μmol), which was used directly in the next reaction without purification.

[0758] Its structural characterization data are as follows:

[0759] MS m / z(ESI):1230.4[M+H] +

[0760] Step 6: Preparation of 1-(N-((7S,10S,13S)-2,7,10,13-tetramethyl-1,6,9,12,15-pentaoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-yl)-17-oxa-2,5,8,11,14-pentaazaoctadecane-18-yl)sulfamoyl)piperidine-4-carboxylic acid (K-11-7)

[0761] 1-(N-((7S,10S,13S)-2,7,10,13-tetramethyl-1,6,9,12,15-pentaoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4 ,6,11-hexahydronaphthacene-2-yl)-17-oxa-2,5,8,11,14-pentaazaoctadecane-18-yl)sulfamoyl)piperidine-4-carboxylic acid allyl ester (75 mg, 61.01 μmol) was dissolved in DMF (3 mL). Tetrahydropyrrole (17.36 mg, 244.05 μmol) was added, and the system instantly changed color. Then, tetrakistriphenylphosphine palladium (35.25 mg, 30.51 μmol) was added. The atmosphere was replaced with nitrogen three times, and the reaction was stirred at room temperature under nitrogen protection for 2 hours. After completion of the reaction, the reaction solution was directly purified by preparative high-performance liquid chromatography and freeze-dried to obtain the title compound (10 mg, 8.41 μmol).

[0762] Its structural characterization data are as follows:

[0763] MS m / z(ESI):1189.3[M+H] +

[0764] The preparation method is as follows:

[0765] Chromatographic column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0766] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate)

[0767] Step 7: Preparation of perfluorophenyl 1-(N-((7S,10S,13S)-2,7,10,13-tetramethyl-1,6,9,12,15-pentaoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-yl)-17-oxa-2,5,8,11,14-pentaazaoctadecane-18-yl)sulfamoyl)piperidine-4-carboxylate (K-11)

[0768] To 1-(N-((7S,10S,13S)-2,7,10,13-tetramethyl-1,6,9,12,15-pentaoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1, To a solution of (10.00 mg, 8.41 μmol) (2,3,4,6,11-hexahydrotetracen-2-yl)-17-oxa-2,5,8,11,14-pentaazaoctadecane-18-yl ...sulfamoyl)piperidine-4-carboxylic

[0769] Its structural characterization data are as follows:

[0770] MS m / z(ESI):1355.3[M+H] +

[0771] The preparation method is as follows:

[0772] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0773] Mobile phase A: acetonitrile; mobile phase B: water

[0774] Example 3: S-((31S,34S,37S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-31,34,37,41-tetramethyl-1,29,32,35,38-pentaoxo-5,8,11,14,17,20,23,26-octaoxa-2,30,33,36,39-pentaazatetradodecane-41-yl)(2S,4S Preparation of 2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4':3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-thiocarbonate (K-17)

[0775] Step 1: Preparation of 2,5-dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine (K-17-2)

[0776] Dissolve (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine (1 g, 2.62 mmol) in dichloromethane (10 mL). Add N-hydroxysuccinimide (300.95 mg, 2.62 mmol) and EDCI (551.43 mg, 2.88 mmol). Stir at room temperature for 2 hours. After completion, the reaction solution was washed twice with 10 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude title compound (1.29 g, 2.42 mmol), which was used directly in the next step without purification.

[0777] Its structural characterization data are as follows:

[0778] MS m / z(ESI):497.3[M+H2O+H] +

[0779] Step 2: Preparation of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine-L-alanine (K-17-3)

[0780] 2,5-Dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine (1.19 g, 2.23 mmol) and L-alanine (199.00 mg, 2.23 mmol) were dissolved in tetrahydrofuran (10 mL) and water (5 mL). Sodium bicarbonate (375.29 mg, 4.47 mmol) was added and stirred at room temperature for 1 hour. After completion of the reaction, 20 mL of water was added to the reaction solution, and the pH was adjusted to 4-5 with 2N HCl. A large amount of white flocculent material precipitated. The mixture was extracted with 20 mL of ethyl acetate x 4. The organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude title compound (1.23 g, 2.03 mmol), which was used directly in the next step without purification.

[0781] Its structural characterization data are as follows:

[0782] MS m / z(ESI):454.1[M+H] +

[0783] Step 3: Preparation of 2,5-dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine-L-alanine (K-17-4)

[0784] (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine-L-alanine (400 mg, 882.05 μmol) and N-hydroxysuccinimide (152.27 mg, 1.32 mmol) were dissolved in THF (4 mL) and DMF (2 mL). EDCI (253.64 mg, 1.32 mmol) was added and stirred at room temperature for 2 hours. After completion of the reaction, the reaction solution was diluted with 100 mL of ethyl acetate and then washed directly with 20 mL of saturated brine. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude title compound (400 mg, 726.53 μmol), which was used directly in the next step without purification.

[0785] Its structural characterization data are as follows:

[0786] MS m / z(ESI):568.3[M+H2O+H] +

[0787] Step 4: Preparation of (9H-fluoren-9-yl)methyl ((S)-1-((S)-1-((S)-1-((2-mercapto-2-methylpropyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carbamate (K-17-5)

[0788] 2,5-Dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine-L-alanine (200 mg, 363.27 μmol) was dissolved in THF (3 mL) and water (1 mL). 1-Amino-2-methylpropane-2-thiol (38.22 mg, 363.27 μmol) and sodium bicarbonate (61.03 mg, 726.53 μmol) were added. The mixture was stirred at room temperature under nitrogen for 2 hours. After completion, the reaction mixture was added with 5 mL of water and extracted with 5 mL of ethyl acetate three times. The organic phase was washed with 5 mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10-100%, methanol / dichloromethane = 5-10% as a final step) and concentrated under reduced pressure to obtain the title compound (93 mg, 172.01 μmol).

[0789] Its structural characterization data are as follows:

[0790] MS m / z(ESI):541.3[M+H] +

[0791] Step 5: Preparation of S-((5S,8S,11S)-1-(9H-fluoren-9-yl)-5,8,11,15-tetramethyl-3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazahexadecane-15-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylic acid thioate (K-17-6)

[0792] (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylic acid (50 mg, 79.67 μmol) and (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((( S-1-((2-mercapto-2-methylpropyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carbamate (43.08 mg, 79.67 μmol) was dissolved in DMF (2 mL), and DIPEA (30.89 mg, 239.01 μmol) was added. PyBOP (49.75 mg, 95.60 μmol) was then added under nitrogen. After complete addition, the mixture was stirred at 25°C under a nitrogen atmosphere for 3 hours. After completion of the reaction, 10 mL of water was added to the reaction solution, followed by extraction with 10 mL of ethyl acetate x 3. The organic phase was washed with 10 mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude title compound (200 mg, 78.24 μmol), which was used directly in the next step without purification.

[0793] Its structural characterization data are as follows:

[0794] MS m / z(ESI):1151.3[M+H] +

[0795] Step 6: Preparation of S-(1-((S)-2-((S)-2-((S)-2-aminopropionamido)propionamido)propionamido)-2-methylpropan-2-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylic acid thioate (K-17-7)

[0796] S-((5S,8S,11S)-1-(9H-fluoren-9-yl)-5,8,11,15-tetramethyl-3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazahexadec-15-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1- Methyl octahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylate (200 mg, 86.94 μmol) was dissolved in DMF (4 mL). Piperidine (60 mg, 704.66 μmol) was added and stirred at room temperature for 10 minutes. After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (15 mg, 16.16 μmol).

[0797] Its structural characterization data are as follows:

[0798] MS m / z(ESI):928.3[M+H] +

[0799] The preparation method is as follows:

[0800] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0801] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate)

[0802] Step 7: S-((31S,34S,37S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-31,34,37,41-tetramethyl-1,29,32,35,38-pentaoxo-5,8,11,14,17,20,23,26-octaoxa-2,30,33,36,39-pentaazatetradecane-41-yl)(2S,4S Preparation of 2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4':3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-thiocarbonate (K-17)

[0803] 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosanetaconic acid-29-carboxylic acid (4.62 mg, 5.39 μmol) and S-(1-((S)-2-((S)-2-((S)-2-aminopropionamido)propionamido)propionamido)-2-methylpropan-2-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S, (9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylate (5 mg, 5.39 μmol) was dissolved in DMF (0.5 mL). DIPEA (2.09 mg, 16.16 μmol) was added dropwise, followed by HATU (2.46 mg, 6.47 μmol). The mixture was stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (4 mg, 2.15 μmol).

[0804] Its structural characterization data are as follows:

[0805] MS m / z(ESI):1769.4[M+H] +

[0806] The preparation method is as follows:

[0807] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0808] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate)

[0809] Example 4: S-((33S,36S,39S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29,33,36,39,43-tetradecyl-1,4,7,10,13,16,19,22,25,28,31,34,37,40-tetradecanoyl-2,5,8,11,14,17,20,23,26,29,32,35, Preparation of 38,41-tetradecane-43-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carbothioate (K-18)

[0810] 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatriacontane-31-carboxylic acid (6.17 mg, 5.39 μmol) and S-(1-((S)-2-((S)-2-aminopropionamido)propionamido)propionamido)-2-methylpropan-2-yl)(2S,4S)-2,5,12- Trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylate (5 mg, 5.39 μmol) was dissolved in DMF (0.5 mL). DIPEA (2.09 mg, 16.16 μmol) was added dropwise, followed by HATU (2.46 mg, 6.47 μmol). The mixture was stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (6.48 mg, 3.12 μmol).

[0811] Its structural characterization data are as follows:

[0812] MS m / z(ESI):1036.6[(M+H2O) / 2] + ,691.5[(M+H2O) / 3] +

[0813] The preparation method is as follows:

[0814] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0815] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate)

[0816] Example 5: 1-(N-((9S,12S,15S)-9,12,15-trimethyl-1,8,11,14,17-pentaoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[ Preparation of pentafluorophenyl 4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-yl)-2,5,19-trioxa-7,10,13,16-tetraazaeicosane-20-yl)sulfamoyl)piperidine-4-carboxylate (K-10)

[0817] Step 1: Preparation of allyl 1-(N-((2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethoxy)carbonyl)sulfamoyl)piperidine-4-carboxylate (K-10-1)

[0818] 1-Hydroxypyrrolidine-2,5-dione (24.64 mg, 214.08 μmol) and 2-((((4-((allyloxy)carbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (50 mg, 142.72 μmol) were dissolved in THF (2 mL). DCC (35.34 mg, 171.26 μmol) was added and stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was filtered, the filter cake was washed with an appropriate amount of THF, and the filtrate was concentrated under reduced pressure to obtain the crude title compound (63 mg, 140.81 μmol), which was used directly in the next step without purification.

[0819] Its structural characterization data are as follows:

[0820] MS m / z(ESI):499.1[M+Na] +

[0821] Step 2: Preparation of (2-((((4-((allyloxy)carbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetyl)-L-alanyl-L-alanine (K-10-2)

[0822] Allyl 1-(N-((2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethoxy)carbonyl)sulfamoyl)piperidine-4-carboxylate (40 mg, 89.40 μmol) and L-alanyl-L-alanine (20.05 mg, 125.16 μmol) were dissolved in DMF (1 mL). DIPEA (23.11 mg, 178.80 μmol) was added and stirred at room temperature for 2 hours. After completion of the reaction, water was added to the reaction solution, which was then acidified with 2N dilute hydrochloric acid to pH 3-4. The solution was extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was further purified by silica gel column chromatography (ethyl acetate-(ethyl acetate:methanol = 1:1)) = 0-80%) and concentrated again under reduced pressure to obtain the title compound (23 mg, 46.70 μmol).

[0823] Its structural characterization data are as follows:

[0824] MS m / z(ESI):493.2[M+H] +

[0825] Step 3: Preparation of (9H-fluoren-9-yl)methyl (S)-(1-(((2-hydroxyethoxy)methyl)amino)-1-oxopropan-2-yl)carbamate (K-10-3)

[0826] Methyl (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)acetate (200 mg, 523.00 μmol) was dissolved in THF (2 mL), and ethylene glycol (324.61 mg, 5.23 mmol) and p-toluenesulfonic acid (18.01 mg, 104.60 μmol) were added. The mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction solution was diluted with 10 mL of ethyl acetate, washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was further purified by silica gel column chromatography (petroleum ether-ethyl acetate = 0-60%) and concentrated again under reduced pressure to obtain the title compound (140 mg, 364.18 μmol).

[0827] Its structural characterization data are as follows:

[0828] MS m / z(ESI):402.3[M+H2O] +

[0829] Step 4: Preparation of (S)-1-(9H-fluoren-9-yl)-5-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-yl 4-methylbenzenesulfonate (K-10-4)

[0830] (9H-fluoren-9-yl)methyl (S)-(1-(((2-hydroxyethoxy)methyl)amino)-1-oxopropan-2-yl)carbamate (490 mg, 1.27 mmol) was dissolved in dichloromethane (5 mL). Pyridine (151.23 mg, 1.91 mmol) was added, followed by p-toluenesulfonyl chloride (291.61 mg, 1.53 mmol). The reaction mixture was stirred at room temperature for 24 hours. Pyridine (151.23 mg, 1.91 mmol) and p-toluenesulfonyl chloride (291.61 mg, 1.53 mmol) were then added. The reaction mixture was stirred at room temperature for another 4 hours, then heated to reflux for 28 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to obtain the crude product. The product was further purified by silica gel column chromatography (petroleum ether-ethyl acetate = 0-60%) and concentrated again under reduced pressure to obtain the title compound (350 mg, 649.82 μmol).

[0831] Its structural characterization data are as follows:

[0832] MS m / z(ESI):556.3[M+H2O] +

[0833] Step 5: Preparation of (S)-1-(9H-fluoren-9-yl)-5-methyl-3,6-dioxo-2,9-dioxa-4,7-diazoundecane-11-yl(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylate (K-10-5)

[0834] (S)-1-(9H-fluoren-9-yl)-5-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-yl 4-methylbenzenesulfonate (300.00 mg, 556.99 μmol) and (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro- 1H-Pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylic acid (60 mg, 95.60 μmol) was dissolved in DMF (2 mL) and sodium bicarbonate (32.13 mg, 382.41 μmol) was added. The mixture was stirred at room temperature for 1 hour and then heated to 45°C for 24 hours. After completion of the reaction, the reaction solution was extracted with water and ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was further purified by silica gel column chromatography (petroleum ether-ethyl acetate = 10-100%) and concentrated again under reduced pressure to obtain the title compound (25 mg, 25.15 μmol).

[0835] Its structural characterization data are as follows:

[0836] MS m / z(ESI):994.4[M+H] +

[0837] Step 6: Preparation of 2-(((S)-2-aminopropionamido)methoxy)ethyl (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylate (K-10-6)

[0838] (S)-1-(9H-fluoren-9-yl)-5-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-yl(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylate (25.00 mg, 25.15 μmol) was dissolved in DMF (2.00 mL), piperidine (16 mg, 187.91 μmol) was added, and the mixture was stirred at room temperature for 5 minutes. After the reaction was completed, the reaction solution was extracted with water and ethyl acetate, and the organic phases were combined and concentrated under reduced pressure to obtain a crude product, which was further purified by preparative HPLC and freeze-dried to obtain the title compound (4 mg, 5.18 μmol).

[0839] Its structural characterization data are as follows:

[0840] MS m / z(ESI):772.3[M+H] +

[0841] The preparation method is as follows:

[0842] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0843] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate)

[0844] Step 7: 1-(N-((9S,12S,15S)-9,12,15-trimethyl-1,8,11,14,17-pentaoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4 Preparation of allyl ',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-yl)-2,5,19-trioxa-7,10,13,16-tetraazaeicosane-20-yl)sulfamoyl)piperidine-4-carboxylate (K-10-7)

[0845] (2-((((4-((allyloxy)carbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetyl)-L-alanyl-L-alanine (5.11 mg, 10.37 μmol) and 2-(((S)-2-aminopropionamido)methoxy)ethyl (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctanoate were added. Hydrogen-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-carboxylate (4 mg, 5.18 μmol) was dissolved in DMF (1 mL). DMTMM (4.58 mg, 15.55 μmol) and DIPEA (2.68 mg, 20.73 μmol) were added and stirred at room temperature for 1 hour. After completion of the reaction, the reaction solution was extracted with water and ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude title compound (12 mg, 1.35 μmol), which was used directly in the next reaction without purification.

[0846] Its structural characterization data are as follows:

[0847] MS m / z(ESI):1247.3[M+H] +

[0848] Step 8: Preparation of 1-(N-((9S,12S,15S)-9,12,15-trimethyl-1,8,11,14,17-pentaoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-yl)-2,5,19-trioxa-7,10,13,16-tetraazaeicosane-20-yl)sulfamoyl)piperidine-4-carboxylic acid (K-10-8)

[0849] 1-(N-((9S,12S,15S)-9,12,15-trimethyl-1,8,11,14,17-pentaoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2 ,3,4,6,11-hexahydro-tetracen-2-yl)-2,5,19-trioxa-7,10,13,16-tetraazaeicosane-20-yl)sulfamoyl)piperidine-4-carboxylic acid allyl ester (12 mg, 9.63 μmol) was dissolved in DMF (0.5 mL), tetrahydropyrrole (1.37 mg, 19.26 μmol) and tetrakistriphenylphosphine palladium (1.11 mg, 9.63e-1 μmol) were added, and the atmosphere was replaced with nitrogen three times. The reaction mixture was stirred at room temperature under nitrogen atmosphere for 2 hours. After the reaction was completed, the reaction solution was freed from the solvent using a freeze dryer, the residue was added with water, and the insoluble matter was removed by filtration. The filtrate was purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (2 mg, 1.66 μmol).

[0850] Its structural characterization data are as follows:

[0851] MS m / z(ESI):1206.3[M+H] +

[0852] The preparation method is as follows:

[0853] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0854] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate)

[0855] Step 9: 1-(N-((9S,12S,15S)-9,12,15-trimethyl-1,8,11,14,17-pentaoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[ Preparation of pentafluorophenyl 4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-yl)-2,5,19-trioxa-7,10,13,16-tetraazaeicosane-20-yl)sulfamoyl)piperidine-4-carboxylate (K-10)

[0856] To-(N-((9S,12S,15S)-9,12,15-trimethyl-1,8,11,14,17-pentaoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dihydro- To a solution of (1,2,3,4,6,11-hexahydronaphthacene-2-yl)-2,5,19-trioxa-7,10,13,16-tetraazaeicosane-20-yl)sulfamoyl)piperidine-4-carboxylic acid (2 mg, 1.66 μmol) was added DMF (0.5 mL) and 2,3,4,5,6-pentafluorophenol (3.05 mg, 16.58 μmol), followed by EDCI (3 mg, 15.65 μmol). The mixture was stirred at room temperature for 1 hour. After completion of the reaction, the solvent was partially removed using a lyophilizer, purified by preparative HPLC, and freeze-dried to obtain the title compound (480.00 μg, 3.15e-1 μmol).

[0857] Its structural characterization data are as follows:

[0858] MS m / z(ESI):1373.2[M+H] +

[0859] The preparation method is as follows:

[0860] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0861] Mobile phase A: acetonitrile; mobile phase B: water

[0862] 2. Antibody Preparation and Binding Activity Assay

[0863] 1. Antibody acquisition and purification

[0864] Based on the amino acid sequences of trastuzumab (IMGT / mAb-DB ID: 97) and pertuzumab (IMGT / mAb-DB ID: 80) in the IMGT database, codon optimization was performed and the encoding genes were synthesized. These genes were then constructed into expression vectors, transfected into CHO cells, and subjected to pressure screening to construct stable expression cell lines. The supernatant was collected and purified using Protein A affinity media to obtain the corresponding antibodies, trastuzumab and pertuzumab.

[0865] 3. Conjugation of Compounds Containing Cellular Bioactive Molecules and Linkers to Antibodies

[0866] The antibodies Trastuzumab and Pertuzumab involved in the antibody-drug conjugates prepared in the following examples are Trastuzumab and Pertuzumab described in the second part above.

[0867] The conjugate preparation of the antibody drug conjugate sample is as follows:

[0868] 1. Preparation of Trastuzumab-K-17

[0869] 0.3086 mL of trastuzumab antibody (16.2 mg / mL) was diluted with 15.43 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 8.0 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 18.95 μL, pH 7.60) solution was added, mixed, and allowed to stand at room temperature for 1.5 hours. K-17 dissolved in dimethyl sulfoxide (17.4 μL, 10 mM, equivalent to 5 times the amount of antibody) was then added, mixed, and allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with 20 mM histidine buffer at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., trastuzumab-K-17). The DAR value, determined by mass spectrometry, was 4.28.

[0870] Table 1: Measured molecular weight and DAR calculation of Trastuzumab-K-17

[0871] 2. Preparation of Trastuzumab-K-18

[0872] 1.234 mL of trastuzumab antibody (16.2 mg / mL) was diluted with 61.73 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 8.0 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 75.8 μL, pH 7.60) solution was added, mixed, and allowed to stand at room temperature for 1.5 hours. K-18 dissolved in dimethyl sulfoxide (62.64 μL, 10 mM, equivalent to 4.5 times the amount of antibody) was then added, mixed, and allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with 20 mM histidine buffer at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., trastuzumab-K-18). The DAR value, determined by mass spectrometry, was 4.44.

[0873] Table 2: Trastuzumab-K-18 Measured Molecular Weight and Calculated DAR

[0874] IV. Detecting the inhibitory effect of compounds on tumor cell proliferation

[0875] 1. Inhibitory effect of the compound on NCI-N87 cell proliferation

[0876] (1) Cell plating: First, culture the tumor cells NCI-N87 in the corresponding culture medium, digest the cells with trypsin, centrifuge, resuspend the cells, count the cells, and adjust the cells to the appropriate concentration for plating. The tumor cell sources are shown in Table 3.

[0877] Table 3. Tumor cell origin

[0878] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the above plate wells and incubated for 72 hours.

[0879] In vitro cell viability assay: After incubation, add 50 μL of Cell Counting-Lite™ 2.0 reagent (Vazyme / Novozyme) to each well, shake and mix thoroughly in the dark. After 10 minutes of reaction, assay can be performed using a microplate reader (Manufacturer: BMG, Model: PHERAStar-FS). Background RLU values ​​are calculated using Cell Counting-Lite™ culture medium without cells, and vehicle RLU values ​​are calculated using Cell Counting-Lite™ culture medium with cells. Cell inhibition rate = 1 - (sample RLU - background RLU) / (vehicle RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated using a four-parameter curve fit. RLU (relative light unit): relative light unit. The assay results are shown in Tables 4-1, 4-2, 4-3, and 4-4.

[0880] (2) Data results

[0881] Table 4-1. Inhibitory activity of compounds on NCI-N87 cell proliferation

[0882] Table 4-2. Inhibitory activity of compounds on NCI-N87 cell proliferation

[0883] Table 4-3. Inhibitory activity of compounds on NCI-N87 cell proliferation

[0884] Table 4-4. Inhibitory activity of compounds on NCI-N87 cell proliferation

[0885] The test results show that the compounds of the present invention in Tables 4-1, 4-4, 4-3 and 4-4 have a significant inhibitory effect on the proliferation of NCI-N87 human gastric cancer cells.

[0886] 2. Inhibitory effect of compounds on HT29 cell proliferation

[0887] (1) Cell plating: First, culture HT29 tumor cells in the corresponding culture medium, digest the cells with trypsin, centrifuge, resuspend the cells, count the cells, and adjust the cells to the appropriate concentration for plating. The sources of tumor cells are shown in Table 5.

[0888] Table 5. Tumor cell origin

[0889] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the above plate wells and incubated for 72 hours.

[0890] In vitro cell viability assay: After incubation, add 50 μL of Cell Counting-Lite™ 2.0 reagent (Vazyme / Novozyme) to each well, shake and mix thoroughly in the dark. After 10 minutes of reaction, assay can be performed using a microplate reader (Manufacturer: BMG, Model: PHERAStar-FS). Background RLU values ​​are calculated using Cell Counting-Lite™ reagents for cell-free medium, and vehicle RLU values ​​are calculated using Cell Counting-Lite™ reagents for cell-containing medium. Cell inhibition rate = 1 - (sample RLU - background RLU) / (vehicle RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated using a four-parameter curve fit. RLU (relative light unit) is the relative light unit. The assay results are shown in Tables 6-1, 6-2, 6-3, and 6-4.

[0891] (2) Data results

[0892] Table 6-1. Inhibitory activity of compounds on HT29 cell proliferation

[0893] Table 6-2. Inhibitory activity of compounds on HT29 cell proliferation

[0894] Table 6-3. Inhibitory activity of compounds on HT29 cell proliferation

[0895] Table 6-4. Inhibitory activity of compounds on HT29 cell proliferation

[0896] The test results show that the compounds of the present invention in Tables 6-1, 6-2, 6-3 and 6-4 have a strong proliferation inhibitory effect on the proliferation of HT29 colon cancer cells.

[0897] 3. Inhibitory effect of the compounds on HCC1806 cell proliferation

[0898] (1) Cell plating: First, culture HCC1806 tumor cells in the appropriate culture medium, digest the cells with trypsin, centrifuge, resuspend the cells, count the cells, and adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 7.

[0899] Table 7. Tumor cell sources

[0900] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the above plate wells and incubated for 72 hours.

[0901] In vitro cell viability assay: After incubation, add 50 μL of Cell Counting-Lite™ 2.0 reagent (Vazyme / Novozyme) to each well, shake and mix in the dark. After 10 minutes of reaction, assays can be performed using a microplate reader (Manufacturer: BMG, Model: PHERAStar-FS). Background RLUs were calculated using the Cell Counting-Lite™ assay for cell-free culture medium, while vehicle RLUs were calculated using the Cell Counting-Lite™ assay for cell-containing culture medium. Cell inhibition rate = 1 - (sample RLU - background RLU) / (vehicle RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated using a four-parameter curve fit. RLU (relative light unit): relative light unit. The assay results are shown in Table 8.

[0902] (2) Data results

[0903] Table 8. Inhibitory activity of compounds on HCC1806 cell proliferation

[0904] The test results show that the compounds of the present invention in Table 8 have a significant inhibitory effect on the proliferation of HCC1806 human breast squamous carcinoma cells.

[0905] 4. Inhibitory effect of compounds on SKOV-3 cell proliferation

[0906] (1) Cell plating: First, culture SKOV-3 tumor cells in the appropriate culture medium, digest the cells with trypsin, centrifuge, resuspend the cells, count the cells, and adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 9.

[0907] Table 9. Tumor cell sources

[0908] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the above plate wells and incubated for 72 hours.

[0909] In vitro cell viability assay: After incubation, add 50 μL of Cell Counting-Lite™ 2.0 reagent (Vazyme / Novozyme) to each well, shake and mix in the dark. After 10 minutes of reaction, assays can be performed using a microplate reader (Manufacturer: BMG, Model: PHERAStar-FS). Background RLUs were calculated using Cell Counting-Lite™ reagents for cell-free medium, and vehicle RLUs were calculated using Cell Counting-Lite™ reagents for cell-containing medium. Cell inhibition rate = 1 - (sample RLU - background RLU) / (vehicle RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated using a four-parameter curve fit. RLU (relative light unit): relative light unit. The assay results are shown in Table 10.

[0910] (2) Data results

[0911] Table 10. Inhibitory activity of compounds on SKOV-3 cell proliferation

[0912] The test results show that the compounds of the present invention in Table 10 have a significant inhibitory effect on the proliferation of SKOV-3 human ovarian cancer cells.

[0913] 5. Inhibitory effect of compounds on NCI-H358 cell proliferation

[0914] (1) Cell plating: First, culture the tumor cells NCI-H358 in the corresponding culture medium, digest the cells with trypsin, centrifuge, resuspend the cells, count the cells, and adjust the cells to the appropriate concentration for plating. The tumor cell sources are shown in Table 11.

[0915] Table 11. Tumor cell sources

[0916] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the above plate wells and incubated for 72 hours.

[0917] In vitro cell viability assay: After incubation, add 50 μL of Cell Counting-Lite™ 2.0 reagent (Vazyme / Novozyme) to each well, shake and mix in the dark. After 10 minutes of reaction, assays can be performed using a microplate reader (Manufacturer: BMG, Model: PHERAStar-FS). Background RLU values ​​were calculated using Cell Counting-Lite™ for cell-free culture medium, and vehicle RLU values ​​were calculated using Cell Counting-Lite™ for cell-containing culture medium. Cell inhibition rate = 1 - (sample RLU - background RLU) / (vehicle RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated using a four-parameter curve fit. RLU (relative light unit): relative light unit. The assay results are shown in Table 12.

[0918] (2) Data results

[0919] Table 12. Inhibitory activity of compounds on NCI-H358 cell proliferation

[0920] The test results show that the compounds of the present invention in Table 12 have a significant inhibitory effect on the proliferation of NCI-H358 human non-small cell lung cancer cells. 6. Inhibitory effect of compounds on the proliferation of KPL-4 cells

[0921] (1) Cell plating: First, culture KPL-4 tumor cells in the appropriate culture medium, digest the cells with trypsin, centrifuge, resuspend the cells, count the cells, and adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 13.

[0922] Table 13. Tumor cell sources

[0923] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the above plate wells and incubated for 72 hours.

[0924] In vitro cell viability assay: After incubation, add 50 μL of Cell Counting-Lite™ 2.0 reagent (Vazyme / Novozyme) to each well, shake and mix in the dark. After 10 minutes of reaction, assays can be performed using a microplate reader (Manufacturer: BMG, Model: PHERAStar-FS). Background RLU values ​​were calculated using Cell Counting-Lite™ for cell-free culture medium, and vehicle RLU values ​​were calculated using Cell Counting-Lite™ for cell-containing culture medium. Cell inhibition rate = 1 - (sample RLU - background RLU) / (vehicle RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated using a four-parameter curve fit. RLU (relative light unit): relative light unit. The assay results are shown in Table 14.

[0925] (2) Data results

[0926] Table 14. Inhibitory activity of compounds on KPL-4 cell proliferation

[0927] The test results show that the compounds of the present invention in Table 14 have a significant inhibitory effect on the proliferation of KPL-4 human breast cancer cells.

[0928] 7. Inhibitory effect of compounds on HCC827 cell proliferation

[0929] (1) Cell plating: First, culture HCC827 tumor cells in the appropriate culture medium, digest the cells with trypsin, centrifuge, resuspend the cells, count the cells, and adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 15.

[0930] Table 15. Tumor cell sources

[0931] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the above plate wells and incubated for 72 hours.

[0932] In vitro cell viability assay: After incubation, add 50 μL of Cell Counting-Lite™ 2.0 reagent (Vazyme / Novozyme) to each well, shake and mix in the dark. After 10 minutes of reaction, assays can be performed using a microplate reader (Manufacturer: BMG, Model: PHERAStar-FS). Background RLU values ​​were calculated using Cell Counting-Lite™ for cell-free medium, and vehicle RLU values ​​were calculated using Cell Counting-Lite™ for cell-containing medium. Cell inhibition rate = 1 - (sample RLU - background RLU) / (vehicle RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated using a four-parameter curve fit. RLU (relative light unit): relative light unit. The assay results are shown in Table 16.

[0933] (2) Data results

[0934] Table 16. Antiproliferation activity of compounds on HCC827 cells

[0935] The test results show that the compounds of the present invention in Table 16 have an inhibitory effect on the proliferation of HCC827 human non-small cell lung cancer cells.

[0936] 8. Inhibitory effect of compounds on HCC1954 cell proliferation

[0937] (1) Cell plating: First, culture HCC1954 tumor cells in the appropriate culture medium, digest the cells with trypsin, centrifuge, resuspend the cells, count the cells, and adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 17.

[0938] Table 17. Tumor cell sources

[0939] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the above plate wells and incubated for 72 hours.

[0940] In vitro cell viability assay: After incubation, add 50 μL of Cell Counting-Lite™ 2.0 reagent (Vazyme / Novozyme) to each well, shake and mix in the dark. After 10 minutes of reaction, assays can be performed using a microplate reader (Manufacturer: BMG, Model: PHERAStar-FS). Background RLUs were calculated using Cell Counting-Lite™ reagents for cell-free medium, and vehicle RLUs were calculated using Cell Counting-Lite™ reagents for cell-containing medium. Cell inhibition rate = 1 - (sample RLU - background RLU) / (vehicle RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated using a four-parameter curve fit. RLU (relative light unit): relative light unit. The assay results are shown in Table 18.

[0941] (2) Data results

[0942] Table 18. Proliferation inhibition activity of compounds on HCC1954 cells

[0943] The test results show that the compounds of the present invention in Table 18 have an inhibitory effect on the proliferation of HCC1954 human non-small cell lung cancer cells.

[0944] 9. Inhibitory effect of compounds on NCI-H1975 cell proliferation

[0945] (1) Cell plating: First, culture the tumor cells NCI-H1975 in the corresponding culture medium, digest the cells with trypsin, centrifuge, resuspend the cells, count the cells, and adjust the cells to the appropriate concentration for plating. The tumor cell sources are shown in Table 19.

[0946] Table 19. Tumor cell sources

[0947] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the above plate wells and incubated for 72 hours.

[0948] In vitro cell viability assay: After incubation, add 50 μL of Cell Counting-Lite™ 2.0 reagent (Vazyme / Novozyme) to each well, shake and mix in the dark. After 10 minutes of reaction, assays can be performed using a microplate reader (Manufacturer: BMG, Model: PHERAStar-FS). Background RLUs were calculated using Cell Counting-Lite™ reagents for cell-free medium, and vehicle RLUs were calculated using Cell Counting-Lite™ reagents for cell-containing medium. Cell inhibition rate = 1 - (sample RLU - background RLU) / (vehicle RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated using a four-parameter curve fit. RLU (relative light unit): relative light unit. The assay results are shown in Table 20.

[0949] (2) Data results

[0950] Table 20. Antiproliferation activity of compounds on NCI-H1975 cells

[0951] The test results show that the compounds of the present invention in Table 20 have an inhibitory effect on the proliferation of NCI-H1975 human non-small cell lung cancer cells.

[0952] 5. Evaluation of the Antibody-Drug Conjugate's Inhibitory Effect on Tumor Growth in a Mouse Subcutaneous Xenograft Tumor Model

[0953] The preparation containing the ADC of the present invention was administered via tail vein injection to a CDX mouse model subcutaneously transplanted with human gastric cancer cells NCI-N87. The tumor volume and animal body weight changes were measured twice a week, and the tumor inhibition efficacy of the ADC of the present invention on tumor-bearing mice was calculated.

[0954] Test drug

[0955] Drug Name, Source, and Preparation: Take an appropriate amount of the ADC of the present invention (Sichuan Kelun Botai Biopharmaceutical Co., Ltd.) and dilute the stock solution with 0.9% NaCl injection to a dosing solution at a 1 mg / kg dose. Use 0.9% NaCl injection as a vehicle control.

[0956] Experimental animals and cell lines

[0957] Balb / c Nude mice (Chengdu Yaokang Biotechnology Co., Ltd., production license number: SCXK (Sichuan) 2020-0034, animal certificate number: 511214900019415)

[0958] Human gastric cancer cells NCI-N87 (ATCC)

[0959] Experimental grouping and evaluation methods

[0960] The average tumor volume was about 150 mm. 3 Tumor-bearing mice were randomly divided into groups (the number of groups was determined based on the sample size). The groups were administered 0.9% NaCl injection (hereinafter referred to as vehicle control) and the ADC of the present invention. The dosing frequency was as described in the specific examples. The administration method was tail vein injection, and the administration volume was 10 ml / kg. Tumor diameter was measured twice weekly with a vernier caliper, and tumor volume was calculated using the following formula: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively. Animal mortality was recorded daily.

[0961] The tumor growth inhibition rate (TGI) was calculated using the following formula to evaluate the tumor inhibition efficacy of the ADC of the present invention:

[0962] V T末 >V T0 ,TGI(%)=[1-(V T末 -V T0 ) / (V C末 -V C0 )]*100% or V T末 ≤V T0 ,TGI(%)=[1-(V T末 -VT0) / VT0]*100%.

[0963] Where V T末 : Mean tumor volume of treatment group at the end of the experiment

[0964] V T0 : Mean tumor volume at the start of drug administration in the treatment group

[0965] V C末 : Mean tumor volume of negative control group at the end of the experiment

[0966] V C0 : Mean tumor volume of negative control group at the beginning of drug administration

[0967] The following formula was used to calculate the tumor relative proliferation rate T / C (%), which was used to evaluate the tumor inhibition efficacy of the ADC of the present invention:

[0968] T / C=(V T末 / V T0 ) / (V C末 / V C0 ).

[0969] (1) Efficacy testing of anti-human Her2 antibody-drug conjugates in the NCI-N87 model

[0970] NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. NCI-N87 cells were collected during the exponential growth phase, resuspended in PBS to an appropriate concentration, and inoculated subcutaneously into female Balb / c-nu mice to establish a gastric cancer model. When the average tumor volume reached approximately 150 mm, the cells were cultured in RPMI 1640 medium at 37°C and 5% CO2. 3 At approximately 14 days, the mice were randomly divided into the following groups according to tumor size: a vehicle control group (i.e., negative control, vehicle group) and a 1 mg / kg trastuzumab-K-18 group of the present invention. The mice were administered by tail vein injection (iv) on Day 0 and Day 7, for a total of two doses. After administration, the mice were weighed once a week and the major and minor diameters of the tumors were measured with a vernier caliper. The tumor volume was calculated according to the following formula: V = 0.5a × b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively. Animal deaths were observed and recorded every day.

[0971] The ADC of this invention demonstrated significant tumor growth inhibition in the NCI-N87 gastric cancer xenograft model. Compared to the vehicle group, the tumor growth inhibition rate (TGI) of the 1 mg / kg trastuzumab-K-18 group was 164.99%. No animals died or experienced significant weight loss on Day 30, and no significant drug toxicity was observed. The ADC of this invention was well tolerated by mice during treatment. Detailed results are shown in Table 21, Figures 1 and 2.

[0972] Table 21 Human gastric cancer cell NCI-N87 CDX model

[0973] Note: TGI is tumor growth inhibition rate, T / C is relative tumor proliferation rate, the same below.

[0974] VI. Evaluation of the Antibody-Drug Conjugate's Inhibitory Effect on Tumor Growth in a Mouse Subcutaneous Xenograft Tumor Model

[0975] The preparations containing the ADC of the present invention were administered via tail vein injection to CDX mouse models subcutaneously transplanted with human breast cancer cells JIMT-1. The tumor volume and animal body weight changes were measured twice a week, and the tumor inhibition efficacy of the ADC of the present invention on tumor-bearing mice was calculated.

[0976] Test drug

[0977] Drug Name, Source, and Preparation: Take an appropriate amount of the ADC of the present invention (Sichuan Kelun Botai Biopharmaceutical Co., Ltd.) and dilute the stock solution with 0.9% NaCl injection to the dosing volume. Use 0.9% NaCl injection as the vehicle control (Vehicle).

[0978] Experimental animals and cell lines

[0979] NOD SCID mice (Chengdu Yaokang Biotechnology Co., Ltd., production license number: SCXK (Sichuan) 2020-0034, animal certificate number: 511214900024561)

[0980] Human breast cancer cells JIMT-1 (Nanjing Kebai)

[0981] Experimental grouping and evaluation methods

[0982] The average tumor volume was about 150 mm. 3 Tumor-bearing mice were randomly divided into groups (the number of groups was determined based on the sample size). The groups were administered 0.9% NaCl injection (hereinafter referred to as vehicle control) and the ADC of the present invention. The dosing frequency was as described in the specific examples. The administration method was tail vein injection, and the administration volume was 10 ml / kg. Tumor diameter was measured twice weekly with a vernier caliper, and tumor volume was calculated using the following formula: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively. Animal mortality was recorded daily.

[0983] The tumor growth inhibition rate (TGI) was calculated using the following formula to evaluate the tumor inhibition efficacy of the ADC of the present invention:

[0984] V T末 >V T0 ,TGI(%)=[1-(V T末 -V T0 ) / (V C末 -V C0 )]*100% or V T末 ≤V T0 ,TGI(%)=[1-(V T末 -VT0) / VT0]*100%.

[0985] Where V T末 : Mean tumor volume of treatment group at the end of the experiment

[0986] V T0 : Mean tumor volume at the start of drug administration in the treatment group

[0987] V C末 : Mean tumor volume of negative control group at the end of the experiment

[0988] V C0 : Mean tumor volume of negative control group at the beginning of drug administration

[0989] The following formula was used to calculate the tumor relative proliferation rate T / C (%), which was used to evaluate the tumor inhibition efficacy of the ADC of the present invention:

[0990] T / C=(V T末 / V T0 ) / (V C末 / V C0 ).

[0991] (1) Efficacy testing of anti-human Her2 antibody-drug conjugates in the JIMT-1 model

[0992] JIMT-1 cells were cultured in DMEM containing 10% fetal bovine serum at 37°C and 5% CO2. JIMT-1 cells in the exponential growth phase were harvested, resuspended in PBS containing 50% Matrigel to an appropriate concentration, and inoculated subcutaneously into female NOD SCID mice to establish a breast cancer model. When the average tumor volume reached approximately 150 mm, the cells were inoculated with a 5% PBS solution containing 50% Matrigel. 3 Around 30 minutes later, the mice were randomly divided into groups according to the size of the tumor, in the following order: vehicle control group (i.e., negative control, Vehicle group), Trastuzumab-K-18 1 mg / kg group of the present invention, Trastuzumab-K-18 3 mg / kg, Trastuzumab-K-17 1 mg / kg group, and Trastuzumab-K-17 3 mg / kg group. Each group was injected by tail vein (iv), and the drug was administered on Day 0, for a total of 1 dose. After administration, the body weight of the mice was measured twice a week, and the long and short diameters of the tumor were measured with a vernier caliper, and the tumor volume was calculated according to the following formula: V = 0.5a × b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively. Animal deaths were observed and recorded every day.

[0993] The ADC of this invention demonstrated significant tumor growth inhibition in the JIMT-1 breast cancer xenograft model. Compared with the vehicle group, the tumor growth inhibition rates (TGI) of the 1 mg / kg trastuzumab-K-18, 3 mg / kg trastuzumab-K-18, 1 mg / kg trastuzumab-K-17, and 3 mg / kg trastuzumab-K-17 groups were 81.64%, 163.54%, 62.33%, and 139.31%, respectively. On Day 20, there were no animal deaths or significant weight loss in any treatment group, and no significant drug toxicity was observed. The ADC of this invention was well tolerated by mice during treatment. Detailed results are shown in Table 22, Figures 3 and 4.

[0994] Table 22 Human breast cancer cell JIMT-1CDX model

[0995] Note: TGI is tumor growth inhibition rate, T / C is relative tumor proliferation rate, the same below.

[0996] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are all within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody-drug conjugate having a structure represented by the formula Ab-[M-L-E-D]x, wherein: Ab is an antibody or an antigen-binding fragment thereof that specifically binds to the epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases: M is the linker site of the antibody or its antigen-binding fragment; L is a structural fragment connecting the linker M and E; E is a structural fragment connecting L and D; D is a cytotoxic drug fragment; X is from 1 to 10.

2. The antibody-drug conjugate according to claim 1, wherein, M is selected from the following substituted or unsubstituted structural fragments:

3. The antibody-drug conjugate according to claim 1, wherein, M is selected from the following substituted or unsubstituted structural fragments: Preferably, M is selected from the following substituted or unsubstituted structural fragments:

4. The antibody-drug conjugate according to any one of claims 1-3, wherein, L is selected from one or more of the following substituted or unsubstituted structural fragments: C 1-6 alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, 9-12 membered nitrogen-containing heterocyclic group, -N(R'), -NH(R'), -N(R')2, carbonyl, -O-, natural amino acid or unnatural amino acid and their analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), Lys(R'), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), where R' represents hydrogen, C 1-6 alkyl, -C 1-6 alkyleneCO2H, -C 1-6 alkyleneSO3H, -SO3H, -PO3H2, -C 1-6 alkylene-NHC 1-6 alkyl, -C 1-6 alkylene-N(C 1-6 alkyl)2, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 alkylene - heterocycle, -CH2NH-SO3H, -CH2N(C 1-6 alkyl)-SO3H, -CH2NHC 1-6 alkylene-SO3H, -CH2N(C 1-6 alkyl)C 1-6 Alkylene-SO3H, -CH2N(C 1-6 Alkylene-SO3H)2, -CH2N + (C 1-6 Alkylene-SO3H)3, -CH2N + (C 1- 6 Alkyl)-C 1-6 Alkylene-SO3H, -CH2N(C 1-6 Alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene-SO3H)3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene-SO3H)3, -CH2N(C 1-6 Alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkyl)3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkyl)3, -CH2N(C 1-6 Alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 Alkyl)3, -CH2N(C 1-6 Alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 Alkyl)2-CH2CO2H, -CH2N(C 1-6 Alkyl)-C 1-6 Alkylene-CO2H, -CH2N + (C 1-6 Alkyl)2-C 1-6 Alkylene-CO2H, Glucosyl, Galactosyl, Glucuronyl, Galacturonyl, -CH2N(C 1-6 Alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl, -CH2N(C 1-6 Alkyl)-C(=O)-(OCH2CH2) r -OC 1-6 Alkyl, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl, or a polyethylene glycol fragment containing 1-10 EO units (i.e., -(CH2CH2O) r -C 1-6 alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), or NOTA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residue), where r is an integer selected from 1 to 20; s is an integer selected from 1 to 20; Preferably, L is a divalent substituted or unsubstituted structural fragment selected from one or more of the following groups: C 1-6 alkylene, 6-10-membered aryl, 5-6-membered heteroaryl, -N(R')-, carbonyl, -O-, natural amino acids or unnatural amino acids and their analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), Lys(R'), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), wherein R' represents hydrogen, C 1-6 alkyl, glucosyl, galactosyl, glucuronyl, galacturonyl, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O) r -C 1-6 alkyl, -(CH2N(Me)-C(=O)) r -C 1-6 alkyl, or a polyethylene glycol fragment containing 1 - 10 EO units (i.e., -(CH2CH2O) r -C 1-6 alkyl), DOTA (1,4,7,10 - tetraazacyclododecane - 1,4,7,10 - tetraacetic acid residue), DOTAGA (1,4,7,10 - tetraazacyclododecane - 1,4,7,10 - tetraacetic acid, α - propionyl), or NOTA (1,4,7 - triazacyclononane - N,N',N'' - triacetic acid residue), wherein r is an integer selected from 1 - 20; s is an integer selected from 1 - 20; Preferably, L is a divalent substituted or unsubstituted structural fragment selected from one or more of the following groups: s is an integer selected from 1 - 20; Preferably, L is a divalent substituted or unsubstituted structural fragment selected from one or more of the following groups: s is an integer selected from 1 - 20, preferably s is an integer selected from 1 - 15, such as integers from 1 - 12, 3 - 12, 5 - 10, 8 - 10, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

5. The antibody-drug conjugate according to any one of claims 1-4, wherein, E is a single bond, substituted or unsubstituted -NH-CH2-, or a substituted or unsubstituted structural fragment selected from the following: Preferably, E is a single bond, substituted or unsubstituted -NH-CH2-, or Preferably, E is a single bond or a substituted or unsubstituted -NH-CH2-.

6. The antibody-drug conjugate according to any one of claims 1-5, wherein, The cytotoxic drug is selected from antimicrotubule agents, DNA intercalating agents, DNA topoisomerase inhibitors, RNA polymerase inhibitors, and gene transcription inhibitors; Preferably, the antimicrotubule agent is an auristatin compound, a maytansine compound, or an eribulin compound; the DNA intercalating agent is a pyrrolobenzodiazepine (PBD) compound, trabectedin, or lurbinectedin; the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, or lurbinectedin) or a topoisomerase II inhibitor (e.g., doxorubicin, adriamycin, PNU-159682 and its analogs, docaramine, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); the RNA polymerase inhibitor is α-amanitin; the gene transcription inhibitor is triptolide and its pharmaceutically acceptable salts, esters, and analogs; Preferably, the cytotoxic drug has a structure represented by the following formula (I): Among them, represents a single bond or no chemical bond; R1, R2, R3, R4, R6, and R7 are each independently selected from hydrogen, -CN, halogen, -OH, -NH2, -NH(C 1-6 alkyl), -N(C 1- 6alkyl)2, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3- to 6-membered heterocyclic group, C 6-10 aryl, and 5- to 10-membered heteroaryl; the alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl, and heteroaryl are optionally substituted by one or more substituents selected from -CN, halogen, -OH, -NH2, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3- to 6-membered heterocyclic group, C 6-10 aryl, and 5- to 10-membered heteroaryl; R5 is -(C=O)-D2-D3-X6, wherein D2 is absent or is -O-, -S-, -NR X -, wherein R X is selected from hydrogen or C 1-6 alkyl, and the C 1-6 alkyl is optionally substituted by one or more substituents selected from halogen, -OH, -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)2, -CN; D3 is C 1-6 alkylene, C 2-6 alkenylene or C 2-6 alkynylene; the alkylene, alkenylene and alkynylene are optionally substituted by one or more substituents selected from -CN, halogen, -OH, -NH2, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3-6 membered heterocyclic group, C 6-10 aryl and 5-10 membered heteroaryl; X6 is selected from hydrogen, halogen, -OH, C 1-6 alkoxy, -SH, -S-C 1-6 alkyl, -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)2, and the C 1-6 alkyl is optionally substituted by one or more substituents selected from halogen, -OH, -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)2, -CN, C 1-6 alkoxy; Y1 and Y2 are -C(R8)2-, and each R8 is independently selected from hydrogen, -CN, halogen, -OH, -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)2, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3- to 6-membered heterocyclic group, C 6-10 aryl and 5- to 10-membered heteroaryl, or two R8s attached to the same carbon atom together form =O; X1, X2, and X5 are each independently selected from substituted or unsubstituted C 1-6 alkylene, -O-, -NR Y -, or -S-; where R Y is, in each occurrence, independently selected from hydrogen or C 1-6 alkyl optionally substituted by one or more substituents selected from halogen, -OH, -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)2, -CN; When When representing a single bond, X4 is selected from substituted or unsubstituted C 1-6 alkylene, -O-, -NR Y -, or -S-; wherein R Y is independently selected from hydrogen or C optionally substituted by one or more substituents selected from halogen, -OH, -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)2, -CN in each occurrence 1-6 alkyl; When When there is no chemical bond, X4 is selected from substituted or unsubstituted C 1-6 alkyl, -OR Y , -N(R Y )2 or -S-; where R Y , each time it appears, is independently selected from hydrogen or C 1-6 alkyl optionally substituted by one or more substituents selected from halogen, -OH, -NH2, -NH(C 1- alkyl), -N(C 1-6 6 alkyl)2, -CN; X3 is selected from CR Z or N, wherein R Z is selected from hydrogen or C 1-6 alkyl optionally substituted by one or more substituents selected from halogen, -OH, -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)2, -CN; Further preferably, R1, R2, R3, R4, R6, and R7 are each independently selected from hydrogen, -CN, halogen, -OH, -NH2, -NH(C 1- 6-alkyl), -N(C 1-6 alkyl)2, C 1-6 alkyl, and C 1-6 alkoxy; the alkyl and alkoxy are optionally substituted by one or more substituents selected from -CN, halogen, -OH, -NH2, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3- to 6-membered heterocyclic group, C 6- 10 aryl, and 5- to 10-membered heteroaryl. More preferably, R1, R2, R3, R4, R6, and R7 are each independently selected from hydrogen, -OH, and C 1-6 alkoxy; the alkoxy is optionally substituted by one or more substituents selected from -CN, halogen, -OH, -NH2, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3- to 6-membered heterocyclic group, C 6-10 aryl, and 5- to 10-membered heteroaryl; Further preferably, R1, R2, R3, R4, R6 and R7 are each independently selected from hydrogen, -OH, C 1-6 alkyl and C 1-6 alkoxy; Further preferably, R1, R2, R3, R4, R6, and R7 are each independently selected from hydrogen, -OH, and C 1-6 alkoxy groups; Further preferably, R2, R3, and R4 are all -OH; More preferably, R1 and R7 are each independently selected from C 1-6 alkoxy groups such as methoxy, ethoxy, n-propoxy or isopropoxy; Further preferably, R1 and R7 are both methoxy; More preferably, R6 is selected from C 1-6 alkyl groups, such as methyl, ethyl, n-propyl or isopropyl; Further preferably, R6 is methyl; Further preferably, R1 and R7 are both methoxy; R2, R3, and R4 are all -OH; R6 is methyl; Further preferably, R5 is -(C=O)-D2-D3-X6, wherein D2 is -O-, -S-, -NR X -, wherein R X is selected from hydrogen or C 1-6 alkyl; D3 is C 1-6 alkylene; the alkylene is optionally substituted by one or more substituents selected from -CN, halogen, -OH, -NH2, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3-6 membered heterocyclic group, C 6-10 aryl and 5-10 membered heteroaryl; X6 is selected from -OH, C 1-6 alkoxy, -SH, -S-C 1-6 alkyl, -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)2, the C 1-6 alkyl is optionally substituted by one or more substituents selected from halogen, -OH, -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)2, -CN, C 1-6 alkoxy; More preferably, R5 is -(C=O)-D2-D3-X6, wherein D2 is -O-, -S-, -NR X -, wherein R X is selected from hydrogen or C 1-6 alkyl; D3 is C 1-6 alkylene; X6 is selected from -OH, C 1-6 alkoxy, -SH, -S-C 1-6 alkyl, -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)2; More preferably, R5 is -(C=O)-D2-D3-X6, wherein D2 is -O-, -S- or -NR X -, wherein R X is selected from hydrogen, methyl and ethyl; D3 is selected from methylene, ethylene, -(CH2)3, -CH2-CH(CH3)- and -CH2-C(CH3)2-; X6 is selected from -OH, methoxy, ethoxy, -SH, -S-CH3, -NH2, -NH(CH3) and -N(CH3)2; Further preferably, R5 is -(C=O)-D2-D3-X6, wherein D2 is selected from -O-, -S-, -NH-, and -N(CH3)-; D3 is selected from methylene, ethylene, -(CH2)3, -CH2-CH(CH3)-, and -CH2-C(CH3)2-; X6 is selected from -OH, methoxy, ethoxy, -SH, -S-CH3, -NH2, -NH(CH3), and -N(CH3)2; More preferably, R5 is -(C=O)-D2-D3-X6, where D2 is -O-, -S-, -NR X -, where R X is selected from hydrogen or C 1-3 alkyl; D3 is C 1-6 alkylene; X6 is selected from -OH, -SH, -NH2; Further preferably, R5 is selected from: More preferably, Y1 and Y2 are -C(R8)2-, and each R8 is independently selected from hydrogen, -CN, halogen, -OH, -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)2, C 1-6 alkyl, C 1-6 alkoxy, or two R8s attached to the same carbon atom together form =O; Preferably, Y1, Y2 are -(C=O)-; Further preferably, X1, X2 and X5 are each independently selected from substituted or unsubstituted C 1-6 alkylene, -O- or -NR Y ; wherein R Y , each occurrence of which is independently selected from hydrogen or C 1-6 alkyl optionally substituted by one or more substituents selected from halogen, -OH, -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)2, -CN; More preferably, X1, X2, X4, and X5 are all -O-; Further preferably, when When representing a single bond, X4 is selected from substituted or unsubstituted C 1-6 alkylene, -O-; preferably, X4 is selected from -O-; Further preferably, when When there is no chemical bond, X4 is selected from substituted or unsubstituted C 1-6 alkyl, -OR Y ; wherein R Y is independently selected from hydrogen or C optionally substituted by one or more substituents selected from halogen, -OH, -NH2, -NH(C 1-6 alkyl), -N(C 1- 6alkyl)2, -CN each time it appears; preferably, X4 is selected from -OH; 1-6 ​ More preferably, X3 is selected from CR Z or N, wherein R Z is selected from hydrogen or C 1-6 alkyl optionally substituted by one or more substituents selected from halogen, -OH, -NH2, -CN; Further preferably, X3 is N; Further preferably, the cytotoxic drug has a structure shown in the following formula (III)-1 or (III)-2: Further preferably, the cytotoxic drug has a structure shown in the following formula (IV)-1 or (IV)-2: Preferably, the cytotoxic drug is selected from the following compounds or their isotope-labeled compounds: Preferably, the cytotoxic drug is linked to E in the antibody-drug conjugate through its -OH, primary amino group, secondary amino group, or tertiary amino group.

7. The antibody-drug conjugate according to any one of claims 1-6, wherein, -M-LED can be obtained through the following structures: K-1 to K-18, K'-17 to K'-18, preferably through the following structure K-1 to K-18 by means of a substitution reaction (such as removing structures such as pentafluorophenoxy groups thereon): Wherein, n is selected from integers of 1 - 20, preferably n is selected from integers of 1 - 15, such as integers of 1 - 12, 3 - 12, 5 - 10, 8 - 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10.

8. The antibody-drug conjugate according to any one of claims 1-7, wherein, The antibody or its antigen-binding fragment comprises: (1) The following heavy-chain variable region (VH) and / or light-chain variable region (VL): (1a) The heavy-chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with a sequence of SEQ ID NO:5 or its variant, CDR-H2 with a sequence of SEQ ID NO:6 or its variant, CDR-H3 with a sequence of SEQ ID NO:7 or its variant; and / or, the light-chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with a sequence of SEQ ID NO:8 or its variant, CDR-L2 with a sequence of SEQ ID NO:9 or its variant, CDR-L3 with a sequence of SEQ ID NO:10 or its variant; or, (1b) The heavy-chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with a sequence of SEQ ID NO:20 or its variant, CDR-H2 with a sequence of SEQ ID NO:21 or its variant, CDR-H3 with a sequence of SEQ ID NO:22 or its variant; and / or, the light-chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with a sequence of SEQ ID NO:23 or its variant, CDR-L2 with a sequence of SEQ ID NO:24 or its variant, CDR-L3 with a sequence of SEQ ID NO:25 or its variant; Wherein, the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution; Or, (2) The following heavy-chain variable region (VH) and / or light-chain variable region (VL): (2a) The heavy-chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with a sequence of SEQ ID NO:18 or its variant, CDR-H2 with a sequence of SEQ ID NO:19 or its variant, CDR-H3 with a sequence of SEQ ID NO:7 or its variant; and / or, the light-chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with a sequence of SEQ ID NO:8 or its variant, CDR-L2 with a sequence of SEQ ID NO:9 or its variant, CDR-L3 with a sequence of SEQ ID NO:10 or its variant; or, (2b) The heavy chain variable region (VH) contains the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO: 33 or its variant, CDR-H2 with the sequence of SEQ ID NO: 34 or its variant, CDR-H3 with the sequence of SEQ ID NO: 22 or its variant; and / or, the light chain variable region (VL) contains the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO: 23 or its variant, CDR-L2 with the sequence of SEQ ID NO: 24 or its variant, CDR-L3 with the sequence of SEQ ID NO: 25 or its variant; Among them, the variant described in any one of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared with the sequence it is derived from, or the variant has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared with the sequence it is derived from; preferably, the substitution is a conservative substitution; Or, (3) The following heavy chain variable region (VH) and / or light chain variable region (VL): (3a) The heavy chain variable region (VH) contains the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO: 11 or its variant, CDR-H2 with the sequence of SEQ ID NO: 12 or its variant, CDR-H3 with the sequence of SEQ ID NO: 7 or its variant; and / or, the light chain variable region (VL) contains the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO: 8 or its variant, CDR-L2 with the sequence of SEQ ID NO: 9 or its variant, CDR-L3 with the sequence of SEQ ID NO: 10 or its variant; or, (3b) The heavy chain variable region (VH) contains the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO: 26 or its variant, CDR-H2 with the sequence of SEQ ID NO: 27 or its variant, CDR-H3 with the sequence of SEQ ID NO: 22 or its variant; and / or, the light chain variable region (VL) contains the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO: 23 or its variant, CDR-L2 with the sequence of SEQ ID NO: 24 or its variant, CDR-L3 with the sequence of SEQ ID NO: 25 or its variant; Among them, the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, At least 98%, at least 99%, or 100% sequence identity, or the variant has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution; Or, (4) The following heavy chain variable region (VH) and / or light chain variable region (VL): (4a) A heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO:13 or its variant, CDR-H2 with the sequence of SEQ ID NO:14 or its variant, CDR-H3 with the sequence of SEQ ID NO:15 or its variant; and / or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO:16 or its variant, CDR-L2 with the sequence of SEQ ID NO:17 or its variant, CDR-L3 with the sequence of SEQ ID NO:10 or its variant; or, (4b) A heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO:28 or its variant, CDR-H2 with the sequence of SEQ ID NO:29 or its variant, CDR-H3 with the sequence of SEQ ID NO:30 or its variant; and / or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO:31 or its variant, CDR-L2 with the sequence of SEQ ID NO:32 or its variant, CDR-L3 with the sequence of SEQ ID NO:25 or its variant; Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution; Preferably, the antibody or its antigen-binding fragment comprises: (1) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system: (1a) The heavy chain variable region (VH) contains the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or its variant, CDR-H2 with the sequence of SEQ ID NO:6 or its variant, CDR-H3 with the sequence of SEQ ID NO:7 or its variant; and / or, the light chain variable region (VL) contains the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or its variant, CDR-L2 with the sequence of SEQ ID NO:9 or its variant, CDR-L3 with the sequence of SEQ ID NO:10 or its variant; or, (1b) The heavy chain variable region (VH) contains the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or its variant, CDR-H2 with the sequence of SEQ ID NO:21 or its variant, CDR-H3 with the sequence of SEQ ID NO:22 or its variant; and / or, the light chain variable region (VL) contains the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or its variant, CDR-L2 with the sequence of SEQ ID NO:24 or its variant, CDR-L3 with the sequence of SEQ ID NO:25 or its variant; Wherein, the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared with the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived; preferably, the substitution is a conservative substitution; Or, (2) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system: (2a) The heavy chain variable region (VH) contains the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO:18 or its variant, CDR-H2 with the sequence of SEQ ID NO:19 or its variant, CDR-H3 with the sequence of SEQ ID NO:7 or its variant; and / or, the light chain variable region (VL) contains the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or its variant, CDR-L2 with the sequence of SEQ ID NO:9 or its variant, CDR-L3 with the sequence of SEQ ID NO:10 or its variant; or, (2b) The heavy chain variable region (VH) contains the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO: 33 or its variant, CDR-H2 with the sequence of SEQ ID NO: 34 or its variant, CDR-H3 with the sequence of SEQ ID NO: 22 or its variant; and / or, the light chain variable region (VL) contains the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO: 23 or its variant, CDR-L2 with the sequence of SEQ ID NO: 24 or its variant, CDR-L3 with the sequence of SEQ ID NO: 25 or its variant; Among them, the variant described in any one of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared with the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived; preferably, the substitution is a conservative substitution; Or, (3) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system: (3a) The heavy chain variable region (VH) contains the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO: 11 or its variant, CDR-H2 with the sequence of SEQ ID NO: 12 or its variant, CDR-H3 with the sequence of SEQ ID NO: 7 or its variant; and / or, the light chain variable region (VL) contains the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO: 8 or its variant, CDR-L2 with the sequence of SEQ ID NO: 9 or its variant, CDR-L3 with the sequence of SEQ ID NO: 10 or its variant; or, (3b) The heavy chain variable region (VH) contains the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO: 26 or its variant, CDR-H2 with the sequence of SEQ ID NO: 27 or its variant, CDR-H3 with the sequence of SEQ ID NO: 22 or its variant; and / or, the light chain variable region (VL) contains the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO: 23 or its variant, CDR-L2 with the sequence of SEQ ID NO: 24 or its variant, CDR-L3 with the sequence of SEQ ID NO: 25 or its variant; Wherein, the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions, or additions (such as 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution; Or, (4) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system: (4a) A heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO:13 or its variant, CDR-H2 with the sequence of SEQ ID NO:14 or its variant, CDR-H3 with the sequence of SEQ ID NO:15 or its variant; and / or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO:16 or its variant, CDR-L2 with the sequence of SEQ ID NO:17 or its variant, CDR-L3 with the sequence of SEQ ID NO:10 or its variant; or, (4b) A heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO:28 or its variant, CDR-H2 with the sequence of SEQ ID NO:29 or its variant, CDR-H3 with the sequence of SEQ ID NO:30 or its variant; and / or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO:31 or its variant, CDR-L2 with the sequence of SEQ ID NO:32 or its variant, CDR-L3 with the sequence of SEQ ID NO:25 or its variant; Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions, or additions (such as 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution; Preferably, the antibody or its antigen-binding fragment comprises: (1) The following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system: (1a) The heavy chain variable region (VH) contains the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO:5, CDR-H2 with the sequence of SEQ ID NO:6, CDR-H3 with the sequence of SEQ ID NO:7; and, the light chain variable region (VL) contains the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO:8, CDR-L2 with the sequence of SEQ ID NO:9, CDR-L3 with the sequence of SEQ ID NO:10; or, (1b) The heavy chain variable region (VH) contains the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO:20, CDR-H2 with the sequence of SEQ ID NO:21, CDR-H3 with the sequence of SEQ ID NO:22; and, the light chain variable region (VL) contains the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO:23, CDR-L2 with the sequence of SEQ ID NO:24, CDR-L3 with the sequence of SEQ ID NO:25; Or, (2) The following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system: (2a) The heavy chain variable region (VH) contains the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO:18, CDR-H2 with the sequence of SEQ ID NO:19, CDR-H3 with the sequence of SEQ ID NO:7; and, the light chain variable region (VL) contains the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO:8, CDR-L2 with the sequence of SEQ ID NO:9, CDR-L3 with the sequence of SEQ ID NO:10; or, (2b) The heavy chain variable region (VH) contains the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO:33, CDR-H2 with the sequence of SEQ ID NO:34, CDR-H3 with the sequence of SEQ ID NO:22; and, the light chain variable region (VL) contains the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO:23, CDR-L2 with the sequence of SEQ ID NO:24, CDR-L3 with the sequence of SEQ ID NO:25; Or, (3) The following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system: (3a) The heavy chain variable region (VH) contains the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11, CDR-H2 with the sequence of SEQ ID NO:12, CDR-H3 with the sequence of SEQ ID NO:7; and, the light chain variable region (VL) contains the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8, CDR-L2 with the sequence of SEQ ID NO:9, CDR-L3 with the sequence of SEQ ID NO:10; or, (3b) The heavy chain variable region (VH) contains the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:26, CDR-H2 with the sequence of SEQ ID NO:27, CDR-H3 with the sequence of SEQ ID NO:22; and, the light chain variable region (VL) contains the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23, CDR-L2 with the sequence of SEQ ID NO:24, CDR-L3 with the sequence of SEQ ID NO:25; Or, (4) The following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system: (4a) The heavy chain variable region (VH) contains the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:13, CDR-H2 with the sequence of SEQ ID NO:14, CDR-H3 with the sequence of SEQ ID NO:15; and, the light chain variable region (VL) contains the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:16, CDR-L2 with the sequence of SEQ ID NO:17, CDR-L3 with the sequence of SEQ ID NO:10; or, (4b) The heavy chain variable region (VH) contains the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:28, CDR-H2 with the sequence of SEQ ID NO:29, CDR-H3 with the sequence of SEQ ID NO:30; and, the light chain variable region (VL) contains the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:31, CDR-L2 with the sequence of SEQ ID NO:32, CDR-L3 with the sequence of SEQ ID NO:

25.

9. The antibody-drug conjugate according to claim 1 or 8, wherein, The antibody or its antigen-binding fragment comprises: (a) VH shown in SEQ ID NO:1 or its variant, and / or, VL shown in SEQ ID NO:2 or its variant; or (b) VH shown in SEQ ID NO:3 or its variant, and / or, VL shown in SEQ ID NO:4 or its variant; Wherein, the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution; Preferably, the antibody or its antigen-binding fragment comprises: (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or (b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO:

4.

10. The antibody-drug conjugate according to claim 8 or 9, wherein, The antibody or its antigen-binding fragment further comprises: (a) The heavy chain constant region (CH) of human immunoglobulin or its variant, which has one or more amino acid substitutions, deletions, or additions (e.g., at most 20, at most 15, at most 10, or at most 5 amino acid substitutions, deletions, or additions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the wild-type sequence from which it is derived; and (b) The light chain constant region (CL) of human immunoglobulin or its variant, which has one or more amino acid substitutions, deletions, or additions (e.g., at most 20, at most 15, at most 10, or at most 5 amino acid substitutions, deletions, or additions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the wild-type sequence from which it is derived; Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as human IgG1 heavy chain constant region or human IgG4 heavy chain constant region; Preferably, the antibody or its antigen-binding fragment comprises the heavy chain constant region (CH) shown in SEQ ID NO: 35 or its variant, and the variant has at most 20 amino acid conservative substitutions (e.g., at most 15, at most 10, or at most 5 amino acid conservative substitutions; e.g., 1, 2, 3, 4, or 5 amino acid conservative substitutions) compared to SEQ ID NO: 35; Preferably, the antibody or its antigen-binding fragment comprises the light chain constant region (CL) shown in SEQ ID NO: 36 or its variant, and the variant has at most 20 amino acid conservative substitutions (e.g., at most 15, at most 10, or at most 5 amino acid conservative substitutions; e.g., 1, 2, 3, 4, or 5 amino acid conservative substitutions) compared to SEQ ID NO: 36; Preferably, the antibody or its antigen-binding fragment comprises the heavy chain constant region (CH) shown in SEQ ID NO: 35 and the light chain constant region (CL) shown in SEQ ID NO:

36.

11. The antibody-drug conjugate according to any one of claims 1 and 8-10, wherein, The antibody or its antigen-binding fragment comprises: (1) A heavy chain comprising the VH sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or (2) A heavy chain comprising the VH sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO:

36.

12. The antibody-drug conjugate according to any one of claims 1-11, wherein M is linked to a thiol group (-SH) or an amino group (-NH2) on the Ab.

13. The antibody-drug conjugate according to any one of claims 1-12, wherein, The antibody or its antigen-binding fragment is selected from the antibody or its antigen-binding fragment according to claim 11; -M-L-E-D is selected from the structural compounds shown in claim 7; x is from 1 to 10; preferably, x is from 1 to 8, or x is from 1 to 4.

14. The antibody-drug conjugate according to any one of claims 8-13, selected from: Among them, In each antibody-drug conjugate, HA represents an antibody or its antigen-binding fragment that specifically binds to the epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases. Preferably, the antibody or its antigen-binding fragment is as described in any one of claims 8-11; n is an integer selected from 1-20, preferably an integer selected from 1-15, such as an integer from 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10; Preferably, the antibody or its antigen-binding fragment comprises: (1) The following heavy chain variable region (VH) and / or light chain variable region (VL): (1a) A heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with a sequence of SEQ ID NO: 5 or its variant, CDR-H2 with a sequence of SEQ ID NO: 6 or its variant, CDR-H3 with a sequence of SEQ ID NO: 7 or its variant; and / or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with a sequence of SEQ ID NO: 8 or its variant, CDR-L2 with a sequence of SEQ ID NO: 9 or its variant, CDR-L3 with a sequence of SEQ ID NO: 10 or its variant; or, (1b) A heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with a sequence of SEQ ID NO: 20 or its variant, CDR-H2 with a sequence of SEQ ID NO: 21 or its variant, CDR-H3 with a sequence of SEQ ID NO: 22 or its variant; and / or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with a sequence of SEQ ID NO: 23 or its variant, CDR-L2 with a sequence of SEQ ID NO: 24 or its variant, CDR-L3 with a sequence of SEQ ID NO: 25 or its variant; Wherein, the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution; Or, (2) The following heavy chain variable region (VH) and / or light chain variable region (VL): (2a) A heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO:18 or its variant, CDR-H2 with the sequence of SEQ ID NO:19 or its variant, CDR-H3 with the sequence of SEQ ID NO:7 or its variant; and / or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or its variant, CDR-L2 with the sequence of SEQ ID NO:9 or its variant, CDR-L3 with the sequence of SEQ ID NO:10 or its variant; or, (2b) A heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO:33 or its variant, CDR-H2 with the sequence of SEQ ID NO:34 or its variant, CDR-H3 with the sequence of SEQ ID NO:22 or its variant; and / or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or its variant, CDR-L2 with the sequence of SEQ ID NO:24 or its variant, CDR-L3 with the sequence of SEQ ID NO:25 or its variant; Wherein, the variant described in any one of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution; Or, (3) The following heavy chain variable region (VH) and / or light chain variable region (VL): (3a) The heavy chain variable region (VH) contains the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or its variant, CDR-H2 with the sequence of SEQ ID NO:12 or its variant, CDR-H3 with the sequence of SEQ ID NO:7 or its variant; and / or, the light chain variable region (VL) contains the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or its variant, CDR-L2 with the sequence of SEQ ID NO:9 or its variant, CDR-L3 with the sequence of SEQ ID NO:10 or its variant; or, (3b) The heavy chain variable region (VH) contains the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO:26 or its variant, CDR-H2 with the sequence of SEQ ID NO:27 or its variant, CDR-H3 with the sequence of SEQ ID NO:22 or its variant; and / or, the light chain variable region (VL) contains the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or its variant, CDR-L2 with the sequence of SEQ ID NO:24 or its variant, CDR-L3 with the sequence of SEQ ID NO:25 or its variant; Wherein, the variant described in any one of (3a), (3b), (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared with the sequence it is derived from, or the variant has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared with the sequence it is derived from; preferably, the substitution is a conservative substitution; Or, (4) The following heavy chain variable region (VH) and / or light chain variable region (VL): (4a) The heavy chain variable region (VH) contains the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO:13 or its variant, CDR-H2 with the sequence of SEQ ID NO:14 or its variant, CDR-H3 with the sequence of SEQ ID NO:15 or its variant; and / or, the light chain variable region (VL) contains the following 3 CDRs: CDR-L1 with the sequence of SEQ ID NO:16 or its variant, CDR-L2 with the sequence of SEQ ID NO:17 or its variant, CDR-L3 with the sequence of SEQ ID NO:10 or its variant; or, (4b) comprises the following 3 heavy-chain variable regions (VH) of CDR: CDR-H1 with the sequence of SEQ ID NO:28 or its variant, CDR-H2 with the sequence of SEQ ID NO:29 or its variant, CDR-H3 with the sequence of SEQ ID NO:30 or its variant; and / or, comprises the following 3 light-chain variable regions (VL) of CDR: CDR-L1 with the sequence of SEQ ID NO:31 or its variant, CDR-L2 with the sequence of SEQ ID NO:32 or its variant, CDR-L3 with the sequence of SEQ ID NO:25 or its variant; Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared with the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived; preferably, the substitution is a conservative substitution; Preferably, the antibody or antigen-binding fragment: (1) comprises a heavy chain with the VH of the sequence shown in SEQ ID NO:1 and the heavy-chain constant region (CH) shown in SEQ ID NO:35, and, a light chain with the VL of the sequence shown in SEQ ID NO:2 and the light-chain constant region (CL) shown in SEQ ID NO:36; or (2) comprises a heavy chain with the VH of the sequence shown in SEQ ID NO:3 and the heavy-chain constant region (CH) shown in SEQ ID NO:35, and, a light chain with the VL of the sequence shown in SEQ ID NO:4 and the light-chain constant region (CL) shown in SEQ ID NO:36; Among them, Represents the specific connection mode of the thiol group in the antibody or its antigen-binding fragment with the linker; Represents the specific connection mode of the amino group in the antibody or its antigen-binding fragment with the linker; Preferably, the antibody-drug conjugate is selected from ADC K-1 to ADC K-18.

15. A composition comprising one or more antibody-drug conjugates according to any one of claims 1-14, wherein the composition has a drug-antibody ratio (DAR) of 1-10, such as: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10, preferably 3-8, such as, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 6.0-6.5, 6.0-7.0, 6.0-7.5, 6.0-8.0, 6.0-8.5, 6.5-7.0, 6.5-7.5, 6.5-8.0, 6.5-8.5, 7.0-7.5, 7.0-8.0 or 7.5-8.0; Alternatively, the DAR value of the composition is from about 1.0 to 6.0, such as from about 1.0 to 5.5, from about 1.0 to 5.0, from about 1.5 to 6.0, from about 1.5 to about 5.5, from about 1.5 to 5.0, from 2.0 to 5.5, from about 2.0 to about 5.0, such as about 1.0, about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.1, about 1.11, about 1.12, about 1.13, about 1.14, about 1.15, about 1.16, about 1.17, about 1.18, about 1.19, about 1.2, about 1.21, about 1.22, about 1.23, about 1.24, about 1.25, about 1.26, about 1.27, about 1.28, about 1.29, about 1.3, about 1.31, about 1.32, about 1.33, about 1.34, about 1.35, about 1.36, about 1.37, about 1.38, about 1.39, about 1.4, about 1.41, about 1.42, about 1.43, about 1.44, about 1.45, about 1.46, about 1.47, about 1.48, about 1.49, about 1.5, about 1.51, about 1.52, about 1.53, about 1.54, about 1.55, about 1.56, about 1.57, about 1.58, about 1.59, about 1.6, about 1.61, about 1.62, about 1.63, about 1.64, about 1.65, about 1.66, about 1.67, about 1.68, about 1.69, about 1.7, about 1.71, about 1.72, about 1.73, about 1.74, about 1.75, about 1.76, about 1.77, about 1.78, about 1.79, about 1.8, about 1.81, about 1.82, about 1.83, about 1.84, about 1.85, about 1.86, about 1.87, about 1.88, about 1.89, about 1.9, about 1.91, about 1.92, about 1.93, about 1.94, about 1.95, about 1.96, about 1.97, about 1.98, about 1.99, about 2.0, about 2.01, about 2.02, about 2.03, about 2.04, about 2.05, about 2.06, about 2.07, about 2.08, about 2.09, about 2.1, about 2.11, about 2.12, about 2.13, about 2.14, about 2.15, about 2.16, about 2.17, about 2.18, about 2.19, about 2.2, about 2.21, about 2.22, about 2.23, about 2.24, about 2.25, about 2.26, about 2.27, about 2.28, about 2.29, about 2.3, about 2.31, about 2.32, about 2.33, about 2.34, about 2.35, about 2.36, about 2.37, about 2.38, about 2.39, about 2.4, about 2.41, about 2.42, about 2.43, about 2.44, about 2.45, about 2.46, about 2.47, about 2.48, about 2.49, about 2.5, about 2.51, about 2.52, about 2.53, approximately 2.54, approximately 2.55, approximately 2.56, approximately 2.57, approximately 2.58, approximately 2.59, approximately 2.6, approximately 2.61, approximately 2.62, approximately 2.63, approximately 2.64, approximately 2.65, approximately 2.66, approximately 2.67, approximately 2.68, approximately 2.69, approximately 2.7, approximately 2.71, approximately 2.72, approximately 2.73, approximately 2.74, approximately 2.75, approximately 2.76, approximately 2.77, approximately 2.78, approximately 2.79, approximately 2.8, approximately 2.81, approximately 2.82, approximately 2.83, approximately 2.84, approximately 2.85, approximately 2.86, approximately 2.87, approximately 2.88, approximately 2.89, approximately 2.9, approximately 2.91, approximately 2.92, approximately 2.93, approximately 2.94, approximately 2.95, approximately 2.96, approximately 2.97, approximately 2.98, approximately 2.99, approximately 3.0, approximately 3.01, approximately 3.02, approximately 3.03, approximately 3.04, approximately 3.05, approximately 3.06, approximately 3.07, approximately 3.08, approximately 3.09, approximately 3.1, approximately 3.11, approximately 3.12, approximately 3.13, approximately 3.14, approximately 3.15, approximately 3.16, approximately 3.17, approximately 3.18, approximately 3.19, approximately 3.2, approximately 3.21, approximately 3.22, approximately 3.23, approximately 3.24, approximately 3.25, approximately 3.26, approximately 3.27, approximately 3.28, approximately 3.29, approximately 3.3, approximately 3.31, approximately 3.32, approximately 3.33, approximately 3.34, approximately 3.35, approximately 3.36, approximately 3.37, approximately 3.38, approximately 3.39, approximately 3.4, approximately 3.41, approximately 3.42, approximately 3.43, approximately 3.44, approximately 3.45, approximately 3.46, approximately 3.47, approximately 3.48, approximately 3.49, approximately 3.5, approximately 3.51, approximately 3.52, approximately 3.53, approximately 3.54, approximately 3.55, approximately 3.56, approximately 3.57, approximately 3.58, approximately 3.59, approximately 3.6, approximately 3.61, approximately 3.62, approximately 3.63, approximately 3.64, approximately 3.65, approximately 3.66, approximately 3.67, approximately 3.68, approximately 3.69, approximately 3.7, approximately 3.71, approximately 3.72, approximately 3.73, approximately 3.74, approximately 3.75, approximately 3.76, approximately 3.77, approximately 3.78, approximately 3.79, approximately 3.8, approximately 3.81, approximately 3.82, approximately 3.83, approximately 3.84, approximately 3.85, approximately 3.86, approximately 3.87, approximately 3.88, approximately 3.89,. About 3.9, about 3.91, about 3.92, about 3.93, about 3.94, about 3.95, about 3.96, about 3.97, about 3.98, about 3.99, about 4.0, about 4.01, about 4.02, about 4.03, about 4.04, about 4.05, about 4.06, about 4.07, about 4.08, about 4.09, about 4.1, about 4.11, about 4.12, about 4.13, about 4.14, about 4.15, about 4.16, about 4.17, about 4.18, about 4.19, about 4.2, about 4.21, about 4.22, about 4.23, about 4.24, about 4.25, about 4.26, about 4.27, about 4.28, about 4.29, about 4.3, about 4.31, about 4.32, about 4.33, about 4.34, about 4.35, about 4.36, about 4.37, about 4.38, about 4.39, about 4.4, about 4.41, about 4.42, about 4.43, about 4.44, about 4.45, about 4.46, about 4.47, about 4.48, about 4.49, about 4.5, about 4.51, about 4.52, about 4.53, about 4.54, about 4.55, about 4.56, about 4.57, about 4.58, about 4.59, about 4.6, about 4.61, about 4.62, about 4.63, about 4.64, about 4.65, about 4.66, about 4.67, about 4.68, about 4.69, about 4.7, about 4.71, about 4.72, about 4.73, about 4.74, about 4.75, about 4.76, about 4.77, about 4.78, about 4.79, about 4.8, about 4.81, about 4.82, about 4.83, about 4.84, about 4.85, about 4.86, about 4.87, about 4.88, about 4.89, about 4.9, about 4.91, about 4.92, about 4.93, about 4.94, about 4.95, about 4.96, about 4.97, about 4.98, about 4.99, about 5.

0.

16. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1-14, the composition according to claim 15, and one or more pharmaceutically acceptable excipients.

17. Use of the antibody-drug conjugate according to any one of claims 1-14, the composition according to claim 15, or the pharmaceutical composition according to claim 16 in the manufacture of a medicament for the treatment of Her2-expressing cancer.

18. The use according to claim 17, wherein the cancer is selected from solid tumors or hematological malignancies; for example, selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) and urothelial cancer.

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