Conjugate with targeting effect as well as preparation method and application thereof

Abstract

The invention discloses a conjugate with a targeting effect and a preparation method and application thereof, the conjugate has a structure shown in a formula Ab-[M-L-E-D] x, a drug is selected from a DNA topoisomerase inhibitor, and the prepared antibody drug conjugate has a better drug-antibody coupling ratio and has an excellent targeting killing effect on solid tumors such as gastric cancer, breast cancer, lung cancer and urothelial cancer.

Description

Conjugate with targeting effect and preparation method and use 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 202311265152.X and application date September 27, 2023, and the application with CN application number 202410173714.6 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 conjugate with a targeting effect, a preparation method thereof, and uses thereof. 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 use of the above-mentioned ADC drugs in the clinical treatment of Her2-expressing tumors, safety issues or drug resistance issues 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). Regarding the hydrophilicity of the linker, disitamab vedotin and trastuzumab deruxtecan utilize the highly hydrophobic valine-citrulline (Val-Cit) and glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), respectively. Literature reports indicate that the hydrophilicity of the linker significantly impacts the hydrophilicity of the ADC, which in turn affects the ADC's aggregation, pharmacokinetic properties, and toxicity (Chemical Linkers in Antibody-Drug Conjugates (ADCs), Drug Discovery Series No. 81, Chapter 3). Regarding the bioactive molecule that exerts its cancer cell-killing effect, trastuzumab emtansine utilizes the microtubule inhibitor DM1 as a cytotoxin, but when paired with a non-cleavable linker, this results in a weak bystander effect. Disitamab vedotin utilizes the auristatin-like toxin MMAE as a bioeffector molecule, which is susceptible to neurotoxicity accumulation after repeated use. Furthermore, both ADCs utilize non-site-specific random conjugation, resulting in 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 results showed that the conjugate had a relatively good drug-antibody coupling ratio, and the conjugate had excellent binding activity and proliferation inhibition on Her2-positive cells, and had a good targeted killing effect on Her2-positive tumors (such as 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 the structural fragment connecting 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, M is selected from the following substituted or unsubstituted structural fragments:

[0028] In some embodiments, M is selected from the following substituted or unsubstituted structural fragments:

[0029] 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, -NHCH(R')-C(=O)-, carbonyl, -O-, natural amino acid or non-natural amino acid and analogs thereof (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)), Glu(R'), 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-S er-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 (GGGGGG, SEQ ID NO: 45)), Wherein R' is composed of one or more of the following groups, including but not limited to hydrogen, C 1-6 Alkyl, C 1-6 Alkylene, amino, hydroxyl, carboxyl, acyl, -O-, -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, -C 1-6Alkylene-heterocycle, -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)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(C1-6 alkyl)-C(=O)-(OCH2CH2) r -OC 1-6 Alkyl, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl, 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), NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue), -C 1-6 Alkylene-N(C 1-6 Alkyl)-DOTA, -C 1-6 Alkyl-N(C 1-6 Alkyl)-DOTAGA, or -C 1-6 Alkyl-N(C 1-6 alkyl)-NOTA, wherein r is selected from an integer of 1-20; s is selected from an integer of 1-20.

[0030] 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, for example, r is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0031] In some embodiments, s is selected from an integer of 1-15, such as an integer of 1-12, 3-12, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0032] In some embodiments, s is represented by n.

[0033] In some embodiments, "-NOTA" refers to

[0034] In some embodiments, "-DOTA" refers to

[0035] In some embodiments, "-DOTAGA" refers to

[0036] 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)), 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)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 1-6 Alkyl, or polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH2CH2O) r -C 1-6alkyl), wherein r is selected from an integer of 1-20; s is selected from an integer of 1-20; 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, 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)), Wherein R' is composed of one or more of the following groups, including but not limited to hydrogen, C 1-6 Alkyl, C 1-6 Alkylene, amino, hydroxyl, carboxyl, acyl, -O-, 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 1-6Alkyl, 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.

[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, 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-L ys, 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, G ly-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 1-6Alkyl, -(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.

[0038] 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 1-20.

[0039] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following: C 1-6Alkylene, Carbonyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Cit, Val-Lys, A la-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 s is selected from an integer of 1-20.

[0040] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following groups: s is an integer selected from 1-20.

[0041] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following groups: 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, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0042] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following groups: s is an integer selected from 1-20.

[0043] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following groups: s is an integer selected from 1-20.

[0044] In some embodiments, L is formed by linking one or more substituted or unsubstituted structural fragments selected from the following Group I and one or more substituted or unsubstituted structural fragments selected from the following Group II:

[0045] Group I: wherein s is an integer selected from 1 to 20;

[0046] Group II:

[0047] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following:

[0048] In some embodiments, L contains the following structural fragment:

[0049] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following:

[0050] In some embodiments, L is selected from the following substituted or unsubstituted structural fragments:

[0051] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following groups: wherein 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, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0052] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following groups:

[0053] wherein 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, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably, s is selected from 5, 8, 10.

[0054] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following groups: wherein 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, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably, s is selected from 5, 8, 10.

[0055] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following groups:

[0056] wherein 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, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably, s is selected from 5, 8, 10.

[0057] In some embodiments, L contains the following structural fragment

[0058] In some embodiments, L is composed of a structural fragment of Group I linked to one or more substituted or unsubstituted structural fragments selected from Group II:

[0059] Group I

[0060] Group II consists of wherein 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, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably, s is selected from 5, 8, 10.

[0061] In some embodiments, Group II consists of wherein 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, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably, s is selected from 5, 8, 10.

[0062] In some embodiments, Group II consists of wherein 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, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably, s is selected from 5, 8, 10.

[0063] 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:

[0064] In some embodiments, E is a single bond, substituted or unsubstituted -NH-CH2- or

[0065] In some embodiments, E is substituted or unsubstituted -NH-CH2- or

[0066] In some embodiments, Selected from the following substituted or unsubstituted structures:

[0067] n is selected from an integer between 1 and 20. In some embodiments, n is selected from an integer between 1 and 15, such as an integer between 1 and 12, or an integer between 3 and 12, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0068] In some embodiments, Selected from the following substituted or unsubstituted structures:

[0069] n is selected from an integer from 1 to 20. In some embodiments, n is selected from an integer from 1 to 15, such as an integer from 1 to 12, 3 to 12, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0070] In some embodiments, Selected from the following substituted or unsubstituted structures:

[0071] n is selected from an integer from 1 to 20. In some embodiments, n is selected from an integer from 1 to 15, such as an integer from 1 to 12, 3 to 12, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0072] In some embodiments, Selected from the following substituted or unsubstituted structures:

[0073] s is selected from an integer from 1 to 20. In some embodiments, s is selected from an integer from 1 to 15, such as an integer from 1 to 12, 3 to 12, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0074] In some embodiments, Selected from the following substituted or unsubstituted structures: n and s are each independently selected from an integer of 1 to 20. In some embodiments, n and s are each independently selected from an integer of 1 to 15, such as an integer of 1 to 12, 3 to 12, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0075] In some embodiments, Selected from the following substituted or unsubstituted structures: n is selected from an integer of 1-20, preferably an integer of 1-15, such as an integer of 1-12, 3-12, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10.

[0076] In some embodiments, Selected from the following substituted or unsubstituted structures: n is selected from an integer of 1-20, such as an integer of 1-12, 3-12, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10.

[0077] In some embodiments, Selected from the following substituted or unsubstituted structures:

[0078] In some embodiments, Selected from the following substituted or unsubstituted structures: wherein n is selected from an integer of 1-20, such as an integer of 1-12, 3-12, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10.

[0079] In some embodiments, Selected from the following substituted or unsubstituted structures:

[0080] In some embodiments, the cytotoxic drug is selected from an anti-tubulin agent, a DNA intercalator, a DNA topoisomerase inhibitor, an RNA polymerase inhibitor, and a gene transcription inhibitor. In some embodiments, the tubulin inhibitor is an auristatin compound, a maytansine compound, or an eribulin compound. In some embodiments, the DNA intercalator is a pyrrolobenzodiazepine (PBD) compound, trabectedin, or rubectedin. In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, isitecan, topotecan, belotecan, rubitecan, diflomotecan, lurtotecan, karenitecin, gimatecan, namitecan, simmitecan, chimmitecan, silatecan, or elomotecan) 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 its pharmaceutically acceptable salts, esters, and analogs.

[0081] In some embodiments, the cytotoxic drug is selected from a topoisomerase I inhibitor (e.g., Camptothecin, Hydroxycamptothecin, 9-aminocamptothecin, SN-38, Irinotecan, Isotecan, Topotecan, Belotecan, Rubitecan, Diflomotecan, Lurtotecan, Karenitecin, Gimatecan, Namitecan, Simmitecan, Chimmitecan, Silatecan, or Elomotecan).

[0082] The cytotoxic drugs disclosed in this application generally contain a variety of functional groups, such as hydroxyl (-OH), carboxyl (-COOH), primary amino (-NH2), secondary amine (-NR1H), tertiary amine (-NR2R3), wherein R1, R2, and R3 here represent only non-hydrogen substituents on N, or sulfhydryl (-SH), and these functional groups can react with appropriate functional groups in the rest of the conjugate to achieve linkage.

[0083] 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.

[0084] In some embodiments, the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, isotope-labeled compounds, solvates, hydrates, isomers, or any crystalline forms or racemates thereof:

[0085] In some embodiments, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0086] In some embodiments, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0087] In some embodiments, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0088] In some embodiments, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0089] In some embodiments, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0090] In some embodiments, the cytotoxic drug is selected from the following formula III and formula IV

[0091] Wherein, R5, R6 are each independently selected from OH, -NH2, -NH(C 1-6 Alkyl), C 1-6 Alkyl and halogen; said C 1-6 The alkyl group is optionally further substituted with one or more radicals selected from halogen, hydroxy, C 1-6 Halogenated alkyl, C 3-6 substituted by a cycloalkyl substituent;

[0092] R7 is selected from H, -OH, -NH2, -NH(C 1-6 alkyl), and -NH-CO-(C 1-6 Alkylene)-OH; the C 1-6 The alkyl group is optionally further substituted with one or more radicals selected from halogen, hydroxy, C 1-6 Halogenated alkyl, C 3-6 substituted by a cycloalkyl substituent;

[0093] q is 1 or 2;

[0094] R8 and R9 are each independently selected from H, halogen and hydroxyl; or R4 and R5 are connected to the connected carbon atom to form a 5-6 membered oxygen-containing heterocyclic ring; the 5-6 membered oxygen-containing heterocyclic ring is optionally substituted with one or more selected from deuterium (D), halogen, hydroxyl, C 1-6 Halogenated alkyl, C 3-6 The cycloalkyl group is substituted with a substituent.

[0095] R 10 Selected from hydrogen or -C 1-4 Alkylene-NR a R b ;

[0096] R 11 Selected from C 1-6 Alkyl and -C 1-4 Alkylene-NR a R b ;and

[0097] where R a 、R b Each occurrence is independently selected from H, C 1-6 Alkyl, C 3-6 Cycloalkyl, -SO2-C1-6 Alkyl and -CO-C 1-6 Alkyl; the C 1-6 Alkyl and C 1-4 The alkylene group is optionally further substituted with one or more selected from halogen, hydroxy, C 1-6 Halogenated alkyl, C 3-6 The cycloalkyl group is substituted with a substituent.

[0098] In some embodiments, R5, R6 are each independently selected from OH, -NH2, C 1-4 Alkyl and halogen; said C 1- 4 alkyl is optionally further substituted with one or more selected from halogen, hydroxy, C 1-4 Halogenated alkyl, C 3-6 The cycloalkyl group is substituted with a substituent.

[0099] In some embodiments, R7 is selected from -H, -NH2, -NH(C 1-4 alkyl), and -NH-CO-(C 1-4 Alkylene)-OH; the C 1-4 The alkyl group is optionally further substituted with one or more radicals selected from halogen, hydroxy, C 1-4 Halogenated alkyl, C 3-6 The cycloalkyl group is substituted with a substituent.

[0100] In some embodiments, R a 、R b Each occurrence is independently selected from H, C 1-4 Alkyl, C 3-6 Cycloalkyl, -SO2-C 1-4 Alkyl and -CO-C 1-4 Alkyl; the C 1-4 The alkyl group is optionally further substituted with one or more selected from halogen, hydroxy, C 1-6 Halogenated alkyl, C 3-6 The cycloalkyl group is substituted with a substituent.

[0101] In some embodiments, R5, R6 are each independently selected from OH, -NH2, C 1-6 Alkyl and halogen;

[0102] R7 is selected from -H, -NH2, -NH(C 1-6 alkyl), and -NH-CO-(C 1-6 alkylene)-OH; and

[0103] q is 2.

[0104] In some embodiments, R5, R6 are each independently selected from OH, -NH2, C 1-4In some embodiments, R5, R6 are each independently selected from OH, -NH2, methyl, ethyl, n-propyl, isopropyl and halogen. In some embodiments, R5, R6 are each independently selected from OH, -NH2, methyl, fluorine and chlorine.

[0105] In some embodiments, R7 is selected from -H, -NH2, -NH(C 1-4 alkyl), and -NH-CO-(C 1-4 In some embodiments, R7 is selected from -H, -NH2, -NH(CH3), -NH(CH2CH3), -NH(CH(CH3)2), -NH-CO-CH2-OH, -NH-CO-CH2CH2-OH, -NH-CO-CH2(CH3)-OH. In some embodiments, R 7A Selected from -H, -NH2, and -NH-CO-CH2-OH.

[0106] In some embodiments, the structure of the cytotoxic drug fragment D is shown in the following formula III':

[0107] Among them, R 5A Selected from -O- or -N-; R 7A Selected from chemical bonds, -O-, -NH-, -N(C 1-6 alkyl)-, and -NH-CO-(C 1-6 Alkylene)-O-; the C 1-6 The alkyl group is optionally further substituted with one or more radicals selected from halogen, hydroxy, C 1-6 Halogenated alkyl, C 3-6 substituted by a cycloalkyl substituent;

[0108] R5 to R7 and q are as described above.

[0109] In some embodiments, R 7A Selected from chemical bonds, -O-, -NH-, -N(C 1-4 alkyl)-, and -NH-CO-(C 1-4 Alkylene)-O-; the C 1-4 The alkyl group is optionally further substituted with one or more radicals selected from halogen, hydroxy, C 1-4 Halogenated alkyl, C 3-6 In some embodiments, R 7Ais selected from the group consisting of a chemical bond, -O-, -NH-, -N(CH3)-, -N(CH2CH3)-, -N(CH(CH3)2)-, -NH-CO-CH2-O-, -NH-CO-CH2CH2-O-, -NH-CO-CH2(CH3)-O-. In some embodiments, R 7A Selected from -NH-, and -NH-CO-CH2-O-.

[0110] In some embodiments, D is selected from the following structures:

[0111] In some embodiments, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof: 1-1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-26, 1-27, 1-28, 1-29, 1-30, 1-31, 1-32, 1 -33, 1-34, 1-35, 1-36, 1-37, 1-38, 1-39, 2-1, 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 2-24, 2-25, 2-26, 2-27, 2-28, 2-29, 2-30, 2-31, 2-32, 2-3 3, 2-34, 2-35, 2-36, 2-37, 2-38, 2-39, 2-40, 2-41, 2-42, 2-43, 2-44, 2-45, 2-46, 2-47, 2-48, 2-49, 2-50, 2-51, 2-52, 2-53, 2-54, 2-55, 2-56, 2-57, 2-58, 2-59, 2-60, 2-61, 2-62, 2-63, 2-64, 2-65, 2-66, 2-67, 2-68, 2-69, 2-70, 2 -71, 2-72, 2-73, 2-74, 2-75, 2-76, 2-77, 2-78, 2-79, 2-80, 2-81, 2-82, 2-83, 2-84, 2-85, 2-86, 2-87, 2-88, 2-89, 2-90, 2-91, 2-92, 2-93, 2-94, 2-95, 2-96, 2-97, 2-98, 2-99, 2-100, 2-101, 2-102, 2-103, 3-1, 3-2, 3-3, 3-4, 3-5.

[0112] In some embodiments, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0113] In some embodiments, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof: 1-1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-26, 1-27, 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34, 1-35, 1-36, 1-37, 1-38, 1- 39, 2-1, 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 2-24, 2-25, 2-26, 2-27, 2-28, 2-29, 2-30, 2-31, 2-32, 2-33, 2-34, 2-35, 2-36, 2-37, 2-38, 2-39, 2-40, 2-41, 3-1, 3-2, 3-3, 3-4, 3-5.

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

[0115] 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.

[0116] In one aspect, the present application provides a compound or a pharmaceutically acceptable salt thereof having a structure shown in formula DEL-M', wherein:

[0117] M' is -M-Lg, wherein Lg is a leaving group for nucleophilic substitution reaction, and M is a structural fragment that binds to the antibody or antigen-binding fragment thereof;

[0118] L is the structural fragment connecting M and E;

[0119] E is a structural fragment connecting L and D;

[0120] D is a cytotoxic drug fragment.

[0121] 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.

[0122] 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.

[0123] In some embodiments, Lg is selected from halogen, substituted or unsubstituted C 1-6 Alkylsulfonyl (C 1-6alkyl-SO2-), halogenated phenoxy, hydroxyl (-OH), mercapto (-SH) or amino (-NH2).

[0124] In some embodiments, Lg is selected from halogen, substituted or unsubstituted methylsulfonyl, halophenoxy, hydroxyl (-OH), thiol (-SH), or amino (-NH2).

[0125] In some embodiments, Lg is selected from C 1-6 Alkylsulfonyl and halophenoxy.

[0126] In some embodiments, Lg is selected from methylsulfonyl or pentafluorophenoxy.

[0127] In some embodiments, the compound or a pharmaceutically acceptable salt thereof is selected from the following E-1 to E-50, F-1 to F-14, G-1 to G-26, and I-1 to I-50:

[0128] wherein n is selected from an integer of 1-20, such as an integer of 1-12, 3-12, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0129] In some embodiments, the present application provides a compound as shown below or a pharmaceutically acceptable salt thereof: E'-1:

[0130] wherein n is selected from an integer of 1-20, such as an integer of 1-12, 3-12, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0131] In some embodiments, a compound selected from E'-1 to E'-50, F'-1 to F'-3, F'-7 to F'-12, G'-1 to G'-4, I'-10 to I'-49, E-1 to E-50, F-1 to F-14, G-1 to G-26, I-1 to I-50, or a pharmaceutically acceptable salt thereof is provided, wherein n is selected from an integer of 1-20, for example, an integer of 1-12, 3-12, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0132] In some embodiments, a compound selected from E-1 to E-50, F-1 to F-14, G-1 to G-24, or a pharmaceutically acceptable salt thereof is provided, wherein n is selected from an integer of 1-20, such as an integer of 1-12, 3-12, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0133] In some embodiments, a compound selected from E-1 to E-50, F-1 to F-12 or a pharmaceutically acceptable salt thereof is provided.

[0134] In some embodiments, a compound selected from E-1 to E-50, I-1 to I-49 or a pharmaceutically acceptable salt thereof is provided.

[0135] In some embodiments, the aforementioned compounds or pharmaceutically acceptable salts thereof may be optionally substituted with one or more suitable substituents.

[0136] In some embodiments, the antibody drug conjugate of the present application has a structure shown in the formula Ab-[MLED]x, wherein:

[0137] 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;

[0138] x is 1-10, preferably, x is 1-4, more preferably, x is 2 or 4;

[0139] -MLED is formed from compounds E-1 to E-50, F-1 to F-14, G-1 to G-26, or I-1 to I-50 described above.

[0140] In some embodiments, the antibody drug conjugate of the present application has a structure shown in the formula Ab-[MLED]x, wherein:

[0141] 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;

[0142] x is 1-10, preferably, x is 1-4, more preferably, x is 4;

[0143] -MLED is formed by removing the methanesulfonyl group from the above-mentioned compounds E-1 to E-50, F-1 to F-14 or G-1 to G-26.

[0144] In some embodiments, the antibody drug conjugate of the present application has a structure shown in the formula Ab-[MLED]x, wherein:

[0145] 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;

[0146] x is 1-10, preferably, x is 1-4, more preferably, x is 2;

[0147] -MLED is formed by removing the pentafluorophenoxy group from the compounds I-1 to I-50 described above.

[0148] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0149] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL):

[0150] (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

[0151] (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;

[0152] 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;

[0153] or,

[0154] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0155] (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

[0156] (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;

[0157] 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;

[0158] or,

[0159] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0160] (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

[0161] (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;

[0162] wherein the variant described in any one of (3a) and (3b) 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;

[0163] or,

[0164] (4) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0165] (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

[0166] (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;

[0167] 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.

[0168] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0169] (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:

[0170] (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

[0171] (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;

[0172] 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;

[0173] or,

[0174] (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:

[0175] (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

[0176] (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;

[0177] 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;

[0178] or,

[0179] (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:

[0180] (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

[0181] (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;

[0182] wherein the variant described in any one of (3a) and (3b) 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;

[0183] or,

[0184] (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:

[0185] (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

[0186] (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;

[0187] 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.

[0188] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0189] (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:

[0190] (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 / or 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

[0191] (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 / or 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;

[0192] or,

[0193] (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:

[0194] (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 / or 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

[0195] (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 / or 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;

[0196] or,

[0197] (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:

[0198] (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 / or 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

[0199] (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 / or 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;

[0200] or,

[0201] (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:

[0202] (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 / or 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

[0203] (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 / or 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.

[0204] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0205] (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:

[0206] (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

[0207] (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;

[0208] or,

[0209] (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:

[0210] (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

[0211] (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;

[0212] or,

[0213] (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:

[0214] (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

[0215] (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;

[0216] or,

[0217] (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:

[0218] (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

[0219] (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.

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

[0221] (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

[0222] (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;

[0223] 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.

[0224] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0225] (a) VH shown in SEQ ID NO: 1, and / or VL shown in SEQ ID NO: 2; or

[0226] (b) VH represented by SEQ ID NO: 3, and / or VL represented by SEQ ID NO: 4.

[0227] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0228] (a) VH or a variant thereof shown in SEQ ID NO: 1, and VL or a variant thereof shown in SEQ ID NO: 2; or

[0229] (b) VH or a variant thereof shown in SEQ ID NO: 3, and VL or a variant thereof shown in SEQ ID NO: 4;

[0230] 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.

[0231] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0232] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0233] (b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4.

[0234] In some embodiments, the antibody or antigen-binding fragment thereof further comprises:

[0235] (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

[0236] (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.

[0237] 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.

[0238] 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).

[0239] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO:35.

[0240] 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).

[0241] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO:36.

[0242] 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.

[0243] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0244] (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

[0245] (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.

[0246] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0247] (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

[0248] (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.

[0249] 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.

[0250] As known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0251] 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.

[0252] 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.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] In some embodiments, M is linked to a sulfhydryl (—SH) or amino (—NH 2 ) group on Ab.

[0257] In some embodiments, M is linked to a sulfhydryl (—SH) group on Ab.

[0258] In some embodiments, in the presence of Ab-[MLED] xIn 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, Ab is as defined above, and x is 1-10.

[0259] In some embodiments, in the presence of Ab-[MLED] x In the antibody-drug conjugate of the structure shown, MLED is formed by the compounds shown by E'-1 to E'-50, F'-1 to F'-3, F'-7 to F'-12, G'-1 to G'-4, I'-10 to I'-49, E-1 to E-50, F-1 to F-14, G-1 to G-26, and I-1 to I-50, preferably by removing the -SO2Me or pentafluorophenoxy group of the compound, Ab is as defined above, and x is 1 to 10.

[0260] 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:

[0261] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0262] 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:

[0263] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0264] 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:

[0265] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0266] 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:

[0267] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0268] 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:

[0269] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0270] 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:

[0271] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0272] 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:

[0273] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0274] In some embodiments, the antibody drug conjugate is selected from ADC E-1 to ADC E-50, ADC F-1 to ADC F-14, ADC G-1 to G-26, and ADC I-1 to I-50 shown below:

[0275] 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, such as an integer of 1-12, 3-12, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

[0276] in, Or it indicates the specific connection mode between the sulfhydryl group in the antibody or antigen-binding fragment thereof and the M fragment; Indicates the specific connection method between the amino group in the antibody or its antigen-binding fragment and the M fragment.

[0277] In some embodiments, the antibody drug conjugate is selected from the group consisting of ADC E'-1 to ADC E'-50, ADC F'-1 to ADC F'-3, ADC F'-7 to ADC F'-12, ADC G'-1 to G'-4, and ADC I'-10 to I'-49 shown below:

[0278] 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, such as an integer of 1-12, 3-12, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

[0279] in, Or it indicates the specific connection mode between the sulfhydryl group in the antibody or antigen-binding fragment thereof and the M fragment; Indicates the specific connection method between the amino group in the antibody or its antigen-binding fragment and the M fragment.

[0280] In some embodiments, the antibody or antigen-binding fragment thereof is as defined above.

[0281] In some embodiments, HA in each antibody drug conjugate represents trastuzumab, pertuzumab, or an antigen-binding fragment thereof.

[0282] In some embodiments, HA in each antibody drug conjugate represents the following antibody or antigen-binding fragment:

[0283] (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

[0284] (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.

[0285] In some embodiments, x in the antibody drug conjugate represented by Ab-[MLED]x is 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.

[0286] In some embodiments, x in the antibody drug conjugate represented by Ab-[MLED]x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, x in the antibody drug conjugate represented by Ab-[MLED]x is about 2. In some embodiments, x in the antibody drug conjugate represented by Ab-[MLED]x is about 4.

[0287] The antibody-drug conjugates of the present invention are optionally substituted with one or more suitable substituents.

[0288] intermediates

[0289] 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:

[0290] in

[0291] PG1 is each independently H or a carboxyl protecting group, such as C 1-6Alkyl, allyl, benzyl, 2,4-dimethoxybenzyl, p-methoxybenzyl, methoxyethoxymethyl, pentafluorophenyl, 4-p-methylbenzyloxybenzyl;

[0292] PG2 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, or p-nitrobenzoyl;

[0293] PG3 is independently H or an amino protecting group, such as an alkoxycarbonyl amino protecting group, for example, benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methyl (or ethyl)oxycarbonyl; an acyl amino protecting group, for example, 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), and benzyl (Bn);

[0294] Lg is a leaving group, as defined above;

[0295] s is selected from an integer of 1-20, preferably an integer of 1-15, such as an integer of 1-12, 3-12, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10.

[0296] 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:

[0297] 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.

[0298] Synthesis method

[0299] The present invention provides a method for synthesizing the following compound:

[0300] The method comprises step A of subjecting the following compound to an oxidation reaction:

[0301] Wherein n is any one of the above items.

[0302] In some embodiments, the oxidation reaction is carried out in the presence of sodium periodate and / or ruthenium trichloride hydrate.

[0303] In some embodiments, n is 10.

[0304] In some embodiments, the method further comprises step B of obtaining compound INT-1-2' by N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methylglycine.

[0305] In some embodiments, step B is a solid phase synthesis method.

[0306] In some embodiments, step B includes the steps of resin preparation, coupling, deprotection, polypeptide cleavage and purification.

[0307] In some embodiments, the reaction materials of Step B are N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methylglycine and 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid.

[0308] In some embodiments, step B uses 2-CTC resin.

[0309] In some embodiments, the coupling is performed in the presence of HATU and / or DIPEA reagents.

[0310] In some embodiments, the deprotection is performed in the presence of a piperidine reagent.

[0311] The present invention provides a method for synthesizing the following compound A-26-3':

[0312] wherein R5 and R6 are as described above; and the method comprises the step of performing a deprotection reaction on the following compound:

[0313] In some embodiments, R5 is selected from methyl, and R6 is selected from Cl;

[0314] In some embodiments, the PG3 is selected from Fmoc.

[0315] In some embodiments, the method further comprises the step of preparing compound A-26-3' from compound 1-4' and compound A-26-1':

[0316] In some embodiments, the method is performed in the presence of HATU and / or DIPEA reagent.

[0317] Composition

[0318] 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.

[0319] For example, in any embodiment, the compositions described herein have a DAR of about 1 to about 10, or any subrange therebetween, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 or about 9 to 10.

[0320] In certain embodiments, the DAR of the ADC compositions described herein is about 3 to 9, e.g., about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 3.0 to 6.0, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5, about 4.0 to 4.5, about 4.0 to 5.0, about 4.0 to 5.5, about 4.0 to 6.0, about 4.0 to 6.5, about 4.0 to 7.0, about 4.0 to 8. .0, about 4.5 to 5.0, about 4.5 to 5.5, about 4.5 to 6.0, about 4.5 to 6.5, about 4.5 to 7.0, about 4.5 to 7.5 about 5.0 to 8.0, 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 8.5, about 7.0 to 7.5.

[0321] In certain embodiments, the DAR of the ADC compositions described herein is about 3.5-5.0, e.g., about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0.

[0322] 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, from 2.0 to 5.5, from about 2.0 to about 5.0, e.g., 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 .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 2.6 7, 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 2.94, 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 3.15, 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 3.3 .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, about 4.61, about 4.62, about 4.63, about 4.64, about 4.65 5, 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.

[0323] In certain embodiments, the DAR of the ADC compositions described herein is about 4.41, about 4.34, about 4.51, about 4.36, about 4.63, about 4.28, about 4.38, about 3.86, about 4.16, about 3.99, or about 2.45.

[0324] In certain embodiments, the DAR of the ADC compositions described herein is 4.41, 4.34, 4.51, 4.36, 4.63, 4.28, 4.38, 3.86, 4.16, 3.99, or 2.45.

[0325] Pharmaceutical composition

[0326] In another aspect, the present application provides a pharmaceutical composition comprising the antibody-drug conjugate or the composition as described above, and one or more pharmaceutical excipients.

[0327] The compounds or conjugates described herein are typically formulated in a unit injectable form 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.

[0328] application

[0329] The antibody drug conjugates, 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.

[0330] Therefore, the present application provides use of any of the above-mentioned antibody-drug conjugates, compositions, or pharmaceutical compositions containing the same in the preparation of drugs for treating Her2-expressing cancers.

[0331] At the same time, the present application provides the antibody-drug conjugate, composition, or pharmaceutical composition containing the same as described in any of the above items, and its use in a drug for treating Her2-expressing cancer.

[0332] 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, compositions, or pharmaceutical compositions containing the same to a subject in need thereof.

[0333] In some embodiments, the antibody drug conjugate, composition, or pharmaceutical composition containing the same is sufficient (e.g., in a subject):

[0334] (1) Inhibit the proliferation of cells (such as tumor cells);

[0335] (2) inhibit tumor growth;

[0336] (3) induce and / or increase antibody-dependent cellular cytotoxicity activity;

[0337] (4) inhibit signal transduction;

[0338] (5) prevention and / or treatment of cancer; or

[0339] (6) Any combination of (1) to (5) above.

[0340] In some embodiments, the cancer disease is selected from solid tumors or hematological malignancies; for example, selected from gastric cancer, breast cancer, lung cancer (eg, non-small cell lung cancer, in particular lung adenocarcinoma) and urothelial carcinoma.

[0341] 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.

[0342] definition

[0343] 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.

[0344] 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.

[0345] 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 readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0346] 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.

[0347] 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.

[0348] 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.

[0349] 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.

[0350] The term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0351] 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.

[0352] 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.

[0353] 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.

[0354] 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.

[0355] 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.

[0356] 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.

[0357] 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.

[0358] 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.

[0359] 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.

[0360] 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.

[0361] 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.

[0362] 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).

[0363] 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.

[0364] 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 an amino acid residue is substituted with an amino acid residue having a similar side chain, for example, a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (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).

[0365] 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.

[0366] 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.

[0367] 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.

[0368] 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).

[0369] 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.

[0370] 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.

[0371] The term "alkylene" refers to a group obtained by removing two hydrogen atoms from a straight or branched hydrocarbon group, for example, "C 1-6 Alkylene", "C 1-5 Alkylene", "C 1-4 Alkylene", "C 1-3 Specific examples include, but are not limited to, methylene, ethylene, propylene, butylene, etc.

[0372] The term "aryl" refers to an unsaturated carbon ring group having a conjugated π electron system, such as a "6-10 membered aryl group", and specific examples include but are not limited to phenyl and naphthyl.

[0373] The term "heteroaryl" refers to an unsaturated group having a conjugated π electron system, wherein at least one (e.g., 1, 2, 3, or 4) of the ring atoms is a heteroatom, such as N, O, and S, wherein the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized. For example, "5-12 membered heteroaryl", "5-11 membered heteroaryl", "5-10 membered heteroaryl", "5-9 membered heteroaryl", "5-6 membered heteroaryl", and specific examples include, but are not limited to, phenyl, naphthylfuryl, pyrrolyl, thienyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, indazolyl, indolyl, quinolinyl, and isoquinolinyl.

[0374] The term "heterocyclyl" refers to a saturated or partially unsaturated cyclic group consisting of ring atoms and at least one (e.g., 1, 2, 3, 4, or 5 heteroatoms) ring atom being a heteroatom, such as N, O, and S, wherein the nitrogen atom is optionally quaternized, the nitrogen and sulfur heteroatoms are optionally oxidized, and the carbon atom is optionally oxoed. The heterocyclyl includes a monocyclic, bicyclic, or polycyclic ring, including a spirocyclic, a paracyclic, or a bridged ring. The term "nitrogen-containing heterocyclyl" refers to a group in which at least one heteroatom is N, such as a "5-12 membered nitrogen-containing heterocyclyl," a "5-9 membered nitrogen-containing heterocyclyl," or a "9-12 membered nitrogen-containing heterocyclyl." Specific examples include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, and 1,2,3,4-tetrahydroquinolinyl.

[0375] 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).

[0376] 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 6-10 Aryl, 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. -C1-6 Alkyl-C(=O)-OC 1-6 alkyl).

[0377] The term "substituted" refers to the replacement of one or more (e.g., 1, 2, 3, 4, or 5) hydrogens on a 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 (sub)alkyl, C1-C6 halo (sub)alkyl, C1-C6 alkoxy, C2-C6 (sub)alkenyl, C2-C6 (sub)alkynyl, C3-C8 (sub)cycloalkyl, 3-8 member (sub)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.

[0378] 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.

[0379] 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

[0380] Figure 1: Efficacy of anti-human HER2 antibody-drug conjugates (ADCs) in the NCI-N87 subcutaneous tumor-bearing mouse model Figure 2: Changes in body weight of mice in each group in the NCI-N87 CDX model of human gastric cancer cells

[0381] Figure 3: Efficacy of anti-human HER2 antibody-drug conjugate ADC in NCI-N87 cell subcutaneous tumor-bearing mouse model

[0382] Figure 4: Efficacy of anti-human HER2 antibody-drug conjugate ADC in the NCI-N87 subcutaneous tumor-bearing mouse model Figure 5: Changes in body weight of mice in each group in the human gastric cancer cell NCI-N87 CDX model DETAILED DESCRIPTION

[0383] 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.

[0384] The information of the sequences involved in the present invention is described in the following table:

[0385] The abbreviations used in this document have the following meanings:

[0386] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0387] 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).

[0388] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the examples are shown below.

[0389] 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.

[0390] Mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0391] Preparation Example 1: Preparation of (R)-N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxybutanamide (1-36)

[0392] (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (30 mg, 52.64 μmol), (R)-3-hydroxybutyric acid (10.96 mg, 105.28 μmol), HATU (30.02 mg, 78.96 μmol) and DIPEA (20.41 mg, 157.93 μmol) were dissolved in DMF (2 mL). The reaction system was stirred at 25°C for 2 h. The reaction solution was prepared by HPLC and freeze-dried to obtain the title compound (17 mg, 31.42 μmol).

[0393] Its separation and purification method is as follows:

[0394] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0395] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0396] Its structural characterization data are as follows:

[0397] ESI-MS (m / z): 542.1 [M+H] +

[0398] 1H NMR (400MHz, DMSOd6) δ8.43 (d, J = 8.8 Hz, 1H), 8.06 (d, J = 10.4 Hz, 1H), 7.33 (s, 1H), 6. 54(s,1H), 5.63-5.55(m,1H), 5.43(s,2H), 5.27(d,J=18.8Hz,1H), 5.21(d,J=18.8Hz ,1H), 4.66(d,J=4.8Hz,1H), 4.09-3.98(m,1H), 3.32-3.26(m,2H), 2.32-2.25(m,1H) , 2.24-2.12(m,3H), 1.93-1.78(m,2H), 1.08(d,J=6.0Hz,3H), 0.87(t,J=7.2Hz,3H).

[0399] Preparation Example 2: Preparation of (S)-N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxybutanamide (1-39)

[0400] (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (30 mg, 0.053 mmol, trifluoroacetate) and (S)-3-hydroxybutyric acid (16.5 mg, 0.158 mmol) were dissolved in DMF (0.5 mL). HATU (40.0 mg, 0.105 mmol) and DIPEA (20.4 mg, 0.158 mmol) were then added and reacted at room temperature for 2 hours. The reaction mixture was directly purified by preparative HPLC and freeze-dried to give the title compound (13.5 mg, 0.024 mmol).

[0401] Its structural characterization data are as follows:

[0402] MS m / z(ESI):542.1[M+H] +

[0403] 1HNMR(400MHz,DMSOd6)δ8.44(d,J=8.8Hz,1H),8.09(d,J=10.4Hz,1H),7.34(s ,1H),6.56(m,1H),5.64-5.60(m,1H),5.44(s,2H),5.32-5.25(m,2H),4.63(d ,J=4.8Hz,1H),4.06-4.01(m,1H),3.31-3.26(m,2H),2.28-2.23(m,1H),2.20 -2.13(m,3H),1.90-1.82(m,2H),1.08(d,J=6.4Hz,3H),0.87(t,J=7.2Hz,3H).

[0404] Its separation and purification method is as follows:

[0405] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0406] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0407] 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):

[0408] 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)

[0409] Use standard solid phase synthesis methods:

[0410] 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.

[0411] 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 through at 20°C for 30 minutes. The resin was washed with DMF (30.0 mL x 5) before proceeding to the next step.

[0412] 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).

[0413] 4) Repeat steps 2 and 3 using the materials and coupling reagents numbered 2-10 in Table 1.

[0414] 5) The resin was then washed with DMF (30.0 mL×5), MeOH (30.0 mL×5), and then dried under vacuum.

[0415] Table 1

[0416] Peptide cleavage and purification:

[0417] 1) Add the cleavage solution (TFA / DCM, 1 / 100, v / v, 200.0 mL) to the flask containing the dried resin obtained in the solid phase synthesis 5) at room temperature, stir for 3 minutes, and filter. Repeat this step twice.

[0418] 2) After filtration, the filtrates were combined and concentrated.

[0419] 3) The crude product was purified by HPLC and freeze-dried to give the title compound (1117.1 mg, TFA salt).

[0420] Its structural characterization data are as follows:

[0421] ESI-MS (m / z): 1010.4 (M+H) + .

[0422] The purification method is as follows:

[0423] 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)

[0424] 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).

[0425] Its structural characterization data are as follows:

[0426] ESI-MS (m / z): 1074.4 (M+H) + .

[0427] 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-azanonane-29-oic acid (INT-2)

[0428] Step 1: Preparation of 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid methyl ester (INT-2-2)

[0429] 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.

[0430] Its structural characterization data are as follows:

[0431] ESI-MS (m / z): 385.1 [M+H] + .

[0432] Step 2: Preparation of 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid (INT-2-3)

[0433] 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.

[0434] Its structural characterization data are as follows:

[0435] ESI-MS (m / z): 371.1 [M+H] + .

[0436] 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)

[0437] 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 mixture was extracted. 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.

[0438] 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)

[0439] 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 and 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).

[0440] Its structural characterization data are as follows:

[0441] ESI-MS (m / z): 794.3 [M+H] + .

[0442] The purification method is as follows:

[0443] Chromatographic column: Phenomenex luna C18 (250mm*70mm*10μm)

[0444] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0445] 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)

[0446] 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).

[0447] Its structural characterization data are as follows:

[0448] ESI-MS (m / z): 858.3 [M+H] + .

[0449] Intermediate Example 3: Preparation of Allyl (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-3)

[0450] Step 1: Preparation of 2-hydroxymethyl-N-methyl-5-nitrobenzamide (INT-3-2)

[0451] Add 6-nitroisobenzofuran-1(3H)-one (50.0 g, 279 mmol) to a 2M solution of methylamine in tetrahydrofuran (500 mL), heat to 75°C, and stir for 5 hours. The reaction mixture was concentrated to obtain the crude title compound (58.0 g), which was used in the next step without purification.

[0452] Its structural characterization data are as follows:

[0453] ESI-MS (m / z): 211.0 [M+H] + .

[0454] Step 2: Preparation of (2-((methylamino)methyl)-4-nitrophenyl)methanol (INT-3-3)

[0455] Dissolve 2-hydroxymethyl-N-methyl-5-nitrobenzamide (25.0 g, 119 mmol) in tetrahydrofuran (500 mL). Cool the reaction mixture to 0°C, then add a 10M borane-dimethyl sulfide solution (89.3 mL) dropwise to the mixture. After the addition is complete, heat the mixture to 70°C and stir for 5 hours. Cool the mixture again to 0°C, then add a 2M hydrogen chloride-methanol solution (100 mL) dropwise to the reaction mixture. Heat the mixture to 65°C and stir for 12 hours. Filter the reaction mixture, and concentrate the filtrate to obtain the crude title compound (44.3 g), which is used in the next step without purification.

[0456] Its structural characterization data are as follows:

[0457] ESI-MS (m / z): 197.0 [M+H] + .

[0458] Step 3: Preparation of 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethanamine (INT-3-4)

[0459] Dissolve (2-((methylamino)methyl)-4-nitrophenyl)methanol (40.3 g, 205 mmol) in dichloromethane (800 mL), cool to 0°C, add imidazole (55.9 g, 822 mmol) and tert-butyldiphenylsilyl chloride (84.0 g, 308 mmol, 78.8 mL), return to 25°C, and continue stirring for 1 hour. Quench the reaction by adding water (200 mL) and extract with dichloromethane three times (300 mL x 3). The combined organic phases are washed with saturated sodium chloride (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purify on a silica gel column (dichloromethane / methanol = 20 / 1 to 5 / 1) and reconcentrate to obtain the title compound (59.7 g, 137 mmol).

[0460] Its structural characterization data are as follows:

[0461] ESI-MS (m / z): 435.1 [M+H] + .

[0462] Step 4: Preparation of allyl (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate (INT-3-5)

[0463] Dissolve 1-(2-(((tert-Butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethanamine (54.9 g, 126 mmol) in dichloromethane (550 mL). Cool to 0°C, add DIPEA (48.9 g, 379 mmol, 66.0 mL) and allyl chloroformate (30.5 g, 253 mmol, 26.8 mL), and return to 25°C. Stirring is continued for 1 hour. Quench the reaction system with water (300 mL). Extract with dichloromethane three times (200 mL x 3). Combine the organic phases, wash with saturated sodium chloride (300 mL), dry over anhydrous sodium sulfate, and filter and concentrate to obtain the crude product. Purify on a silica gel column (petroleum ether / ethyl acetate = 20 / 1 to 3 / 1) and concentrate again to obtain the title compound (65.5 g, 126 mmol).

[0464] Its structural characterization data are as follows:

[0465] ESI-MS (m / z): 519.1 [M+H] + .

[0466] Step 5: Preparation of allyl (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-3)

[0467] Allyl (2-(((tert-Butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate (62.8 g, 121 mmol) was dissolved in a mixed solvent of ethanol (300 mL) and water (300 mL). Iron powder (33.8 g, 605 mmol) and ammonium chloride (64.8 g, 1.21 mol) were added, and the mixture was heated to 80°C and stirred for 2 hours. The reaction solution was filtered, and the filtrate was extracted with dichloromethane three times (100 mL x 3). The organic phases were combined, washed with saturated aqueous sodium chloride (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (49.3 g).

[0468] Its structural characterization data are as follows:

[0469] ESI-MS (m / z): 511.7 [M+H] + .

[0470] Intermediate Example 4 (S)-2-amino-N-((S)-1-(((S)-1-)((2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropan-2-yl)amine)-1-propanamide (A-26-3)

[0471] Step 1:

[0472] Compound A-26-1 (657 mg, 1.22 mmol) and compound 1-4 (500 mg, 1.11 mmol) were dissolved in N,N-dimethylformamide (10 mL). HATU (630.67 mg, 1.66 mmol) and N,N-diisopropylethylamine (428 mg, 3.32 mmol) were then added and stirred at room temperature for 1 hour. After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain 700 mg of compound A-26-2.

[0473] The preparation method is as follows:

[0474] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0475] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0476] Step 2:

[0477] Compound A-26-2 (500 mg, 0.513 mmol) was dissolved in N,N-dimethylformamide (2 mL), and diethylamine (75.05 mg, 1.03 mmol) was added. The mixture was reacted at room temperature for 1 hour. After the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain 307 mg of compound A-26-3.

[0478] The preparation method is as follows:

[0479] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0480] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0481] Example 1: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,1,13-trimethyl-1,6,9,12,15-pentaoxo-3,18,21,24,27,30,33,36,39-nonyloxy-5,8,11,14-tetraazacyclotetradecane-41-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (E-5)

[0482] Step 1:

[0483] 3,5-Bis(2-methylsulfonylpyrimidin-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). HOBt (3.28 g, 24.3 mmol), EDCI (4.66 g, 24.3 mmol), and DIEA (4.19 g, 32.4 mmol, 5.64 mL) were added sequentially, and the mixture was stirred at 60°C for 2 hours. Water (100 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (60 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound E-5-3 (4.20 g, 4.14 mmol), which was used directly in the next step without purification.

[0484] Step 2:

[0485] 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-azanonane-29-oate (E-5-3, 3.60 g, 4.24 mmol) in dichloromethane (30 mL). Add TFA (15.3 g, 134 mmol, 10 mL) and stir at 25°C for 6 hours. Add water (60 mL) to the reaction mixture, and extract with ethyl acetate (40 mL x 3). The combined organic phases are dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product, which is purified by preparative HPLC and freeze-dried to obtain compound E-5-4 (2.93 g, 3.63 mmol).

[0486] The separation and purification method is as follows:

[0487] Chromatographic column: Phenomenex luna C18 (250mm*70mm*10m)

[0488] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0489] Step 3:

[0490] 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonane-29-oic acid (E-5-4, 148 mg, 186.41 μmol) was added to acetonitrile (15 mL) and water (7.5 mL), and then sodium periodate (398.71 mg, 1.86 mmol) and ruthenium trichloride hydrate (15.47 mg, 74.56 μmol) were added to the reaction system. The reaction was stirred at 25 ° C for 30 min and monitored by LC-MS. Water and EA were added for extraction and concentrated to obtain 155 mg of light yellow solid E-5-5 compound.

[0491] Step 4:

[0492] (S)-2-amino-N-((S)-1-(((S)-1-)((2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl (A-26-3, 27.91 mg, 34.97 μmol), E-5-5 (30 mg, 34.97 μmol), HATU (26.59 mg, 69.93 μmol), and DIPEA (22.60 mg, 174.84 μmol) were added to DMF (3 mL), and the reaction system was reacted at 25°C for 1 hour. The reaction solution was purified by preparative HPLC and freeze-dried to obtain 15 mg of the title compound.

[0493] Its structural characterization data are as follows:

[0494] ESI-MS (m / z): 1591.7 [M+H]+

[0495] The separation and purification method is as follows:

[0496] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0497] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0498] Example 2: 2,2',2"-(10-(2-((5-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amide)-2-((((1S,9S)-9-ethyl-5- (F-1)

[0499] Step 1: Preparation of 2,5-dioxopyrrolidin-1-yl 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoate (F-1-2): Dissolve 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid (0.6 g, 1.62 mmol) in THF (15 mL). Add N-hydroxysuccinimide (278.89 mg, 2.43 mmol) and then dicyclohexylcarbodiimide (0.4 g, 1.94 mmol). Stir at room temperature for 2 hours. After completion of the reaction, filter the mixture, collect the filtrate, and concentrate under reduced pressure to obtain the crude title compound (1.5 g, 3.42 mmol), which is used directly in the next reaction without purification.

[0500] Step 2: Preparation of 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosane-20-carboxylic acid (F-1-3)

[0501] Dissolve 2,5-dioxopyrrolidin-1-yl 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoate (331.5 mg, 0.99 mmol) in DMF (4 mL). Add 1-amino-3,6,9,12,15-pentaoxaoctadecane-18-carboxylic acid (0.3 g, 0.67 mmol) and DIPEA (588.24 mg, 4.56 mol). Stir at room temperature for 2 hours. After completion of the reaction, flash column chromatography (C18, water / acetonitrile = 0.5) afforded the title compound (315.50 mg, 0.44 mmol).

[0502] Its structural characterization data are as follows:

[0503] MS m / z(ESI):662.2[M+H]+

[0504] Step 3: Preparation of 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosane-20-carboxylic acid (F-1-4)

[0505] Dissolve 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosane-20-carboxylic acid (315.50 mg, 0.44 mmol) in acetonitrile (3 mL) and water (1.5 mL). Add sodium periodate (470.50 mg, 2.20 mmol) and ruthenium trichloride monohydrate (9.11 mg, 0.04 mmol). Stir at room temperature for 0.5 hour. After completion of the reaction, the product was purified by flash column chromatography (C18, water / acetonitrile = 2 / 1) and freeze-dried to obtain the title compound (115.50 mg, 0.16 mmol).

[0506] Its structural characterization data are as follows:

[0507] MS m / z(ESI):726.1[M+H] +

[0508] Step 4: Preparation of (1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosanoyl)-L-valine (F-1-5)

[0509] 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosane-20-carboxylic acid (115.50 mg, 0.16 mmol) was dissolved in DMF (4 mL). 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (122.50 mg, 0.32 mmol) and DIPEA (62.0 mg, 0.48 mmol) were added. After stirring at room temperature for 0.5 hour, L-valine (50.0 mg, 0.48 mmol) was added. The reaction was stirred at room temperature for 15 hours. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 2 / 1) and freeze-dried to obtain the title compound (30.50 mg, 0.04 mmol).

[0510] Its structural characterization data are as follows:

[0511] MS m / z(ESI):825.2[M+H] +

[0512] Step 5: Preparation of allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (F-1-6) Allyl (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (150.0 mg, 0.31 mmol), (((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (F-1-6) )carbonyl)-L-alanine (115.1 mg, 0.37 mmol) was dissolved in DCM (8 mL) and MeOH (2 mL), and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (229.7 mg, 0.93 mmol) was added. The mixture was stirred at room temperature for 15 hours and concentrated under reduced pressure to give a 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 (194.2 mg, 0.25 mmol).

[0513] Its structural characterization data are as follows:

[0514] MS m / z(ESI):782.2[M+H] +

[0515] Step 6: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (F-1-7)

[0516] Allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (194.2 mg, 0.25 mmol) was dissolved in DMF (5 mL), and pyridine hydrofluoride (390.2 mg, 3.93 mmol) was added. The mixture was stirred at room temperature for 15 hours. After completion of the reaction, 20 mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (10 mL x 3), washed with 10 mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude title compound. The crude product was purified by flash column chromatography (C18, water / acetonitrile = 2 / 1) and freeze-dried to obtain the title compound (109.3 mg, 0.21 mmol).

[0517] Its structural characterization data are as follows:

[0518] MS m / z(ESI):566.1[M+Na] +

[0519] Step 7: Preparation of allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (F-1-8)

[0520] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (109.3 mg, 0.21 mmol) was dissolved in DMF (5 mL), and DIPEA (81.3 mg, 0.63 mmol) and p-nitrophenyl chloroformate (50.8 mg, 0.25 mmol) were added. The mixture was stirred at room temperature for 2 hours. After completion of the reaction, the product was purified by flash column chromatography (C18, water / acetonitrile = 2 / 1) and freeze-dried to obtain the title compound (134.50 mg, 0.19 mmol).

[0521] Its structural characterization data are as follows:

[0522] MS m / z(ESI):731.2[M+Na] +

[0523] Step 8: Preparation of allyl(5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizinyl[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (F-1-9)

[0524] Allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (134.50 mg, 0.19 mmol), (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13 H-Benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (165.50 mg, 0.38 mmol) was dissolved in DMF (5 mL), and 1-hydroxybenzotriazole (30.80 mg, 0.23 mmol), pyridine (16.5 mg, 0.21 mmol), and DIPEA (73.5 mg, 0.57 mmol) were added. The mixture was stirred at room temperature for 2 hours. After completion of the reaction, the product was purified by flash column chromatography (C18, water / acetonitrile = 0.7) and freeze-dried to obtain the title compound (114.70 mg, 0.12 mmol).

[0525] Its structural characterization data are as follows:

[0526] MS m / z(ESI):1005.2[M+H] +

[0527] Step 9: Preparation of allyl (5-((S)-2-aminopropionamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (F-1-10)

[0528] Allyl(5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (114.70 mg, 0.12 mmol) was dissolved in DMF (3 mL), diethylamine (87.8 mg, 1.2 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the residue was purified by flash column chromatography (C18, water / acetonitrile = 0.7) and freeze-dried to obtain the title compound (62.6 mg, 0.08 mmol).

[0529] Its structural characterization data are as follows:

[0530] MS m / z(ESI):783.1[M+H] +

[0531] Step 10: Preparation of allyl (5-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amido)-2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (F-1-11)

[0532] (1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosanoyl)-L-valine (30.50 mg, 0.04 mmol), allyl(5-((S)-2-aminopropionamido)-2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2- b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)methyl)carbamate (39.10 mg, 0.05 mmol) was dissolved in DMF (4 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (22.80 mg, 0.06 mmol) and DIPEA (15.5 mg, 0.12 mmol) were added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 0.6) and freeze-dried to obtain the title compound (31.7 mg, 0.02 mmol).

[0533] Its structural characterization data are as follows:

[0534] MS m / z(ESI):1589.3[M+H] +

[0535] Step 11: Preparation of 4-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amido)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (F-1-12)

[0536] Allyl (5-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amido)-2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-

[0366] To the solution of (4-(2-(2-hydroxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)methyl)carbamate (31.7 mg, 0.02 mmol) in DMF (2 mL) was added tetrakistriphenylphosphine palladium (5.2 mg, 4.2 μmol), 15 μL formic acid, and 30 μL N-methylmorpholine. The mixture was stirred at room temperature under nitrogen for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 0.6) and freeze-dried to give the title compound (12.1 mg, 0.008 mmol).

[0537] Its structural characterization data are as follows:

[0538] MS m / z(ESI):1505.2[M+H] +

[0539] Step 12: 2,2',2"-(10-(2-((5-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amide)-2-((((1S,9S)-9-ethyl-5-fluoro Preparation of (9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (F-1)

[0540] 4-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amido)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (12.1 mg, 0.008 mmol) was dissolved in DMF (1 mL), and DIPEA (5.2 mg, 0.04 mmol) was added. 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (20.1 mg, 0.04 mmol) was added. After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (1.0 mg, 0.5 μmol).

[0541] Its structural characterization data are as follows:

[0542] MS m / z(ESI):1892.2[M+H] +

[0543] The preparation method is as follows:

[0544] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0545] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0546] Example 3: Preparation of N-((20S,23S)-24-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-20-isopropyl-23-methyl-18,21,24-trioxo-3,6,9,12,15-pentaoxa-19,22-diazatetracosyl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (G-1)

[0547] F-1-4 (25.3 mg, 0.03 mmol), (S)-2-amino-N-((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro)-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (20.0 mg) were added. , 0.03 mmol) was dissolved in DMF (2 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (19.9 mg, 0.05 mmol) and DIPEA (13.5 mg, 0.1 mmol) were added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (26.9 mg, 0.02 mmol).

[0548] Its structural characterization data are as follows:

[0549] MS m / z(ESI):1281.3[M+H] +

[0550] The preparation method is as follows:

[0551] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0552] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0553] Example 4: Preparation of N-((29S,32S)-33-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-29-isopropyl-32-methyl-27,30,33-trioxo-3,6,9,12,15,18,21,24-octaoxa-28,31-diazatriacontanoyl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (G-2)

[0554] E-5-5 (25.7 mg, 0.03 mmol) and G-1-1 (20.0 mg, 0.03 mmol) were dissolved in DMF (2 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (19.9 mg, 0.05 mmol) and DIPEA (13.5 mg, 0.1 mmol) were added. The mixture 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 (24.5 mg, 0.02 mmol).

[0555] Its structural characterization data are as follows:

[0556] MS m / z(ESI):1413.3[M+H] +

[0557] The preparation method is as follows:

[0558] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0559] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0560] Example 5: Preparation of N-(2-((2-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (G-5)

[0561] Step 1: Preparation of (9H-fluoren-9-yl)methyl (2-((2-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13)-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (G-5-1)

[0562] (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine (10.6 mg, 0.03 mmol) and G-1-1 (20.0 mg, 0.03 mmol) were dissolved in DMF (2 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (19.9 mg, 0.05 mmol) and DIPEA (13.5 mg, 0.1 mmol) were added. The mixture was stirred at room temperature for 1 hour. After completion, 10 mL of water was added, and a solid precipitated. The filter cake was filtered and the water was lyophilized to obtain the title compound (23.5 mg, 0.025 mmol).

[0563] Its structural characterization data are as follows:

[0564] MS m / z(ESI):910.3[M+H] +

[0565] Step 2: Preparation of (S)-2-(2-(2-aminoacetamido)acetamido)-N-((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropane-2-yl)-3-methylbutanamide (G-5-2)

[0566] Dissolve G-5-1 (23.5 mg, 0.025 mmol) in DMF (2 mL), add diethylamine (18.3 mg, 0.25 mmol), and stir at room temperature for 1 hour. After completion of the reaction, purify by flash column chromatography (C18, water / acetonitrile = 0.5) and freeze-dry to obtain the title compound (8.2 mg, 0.012 mmol).

[0567] Its structural characterization data are as follows:

[0568] MS m / z(ESI):688.2[M+H] +

[0569] Step 3: Preparation of N-(2-((2-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (G-5)

[0570] G-5-2 (8.2 mg, 0.012 mmol) was dissolved in DMF (1 mL), and DIPEA (7.8 mg, 0.06 mmol) was added, followed by 2,5-dioxopyrrolidin-1-yl 3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoate (19.1 mg, 0.036 mmol). After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (1.45 mg, 1.3 μmol).

[0571] Its structural characterization data are as follows:

[0572] MS m / z(ESI):1104.2[M+H] +

[0573] The preparation method is as follows:

[0574] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0575] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0576] Example 6: N-((31S,34S)-35-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-31-isopropyl-3,6,9,12,15,18 Preparation of 2,5,8,11,14,17,20,23,26,29,32,35-dodecanoyl-3,6,9,12,15,18,21,24,27,30,33-undecaazapentatriacontyl)-N-methyl-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (G-3)

[0577] INT-1 (20.0 mg, 17.46 μmol) and G-1-1 (10.0 mg, 17.46 μmol) were dissolved in DMF (1 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (8.0 mg, 20.0 μmol) and DIPEA (6.8 mg, 52.39 μmol) were added. The mixture 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 (3.2 mg, 1.86 μmol).

[0578] Its structural characterization data are as follows:

[0579] MS m / z(ESI):1700.6[M+H] +

[0580] The preparation method is as follows:

[0581] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0582] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0583] Example 7: 2,2',2"-(10-(2-((5-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amido)-2-((((1S,9S)-5-chloro-9-ethyl Preparation of (9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (F-2)

[0584] Step 1: Preparation of allyl (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizinyl[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (F-2-1)

[0585] F-1-8 (200.0 mg, 0.28 mmol) and (1S,9S)-1-amino-9-ethyl-5-chloro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (253.0 mg, 0.56 mmol) were dissolved in DMF (6 mL). 1-Hydroxybenzotriazole (45.5 mg, 0.34 mmol), pyridine (24.5 mg, 0.31 mmol) and DIPEA (108.5 mg, 0.57 mmol) were added and stirred at room temperature for 2 hours. After the reaction was completed, the residue was purified by flash column chromatography (C18, water / acetonitrile = 0.7) and freeze-dried to obtain the title compound (183.70 mg, 0.18 mmol).

[0586] Its structural characterization data are as follows:

[0587] MS m / z(ESI):1022.2[M+H] +

[0588] Step 2: Preparation of allyl (5-((S)-2-aminopropionamido)-2-(((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (F-2-2)

[0589] Dissolve F-2-1 (183.70 mg, 0.18 mmol) in DMF (3 mL), add diethylamine (131.8 mg, 1.8 mmol), and stir at room temperature for 1 hour. After the reaction is complete, purify by flash column chromatography (C18, water / acetonitrile = 0.6) and freeze-dry to obtain the title compound (136.6 mg, 0.17 mmol).

[0590] Its structural characterization data are as follows:

[0591] MS m / z(ESI):800.1[M+H] +

[0592] Step 3: Preparation of (5-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amido)-2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (F-2-3)

[0593] F-1-5 (140.13 mg, 0.17 mmol) and F-2-2 (136.6 mg, 0.17 mmol) were dissolved in DMF (5 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (97.40 mg, 0.26 mmol) and DIPEA (531.9 mg, 0.51 mmol) were added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 0.6) and freeze-dried to obtain the title compound (81.7 mg, 0.05 mmol).

[0594] Its structural characterization data are as follows:

[0595] MS m / z(ESI):1606.3[M+H] +

[0596] Step 4: Preparation of 4-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amido)-2-((methylamino)methyl)benzyl((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (F-2-4)

[0597] F-2-3 (81.7 mg, 0.05 mmol) was dissolved in DMF (3 mL), and tetrakistriphenylphosphine palladium (11.6 mg, 10.0 μmol) was added, along with 30 μL of formic acid and 60 μL of N-methylmorpholine. The mixture was stirred at room temperature under nitrogen for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 0.5) and freeze-dried to obtain the title compound (32.1 mg, 0.02 mmol).

[0598] Its structural characterization data are as follows:

[0599] MS m / z(ESI):1522.3[M+H]

[0600] Step 5: 2,2',2"-(10-(2-((5-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amido)-2-((((1S,9S)-5-chloro-9-ethyl Preparation of (9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (F-2)

[0601] F-2-4 (32.1 mg, 0.02 mmol) was dissolved in DMF (2 mL), and DIPEA (12.9 mg, 0.1 mmol) was added, followed by 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (30.1 mg, 0.06 mmol). After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (5.91 mg, 3.1 μmol).

[0602] Its structural characterization data are as follows:

[0603] MS m / z(ESI):1908.1[M+H] +

[0604] The preparation method is as follows:

[0605] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0606] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0607] Example 8: Preparation of 2,2',2"-(10-(2-((5-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl))benzamido)-3-methylbutanamido)propionamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizinyl[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (F-13)

[0608] Step 1: Preparation of 3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoic acid (F-13-1)

[0609] Dissolve 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid (0.5 g, 1.35 mmol) in a mixture of ACN (20 mL) and water (10 mL). Add sodium periodate (470.50 mg, 2.20 mol) and ruthenium trichloride monohydrate (9.11 mg, 0.04 mmol). Stir at room temperature for 0.5 hour. After completion of the reaction, purify by flash column chromatography (C18, water / acetonitrile = 5 / 1) and freeze-dry to obtain the title compound (350.50 mg, 0.81 mmol).

[0610] Its structural characterization data are as follows:

[0611] MS m / z(ESI):435.0[M+H] +

[0612] Step 2: Preparation of (3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoyl)-L-valine (F-13-2)

[0613] F-13-1 (350.50 mg, 0.81 mmol) was dissolved in DMF (3 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (342 mg, 0.9 mmol) and DIPEA (313.5 mg, 2.43 mmol) were added. After reacting for 10 min, L-valine (189.5 mg, 1.62 mmol) was added and stirred at room temperature for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 0.5) and freeze-dried to obtain the title compound (120.2 mg, 0.22 mmol).

[0614] Its structural characterization data are as follows:

[0615] MS m / z(ESI):534.1[M+H] +

[0616] Step 3: Preparation of (5-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3-methylbutanamido)propionamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (F-13-3)

[0617] F-13-2 (50.0 mg, 0.094 mmol) and F-1-10 (86.0 mg, 0.11 mmol) were dissolved in DMF (2 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (41.8 mg, 0.11 mmol) and DIPEA (36.8 mg, 0.28 mmol) were added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 2:1) and freeze-dried to obtain the title compound (62.3 mg, 0.047 mmol).

[0618] Its structural characterization data are as follows:

[0619] MS m / z(ESI):1298.4[M+H] +

[0620] Step 4: Preparation of 4-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3-methylbutanamido)propionamido)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (F-13-4)

[0621] F-13-3 (62.3 mg, 0.047 mmol) was dissolved in DMF (3 mL), and tetrakistriphenylphosphine palladium (11.6 mg, 10.0 μmol) was added, along with 30 μL of formic acid and 60 μL of N-methylmorpholine. The mixture was stirred at room temperature under nitrogen for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 0.5) and freeze-dried to obtain the title compound (40.6 mg, 0.033 mmol).

[0622] Its structural characterization data are as follows:

[0623] MS m / z(ESI):1214.4[M+H] +

[0624] Step 5: Preparation of 2,2',2"-(10-(2-((5-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl))benzamido)-3-methylbutanamido)propionamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizinyl[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (F-13)

[0625] F-13-4 (40.6 mg, 0.033 mmol) was dissolved in DMF (2 mL), and DIPEA (12.9 mg, 0.1 mmol) was added, followed by 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (30.1 mg, 0.06 mmol). After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to give the title compound (1.37 mg, 0.85 μmol).

[0626] Its structural characterization data are as follows:

[0627] MS m / z(ESI):1600.6[M+H] +

[0628] The preparation method is as follows:

[0629] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0630] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0631] Example 9: N-((35S,38S)-39-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-35-isopropyl-3,6,9,12,15,18,21 Preparation of 2,5,8,11,14,17,20,23,26,29,33,36,39-tridecahydro-3,6,9,12,15,18,21,24,27,30,34,37-dodecaazanonatriadecyl)-N-methyl-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (G-4)

[0632] Step 1: Preparation of (9H-fluoren-9-yl)methyl (3-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)amino)-3-oxopropyl)carbamate (G-4-2)

[0633] 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionic acid (13.03 mg, 41.84 μmol), (S)-2-amino-N-((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro)-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)-3-methylbutyryl Amine (20.0 mg, 34.87 μmol) was dissolved in DMF (2 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (15.90 mg, 41.84 μmol) and DIPEA (13.52 mg, 104.60 μmol) were added. The mixture was stirred at room temperature for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 1:1) and freeze-dried to give the title compound (26.0 mg, 29.99 μmol).

[0634] Its structural characterization data are as follows:

[0635] MS m / z(ESI):867.4[M+H] +

[0636] Step 2: Preparation of (S)-2-(3-aminopropionamido)-N-((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (G-4-3)

[0637] (9H-fluoren-9-yl)methyl(3-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropyl-2-yl)amino)-3-[((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-3-[((S)-1- ...9H-fluoren-9-yl)methyl]methyl 1-Methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)carbamate (30.0 mg, 34.60 μmol) was dissolved in DMF (1 mL), diethylamine (0.5 mL) was added, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, it was purified by flash column chromatography (C18, water / acetonitrile = 2:1) and freeze-dried to obtain the title compound (22.31 mg, 34.60 μmol).

[0638] Its structural characterization data are as follows:

[0639] MS m / z(ESI):645.6[M+H] +

[0640] Step 3: N-((35S,38S)-39-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-35-isopropyl-3,6,9,12,15,18,21 Preparation of 2,5,8,11,14,17,20,23,26,29,33,36,39-tridecahydro-3,6,9,12,15,18,21,24,27,30,34,37-dodecaazanonatriadecyl)-N-methyl-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (G-4)

[0641] (S)-2-(3-Aminopropionamido)-N-((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (22.31 mg, 34.60 μmol) and 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 (22.31 mg, 34.60 μmol) was dissolved in DMF (2 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (15.78 mg, 41.53 μmol) and DIPEA (13.42 mg, 103.81 μmol) were added. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (7.90 mg, 4.41 μmol).

[0642] Its structural characterization data are as follows:

[0643] MS m / z(ESI):1771.6[M+H] +

[0644] The preparation method is as follows:

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

[0646] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0647] Example 10: 2,2',2"-(10-(2-((5-((9S,12S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-9-isopropyl-12-methyl-1,4,7,10-tetraoxo-2,5,8,11-tetraazatridecane-13-amino)-2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl Preparation of triacetic acid (F-4)

[0648] Step 1: Preparation of 2,5-dioxopyrrolidin-1-yl 3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoate (F-4-1)

[0649] Dissolve 3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoic acid (0.1 g, 230.18 μmol) in DMF (3 mL), add EDCI (132.58 mg, 690.54 μmol), and stir at room temperature for 2 hours. After the reaction is complete, add ethyl acetate (20 mL) and water (15 mL) for separation. The organic phase is evaporated to dryness under reduced pressure to obtain the title compound (80 mg, 150.51 μmol).

[0650] Its structural characterization data are as follows:

[0651] MS m / z(ESI):532.3[M+H] +

[0652] Step 2: Preparation of (S)-2-(((3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoyl)glycylglycyl)oxy)-3-methylbutanoic acid (F-4-2)

[0653] 2,5-Dioxopyrrolidin-1-yl 3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoate (30 mg, 56.44 μmol) was dissolved in DMF (1 mL), and (2-hydroxyacetyl)glycyl-L-valine (26.22 mg, 112.88 μmol) and DIPEA (14.59 mg, 112.88 μmol) were added. The mixture was stirred at room temperature for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 0.5) and freeze-dried to obtain the title compound (25 mg, 38.54 μmol).

[0654] Its structural characterization data are as follows:

[0655] MS m / z(ESI):649.4[M+H] +

[0656] Step 3: Preparation of (5-((9S,12S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-9-isopropyl-12-methyl-1,4,7,10-tetraoxo-2,5,8,11-tetraazatridecane-13-amino)-2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (F-4-3)

[0657] (S)-2-(((3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoyl)glycylglycyl)oxy)-3-methylbutanoic acid (25 mg, 38.54 μmol), (5-((S)-2-aminopropionamido)-2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy To the mixture of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (21.09 mg, 55.50 μmol) and DIPEA (17.93 mg, 138.75 μmol) was added 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (21.09 mg, 55.50 μmol) and DIPEA (17.93 mg, 138.75 μmol). The mixture was stirred at room temperature for 1 hour. After the reaction was completed, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 2:1) and freeze-dried to obtain the title compound (47 mg, 35.26 μmol).

[0658] Its structural characterization data are as follows:

[0659] MS m / z(ESI):1413.6[M+H] +

[0660] Step 4: Preparation of 4-((9S,12S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-9-isopropyl-12-methyl-1,4,7,10-tetraoxo-2,5,8,11-tetraazatridecane-13-amido)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (F-4-4)

[0661] (5-((9S,12S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-9-isopropyl-12-methyl-1,4,7,10-tetraoxo-2,5,8,11-tetraazatridecane-13-amino)-2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13 ,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (30 mg, 21.23 μmol) was dissolved in DMF (2 mL), and tetrakistriphenylphosphine palladium (2.45 mg, 2.12 μmol) was added, 30 μL of formic acid and 60 μL of N-methylmorpholine. The mixture was stirred at room temperature under nitrogen for 1 hour. After completion of the reaction, it was purified by flash column chromatography (C18, water / acetonitrile = 0.5) and freeze-dried to obtain the title compound (28.22 mg, 21.23 μmol).

[0662] Its structural characterization data are as follows:

[0663] MS m / z(ESI):1329.4[M+H] +

[0664] Step 5: 2,2',2"-(10-(2-((5-((9S,12S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-9-isopropyl-12-methyl-1,4,7,10-tetraoxo-2,5,8,11-tetraazatridecane-13-amino)-2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl Preparation of triacetic acid (F-4)

[0665] 4-((9S,12S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-9-isopropyl-12-methyl-1,4,7,10-tetraoxo-2,5,8,11-tetraazatridecan-13-amido)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl) Carbamate (28.22 mg, 21.23 μmol) was dissolved in DMF (2 mL), and DIPEA (12.9 mg, 0.1 mmol) was added. 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (83.20 mg, 63.72 μmol) was added. After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (5.7 mg, 3.29 μmol).

[0666] Its structural characterization data are as follows:

[0667] MS m / z(ESI):1716.8[M+H] +

[0668] The preparation method is as follows:

[0669] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0670] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0671] Example 11: Preparation of 2,2',2"-(10-(2-((5-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3-methylbutanamido)propionamido)-2-(((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (F-14)

[0672] Step 1: Preparation of (5-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3-methylbutanamido)propionamido)-2-(((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (F-14-1)

[0673] (3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoyl)-L-valine (14.80 mg, 27.84 μmol), (5-((S)-2-aminopropionamido)-2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl Allyl (methyl)carbamate (20.84 mg, 23.20 μmol) was dissolved in DMF (2 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12.70 mg, 33.41 μmol) and DIPEA (8.99 mg, 69.59 μmol) were added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 2:1) and freeze-dried to obtain the title compound (26 mg, 19.77 μmol).

[0674] Its structural characterization data are as follows:

[0675] MS m / z(ESI):1315.6[M+H] +

[0676] Step 2: Preparation of 4-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3-methylbutanamido)propionamido)-2-((methylamino)methyl)benzyl((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (F-14-2)

[0677] Allyl (5-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3-methylbutanamido)propionamido)-2-(((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (26 mg, 19.77 μmol) was dissolved in DMF (1 mL), tetrakistriphenylphosphine palladium (2.28 mg, 1.98 μmol) was added, 30 μL formic acid, and 60 μL N-methylmorpholine was stirred at room temperature under nitrogen for 1 hour. After completion of the reaction, the residue was purified by flash column chromatography (C18, water / acetonitrile = 2:1) and freeze-dried to obtain the title compound (20 mg, 16.25 μmol).

[0678] Its structural characterization data are as follows:

[0679] MS m / z(ESI):1231.5[M+H] +

[0680] Step 3: Preparation of 2,2',2"-(10-(2-((5-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3-methylbutanamido)propionamido)-2-(((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (F-14)

[0681] 4-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3-methylbutanamido)propionamido)-2-((methylamino)methyl)benzyl((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (20 mg, 16. The product was dissolved in DMF (1 mL) and DIPEA (6.30 mg, 48.75 μmol) was added. Then, 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (24.45 mg, 48.75 μmol) was added. After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (1.14 mg, 0.70 μmol).

[0682] Its structural characterization data are as follows:

[0683] MS m / z(ESI):1618.1[M+H] +

[0684] The preparation method is as follows:

[0685] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0686] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0687] Example 12: 2,2',2"-(10-(2-((5-((9S,12S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-9-isopropyl-12-methyl-1,4,7,10-tetraoxo-2,5,8,11-tetraazatridecane-13-amino)-2-((((1S,9S)-9-ethyl-5-chloro-9-hydroxy-4-methyl Preparation of triacetic acid (F-5)

[0688] Step 1: Preparation of (5-((9S,12S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-9-isopropyl-12-methyl-1,4,7,10-tetraoxo-2,5,8,11-tetraazatridecane-13-amino)-2-((((1S,9S)-9-ethyl-5-chloro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (F-5-1)

[0689] (S)-2-(((3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoyl)glycylglycyl)oxy)-3-methylbutanoic acid (30 mg, 46.32 μmol), (5-((S)-2-aminopropionamido)-2-((((1S,9S)-9-ethyl-5-chloro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy To the mixture of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (26.40 mg, 69.48 μmol) and DIPEA (17.96 mg, 138.96 μmol) was added 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (26.40 mg, 69.48 μmol) and DIPEA (17.96 mg, 138.96 μmol). The mixture was stirred at room temperature for 1 hour. After the reaction was completed, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 2:1) and freeze-dried to obtain the title compound (30 mg, 20.99 μmol).

[0690] Its structural characterization data are as follows:

[0691] MS m / z(ESI):1429.3[M+H] +

[0692] Step 2: Preparation of 4-((9S,12S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-9-isopropyl-12-methyl-1,4,7,10-tetraoxo-2,5,8,11-tetraazatridecane-13-amido)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-chloro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (F-5-2)

[0693] (5-((9S,12S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-9-isopropyl-12-methyl-1,4,7,10-tetraoxo-2,5,8,11-tetraazatridecane-13-amino)-2-((((1S,9S)-9-ethyl-5-chloro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (F-5-1) (30 mg, 20.99 μmol) was dissolved in DMF (2 mL), and tetrakistriphenylphosphine palladium (2.32 mg, 2.01 μmol) was added, followed by 30 μL of formic acid and 60 μL of N-methylmorpholine. The mixture was stirred at room temperature under nitrogen for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 0.5) and freeze-dried to give the title compound (25 mg, 18.59 μmol).

[0694] Its structural characterization data are as follows:

[0695] MS m / z(ESI):1331.6[M+H] +

[0696] Step 3: 2,2',2"-(10-(2-((5-((9S,12S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-9-isopropyl-12-methyl-1,4,7,10-tetraoxo-2,5,8,11-tetraazatridecane-13-amino)-2-((((1S,9S)-9-ethyl-5-chloro-9-hydroxy-4-methyl Preparation of triacetic acid (F-5)

[0697] 4-((9S,12S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-9-isopropyl-12-methyl-1,4,7,10-tetraoxo-2,5,8,11-tetraazatridecane-13-amido)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-chloro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino Acid ester (F-5-2) (10 mg, 7.44 μmol) was dissolved in DMF (2 mL), and DIPEA (2.88 mg, 22.31 μmol) was added. 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (11.19 mg, 22.31 μmol) was added. After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (1.01 mg, 3.29 μmol).

[0698] Its structural characterization data are as follows:

[0699] MS m / z(ESI):1732.2[M+H] +

[0700] The preparation method is as follows:

[0701] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0702] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0703] Example 13: Preparation of 2,2',2"-(10-(2-(((S)-5-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-6-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)amino)-6-oxohexyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (G-25)

[0704] Step 1: Preparation of tert-butyl ((S)-5-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-6-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)amino)-6-oxohexyl)carbamate (G-25-1)

[0705] N 2 -(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoyl)-N 6 -(tert-Butoxycarbonyl)-L-lysine (64.12 mg, 96.75 μmol) and (S)-2-amino-N-((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (G-1-1) (37 mg, 64.50 μmol) were dissolved in DMF (2 mL), and DIPEA (25.01 mg, 193.50 μmol) and HATU (36.79 mg, 96.75 μmol) were added and reacted at room temperature for one hour. After the reaction was completed, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (20 mg, 16.42 μmol).

[0706] Its structural characterization data are as follows:

[0707] MS m / z(ESI):1219.3[M+H] +

[0708] The preparation method is as follows:

[0709] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0710] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0711] Step 2: Preparation of tert-butyl ((S)-5-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-6-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-6-oxohexyl)carbamate (G-25-2)

[0712] ((S)-5-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-6-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxo- To tert-butyl 1-oxopropyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)amino)-6-oxohexyl)carbamate (G-25-1) (20 mg, 16.42 μmol) was dissolved in DCM (1 mL) and TFA (0.1 mL) was added. The mixture was reacted at room temperature for two hours. After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (20 mg, 16.42 μmol).

[0713] Its structural characterization data are as follows:

[0714] MS m / z(ESI):1119.3[M+H] +

[0715] The preparation method is as follows:

[0716] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0717] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0718] Step 3: Preparation of 2,2',2"-(10-(2-(((S)-5-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-6-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-6-oxohexyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (G-25)

[0719] 2,2′,2″-(10-(2-(2,5-dioxopyrrolidin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (2.02 mg, 4.02 μmol) and ((S)-5-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-6-(((S)-1-(((S)-1-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3 Tert-butyl (1,2-b]quinolin-4-yl)-(1,4':6,7)-indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-6-oxohexyl)carbamate (G-25-2) (3.00 mg, 2.68 μmol) was dissolved in DMF (1 mL), and DIPEA (346.72 μg, 2.68 μmol) was added. The mixture was allowed to react at room temperature for one hour. After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (1.06 mg, 6.55e-1 μmol).

[0720] Its structural characterization data are as follows:

[0721] MS m / z(ESI):1504.8[M+H] +

[0722] The preparation method is as follows:

[0723] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0724] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0725] Example 14: Preparation of 2,2',2"-(10-((2S,5S,14S)-14-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-5-isopropyl-2-methyl-1,4,7,10,13,20-hexaoxo-3,6,9,12,19-pentaazaheneicosane-21-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (G-26)

[0726] Step 1: Preparation of (9H-fluoren-9-yl)methyl (2-((2-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13)-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (G-26-1)

[0727] (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine (31.88 mg, 89.95 μmol) and (S)-2-amino-N-((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro)-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (G-1-1) (43 mg, 74.96 μmol) were dissolved in DMF (1 mL), and HATU (34.20 mg, 89.95 μmol) was added, followed by DIPEA (29.06 mg, 224.88 μmol), and the mixture was reacted at room temperature for one hour. The solvent was then removed under reduced pressure in vacuo to afford the crude title compound, which was directly carried to the next step without purification.

[0728] Its structural characterization data are as follows:

[0729] MS m / z(ESI):910.5[M+H] +

[0730] Step 2: Preparation of (S)-2-(2-(2-aminoacetamido)acetamido)-N-((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (G-26-2)

[0731] The crude (9H-fluoren-9-yl)methyl (2-((2-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13)-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (G-26-1) was dissolved in DMF (2.42 mL) and DEA (54.65 mg, 747.27 μmol, 77.30 μL) was added. The mixture was allowed to react at room temperature for one hour. The solvent was then removed under vacuum to obtain the crude product. The crude product was purified by flash column chromatography (C18, water / acetonitrile = 0.7) and freeze-dried to give the title compound (38 mg, 55.25 μmol).

[0732] Its structural characterization data are as follows:

[0733] MS m / z(ESI):689.2[M+H] +

[0734] Step 3: Preparation of tert-butyl ((2S,5S,14S)-14-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-5-isopropyl-2-methyl-1,4,7,10,13-pentaoxo-3,6,9,12-tetraazaoctadec-18-yl)carbamate (G-26-3)

[0735] N 2 -(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoyl)-N 6-(tert-Butoxycarbonyl)-L-lysine (54.93 mg, 82.88 μmol) and (S)-2-(2-(2-aminoacetamido)acetamido)-N-((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (G-26-2) (38 mg, 55.25 μmol) were dissolved in DMF (2 mL) and HATU (31.51 mg, 82.88 μmol) and DIPEA (14.28 mg, 110.51 μmol) were added and reacted at room temperature for four hours. After the reaction was completed, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (27 mg, 20.26 μmol).

[0736] Its structural characterization data are as follows:

[0737] MS m / z(ESI):1333.4[M+H] +

[0738] The preparation method is as follows:

[0739] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0740] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0741] Step 4: Preparation of N-((2S,5S,14S)-18-amino-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-5-isopropyl-2-methyl-1,4,7,10,13-pentaoxo-3,6,9,12-tetraazaoctadec-14-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (G-26-4)

[0742] Tert-butyl ((2S,5S,14S)-14-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-5-isopropyl-2-methyl-1,4,7,10,13-pentaoxo-3,6,9,12-tetraazaoctadec-18-yl)carbamate (G-26-3) (27 mg, 20.26 μmol) was dissolved in DCM (2 mL) and TFA (0.2 mL) was added. The mixture was reacted at room temperature for two hours. The solvent was then removed to give the crude title compound, which was directly used for the next step without purification.

[0743] Its structural characterization data are as follows:

[0744] MS m / z(ESI):1233.4[M+H] +

[0745] Step 5: Preparation of 2,2',2"-(10-((2S,5S,14S)-14-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-5-isopropyl-2-methyl-1,4,7,10,13,20-hexaoxo-3,6,9,12,19-pentaazaheneicosane-21-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (G-26)

[0746] 2,2',2"-(10-(2-(2,5-dioxopyrrolidin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (14.18 mg, 29.21 μmol) and N-((2S,5S,14S)-18-amino-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]) The crude product of (quinolin-4-yl)amino)-5-isopropyl-2-methyl-1,4,7,10,13-pentaoxo-3,6,9,12-tetraazaoctadec-14-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (G-26-4) was dissolved in DMF (2 mL) and DIPEA (7.55 mg, 58.43 μmol) was added. The mixture was allowed to react at room temperature for two hours. After completion of the reaction, the reaction mixture was directly purified by preparative HPLC and freeze-dried to obtain the title compound (15.30 mg, 8.79 μmol).

[0747] Its structural characterization data are as follows:

[0748] MS m / z(ESI):1619.9[M+H] +

[0749] The preparation method is as follows:

[0750] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0751] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0752] Example 15: 1-(N-((S)-1-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)carbonyl)aminosulfonyl)piperidine-4-carboxylic acid pentafluorophenol ester (I-50)

[0753] Step 1:

[0754] Chlorosulfonyl isocyanate (2.14 g, 15.13 mmol) 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) 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%) and concentrated again to obtain compound I-50-2 (1.87 g, 4.19 mmol).

[0755] Its structural characterization data are as follows:

[0756] 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).

[0757] Step 2:

[0758] Allyl 1-(N-((2-(tert-butoxy)-2-oxoethoxy)carbonyl)aminosulfonyl)piperidine-4-carboxylate (I-50-2) (1.75 g, 4.31 mmol) 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 adjusted to pH about 8 with aqueous sodium bicarbonate solution. Some impurities were removed by extraction, and then adjusted to pH about 3 with 3N dilute hydrochloric acid. The mixture was extracted with ethyl acetate three times (30 mL x 3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound I-50-3 (1.4 g, 4.00 mmol).

[0759] Its structural characterization data are as follows:

[0760] ESI-MS (m / z): 351.1 [M+H] + .

[0761] Step 3:

[0762] 2-((((4-((allyloxy)carbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (I-50-3) (18.32 mg, 52.30 μmol), (S)-2-amino-N-((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropane-2 -yl)-3-methylbutanamide (30 mg, 52.30 μmol) was dissolved in DMF (1 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (23.85 mg, 62.76 μmol) and DIPEA (20.28 mg, 156.89 μmol) were added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, it was purified by flash column chromatography (C18, water / acetonitrile = 1 / 2) and freeze-dried to obtain compound I-50-4 (40 mg, 44.15 μmol).

[0763] Its structural characterization data are as follows:

[0764] MS m / z(ESI):906.4[M+H] +

[0765] Step 4:

[0766] Allyl 1-((2-(((S)-1-(((S)-1-(((S)-1-((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)carbonyl)aminosulfonyl)piperidine-4-carboxylate (I-50-4) (40 mg, 44.15 μmol) was dissolved in DMF (1 mL), tetrakistriphenylphosphine palladium (5.1 mg, 4.41 μmol) was added, 15 μL formic acid, 30 μL N-methylmorpholine was stirred at room temperature for 1 hour under nitrogen protection. After the reaction was completed, it was purified by flash column chromatography (C18, water / acetonitrile = 3 / 1) and freeze-dried to obtain compound I-50-5 (20 mg, 23.10 μmol).

[0767] Its structural characterization data are as follows:

[0768] MS m / z(ESI):867.4[M+H] +

[0769] Step 5:

[0770] 1-(N-((2-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)carbonyl)aminosulfonyl )Piperidine-4-carboxylic acid (I-50-5) (20 mg, 23.10 μmol) was dissolved in DMF (1 mL), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (27.72 mg, 144.36 μmol) and pentafluorophenol (15.94 mg, 86.61 μmol) were added. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain compound I-50 (12.53 mg, 12.02 μmol).

[0771] Its structural characterization data are as follows:

[0772] MS m / z(ESI):1033.4[M+H] +

[0773] The preparation method is as follows:

[0774] Chromatographic column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0775] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0776] 2. Antibody Preparation and Binding Activity Assay

[0777] Antibody acquisition and purification

[0778] According to the amino acid sequence of Trastuzumab (IMGT / mAb-DB ID: 97) and Pertuzumab (IMGT / mAb-DB ID: 80) in the IMGT database, codon optimization was performed and the coding gene was synthesized. The gene was constructed into an expression vector, transfected into CHO cells, and pressure screening was performed to construct a stable expression cell line. The supernatant was expressed and collected, and the corresponding antibodies Trastuzumab and Pertuzumab were purified by Protein A affinity filler. 3. Conjugation of compounds containing cell bioactive molecules and linkers with antibodies

[0779] 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.

[0780] The conjugate preparation of the antibody drug conjugate sample is as follows:

[0781] 1. Preparation of Trastuzumab-E-5

[0782] 0.617 mL of trastuzumab antibody (16.2 mg / mL) was diluted with 30.86 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 19.83 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60, 19.1 μL) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 5.5-fold amount of E-5 dissolved in dimethyl sulfoxide (38.28 μL, 10 mM) 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, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., trastuzumab-E-5). The DAR value was determined by mass spectrometry to be 4.41.

[0783] 2. Preparation of Trastuzumab-F-1

[0784] 0.884 mL of trastuzumab (14.7 mg / mL) was diluted with 44 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then the pH was adjusted to 8.0 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 49.26 μL, pH 7.60) solution was added and mixed, and the mixture was allowed to stand at room temperature for 1.5 hours. Then, a solution of F-1 (47.65 μL, 10 mM) dissolved in dimethyl sulfoxide (5 times the amount of the antibody) was slowly added and mixed, and the mixture was allowed to stand at room temperature for 2 hours. After completion, the buffer was replaced with a 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain an antibody-drug conjugate (i.e., Trastuzumab-F-1). The DAR value determined by mass spectrometry was 4.34.

[0785] 3. Preparation of Trastuzumab-F-2

[0786] 2.333mL of trastuzumab (15mg / mL) was diluted with 117μL of 20mM PB + 0.1M EDTA (pH 7.60), then the pH was adjusted to 7.60 with 1M Na2HPO4 solution. 10mM TCEP (tris(2-carboxyethyl)phosphine, 132.6μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. A solution of F-2 dissolved in dimethyl sulfoxide (DMSO) (171.3μL, 10mM) was then added in a 6.5-fold amount of the antibody, mixed thoroughly, and allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., Trastuzumab-F-2). The DAR value was determined by mass spectrometry to be 4.51.

[0787] 4. Preparation of Trastuzumab-F-4

[0788] 0.238 mL of trastuzumab (14.7 mg / mL) was diluted with 12 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 13.26 μL, pH 7.60) solution was added, mixed, and allowed to stand at room temperature for 1.5 hours. A solution of F-4 dissolved in dimethyl sulfoxide (12.18 μL, 10 mM) was then added (5 times the amount of the antibody) and mixed. The mixture was allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., Trastuzumab-F-4). The DAR value was determined by mass spectrometry to be 4.36.

[0789] 5. Preparation of Trastuzumab-F-13

[0790] 0.204 mL of trastuzumab (14.7 mg / mL) was diluted with 10 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 11.4 μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. Six times the amount of the antibody, dissolved in dimethyl sulfoxide (DMSO), F-13 (12.5 μL, 10 mM) solution, was then added and mixed thoroughly. The mixture was allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., Trastuzumab-F-13). The DAR value was determined by mass spectrometry to be 4.63.

[0791] 6. Preparation of Trastuzumab-G-1

[0792] 2.041 mL of trastuzumab (14.7 mg / mL) was diluted with 102 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, 113.7 μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. A solution of G-1 (155.4 μL, 10 mM) dissolved in dimethyl sulfoxide (DMSO) (7.5 times the amount of the antibody) was then added, mixed thoroughly, and allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., Trastuzumab-G-1). The DAR value was determined by mass spectrometry to be 4.28.

[0793] 7. Preparation of Trastuzumab-G-2

[0794] 2.041 mL of trastuzumab (14.7 mg / mL) was diluted with 102 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, 113.7 μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. Six times the amount of the antibody, G-2 (126.2 μL, 10 mM) dissolved in dimethyl sulfoxide (DMSO) was then added, mixed thoroughly, and allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., Trastuzumab-G-2). The DAR value was determined by mass spectrometry to be 4.38.

[0795] 8. Preparation of Trastuzumab-G-3

[0796] 2.041 mL of trastuzumab (14.7 mg / mL) was diluted with 102 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, 113.7 μL, pH 7.60) solution was added, mixed, and allowed to stand at room temperature for 1.5 hours. Six times the amount of the antibody, G-3 (125.2 μL, 10 mM) dissolved in dimethyl sulfoxide (DMSO) was then added, mixed, and allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., Trastuzumab-G-3). The DAR value, determined by mass spectrometry, was 3.86.

[0797] 9. Preparation of Trastuzumab-G-4

[0798] 2.041 mL of trastuzumab (14.7 mg / mL) was diluted with 102 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 113.7 μL, pH 7.60) solution was added, mixed, and allowed to stand at room temperature for 1.5 hours. Six times the amount of the antibody, G-4 (125.3 μL, 10 mM) dissolved in dimethyl sulfoxide (DMSO) was then added, mixed, and allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., Trastuzumab-G-4). The DAR value was determined by mass spectrometry to be 4.16.

[0799] 10. Preparation of Trastuzumab-G-5

[0800] 0.884 mL of trastuzumab (14.7 mg / mL) was diluted with 44.2 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 49.3 μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. Six times the amount of the antibody, G-5 (54 μL, 10 mM) dissolved in dimethyl sulfoxide (DMSO) was then added, mixed thoroughly, and allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., Trastuzumab-G-5). The DAR value was determined by mass spectrometry to be 3.99.

[0801] 11. Preparation of Trastuzumab-I-50

[0802] 2.04 mL of trastuzumab (14.7 mg / mL) was adjusted to pH 7.40 with 1 M Na2HPO4 solution. I-50 (146 μL, 10 mM, equivalent to 7 times the amount of antibody) dissolved in dimethyl sulfoxide was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., trastuzumab-I-50). The average loading (DAR value) determined by mass spectrometry was 2.45.

[0803] IV. Evaluation of the Antibody-Drug Conjugate's Inhibitory Effect on Tumor Growth in a Mouse Subcutaneous Xenograft Tumor Model

[0804] The ADC-containing preparations were injected into the CDX mouse model with subcutaneous transplantation of human gastric cancer cells NCI-N87 via tail vein injection. The tumor volume and animal body weight changes were measured twice a week to calculate the tumor inhibitory efficacy of ADC on tumor-bearing mice.

[0805] Test drug

[0806] Drug name, source, and preparation method: Take an appropriate amount of ADC and administer at a dose of 1 mg / kg. Dilute the stock solution with 0.9% NaCl injection. Use 0.9% NaCl injection as the vehicle control (Vehicle).

[0807] Experimental animals and cell lines

[0808] Balb / c Nude mice (Chengdu Yaokang Biotechnology Co., Ltd., production license number: SCXK (Sichuan) 2020-0034, animal certificate number: 511214900025102)

[0809] Human gastric cancer cells NCI-N87 (ATCC)

[0810] Experimental grouping and evaluation methods

[0811] 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). Groups were administered 0.9% NaCl injection (hereinafter referred to as vehicle control) and ADC, with dosing frequency as described in the specific examples. The administration method was tail vein injection, with a volume of 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.

[0812] The tumor growth inhibition rate (TGI) was calculated using the following formula to evaluate the tumor inhibition efficacy of ADC:

[0813] 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%.

[0814] Where VT末 : Mean tumor volume at the end of the treatment group experiment

[0815] V T0 : Mean tumor volume at the start of drug administration in the treatment group

[0816] V C末 : Mean tumor volume of negative control group at the end of the experiment

[0817] V C0 : Mean tumor volume of negative control group at the beginning of drug administration

[0818] The following formula was used to calculate the tumor relative proliferation rate T / C (%), which was used to evaluate the tumor inhibition efficacy of ADC: T / C = (V T末 / V T0 ) / (V C末 / V C0 ).

[0819] Efficacy testing of anti-human Her2 antibody-drug conjugates in the NCI-N87 model

[0820] NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. NCI-N87 cells were harvested during the exponential growth phase, resuspended in PBS to an appropriate concentration, and inoculated subcutaneously into female Balb / c-Nude mice to establish a gastric cancer model. The average tumor volume was approximately 150 mm. 3 Around 24 hours after the onset of leukemia, the patients were randomly divided into the following groups according to tumor size: a vehicle control group (i.e., negative control, vehicle group), a 1 mg / kg trastuzumab-F-2 group and a 1 mg / kg trastuzumab-G-1 group of the present invention, and a control group of 1 mg / kg DS8201 (Note: DS8201 is an ADC targeting human HER2 developed by Daiichi Sankyo; the sample used in this experiment was prepared by Kelun Botai). The patients were administered via tail vein (iv) injection on Day 0, Day 7, and Day 14, for a total of three doses.

[0821] The ADC of the present invention demonstrated significant tumor growth inhibition in the NCI-N87 gastric cancer xenograft model. Compared to the vehicle group, the tumor growth inhibition rates (TGI) of the 1 mg / kg trastuzumab-F-2, 1 mg / kg trastuzumab-G-1, and 1 mg / kg DS820 control groups were 72.86%, 167.97%, and 69.02%, respectively. No animals died or experienced significant weight loss on Day 27 in the treatment groups, and no significant drug toxicity was observed. The ADC of the present invention was well tolerated by mice during treatment. Specific results are shown in Table 1, Figures 1 and 2.

[0822] Table 1 Human gastric cancer cell NCI-N87 CDX model

[0823] Note: TGI is tumor growth inhibition rate, T / C is relative tumor proliferation rate, the same below.

[0824] 5. Evaluation of the Antibody-Drug Conjugate's Inhibitory Effect on Tumor Growth in a Mouse Subcutaneous Xenograft Tumor Model

[0825] The ADC-containing preparations were injected into the CDX mouse model with subcutaneous transplantation of human gastric cancer cells NCI-N87 via tail vein injection. The tumor volume and animal body weight changes were measured twice a week to calculate the tumor inhibitory efficacy of ADC on tumor-bearing mice.

[0826] Test drug

[0827] Drug name, source, and preparation method: Take an appropriate amount of ADC and administer at doses of 1 mg / kg and 2 mg / kg, dilute the stock solution with 0.9% NaCl injection, and use 0.9% NaCl injection as the vehicle control (Vehicle).

[0828] Experimental animals and cell lines

[0829] Balb / c Nude mice (Chengdu Yaokang Biotechnology Co., Ltd., production license number: SCXK (Sichuan) 2020-0034, animal certificate number: 511214900026661)

[0830] Human gastric cancer cells NCI-N87 (ATCC)

[0831] Experimental grouping and evaluation methods

[0832] 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). Groups were administered 0.9% NaCl injection (hereinafter referred to as vehicle control) and ADC, with dosing frequency as described in the specific examples. The administration method was tail vein injection, with a volume of 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.

[0833] The tumor growth inhibition rate (TGI) was calculated using the following formula to evaluate the tumor inhibition efficacy of ADC:

[0834] V T末 >V T0 , TGI (%) = [1-(V T末 -VT0 ) / (V C末 -V C0 )]*100% or V T末 ≤V T0 , TGI (%) = [1-(V T 末 -VT0) / VT0]*100%.

[0835] Where V T末 : Mean tumor volume at the end of the treatment group experiment

[0836] V T0 : Mean tumor volume at the start of drug administration in the treatment group

[0837] V C末 : Mean tumor volume of negative control group at the end of the experiment

[0838] V C0 : Mean tumor volume of negative control group at the beginning of drug administration

[0839] The following formula was used to calculate the tumor relative proliferation rate T / C (%), which was used to evaluate the tumor inhibition efficacy of ADC: T / C = (V T末 / V T0 ) / (V C末 / V C0 ).

[0840] Efficacy testing of anti-human Her2 antibody-drug conjugates in the NCI-N87 model

[0841] NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. NCI-N87 cells were harvested during the exponential growth phase, resuspended in PBS to an appropriate concentration, and inoculated subcutaneously into female Balb / c-Nude mice to establish a gastric cancer model. The average tumor volume was approximately 150 mm. 3 At approximately 1:10 pm, the patients were randomly divided into the following groups according to tumor size: a vehicle control group (i.e., a negative control, the Vehicle group), a 2 mg / kg group of trastuzumab-F-1 according to the present invention, a 1 mg / kg group of trastuzumab-G-5, a 1 mg / kg group of trastuzumab-G-2, a 1 mg / kg group of trastuzumab-G-3, a 1 mg / kg group of trastuzumab-G-4, and a control DS8201 1 mg / kg group (Note: DS8201 is an ADC targeting human HER2 developed by Daiichi Sankyo. The samples used in this experiment were prepared internally by Kelun Botai). The patients were injected intravenously (iv) on Day 0, Day 7, and Day 14, for a total of 3 doses.

[0842] The ADC of the present invention exhibited significant tumor growth inhibition in the NCI-N87 gastric cancer xenograft model. Compared with the vehicle group, the tumor growth inhibition rates (TGI) of the 2 mg / kg trastuzumab-F-1 group, 1 mg / kg trastuzumab-G-5 group, 1 mg / kg trastuzumab-G-2 group, 1 mg / kg trastuzumab-G-3 group, 1 mg / kg trastuzumab-G-4 group, and the control DS8201 1 mg / kg group were 156.31%, 80.06%, 122.21%, 99.55%, 130.19%, and 60.21%, respectively. No animal mortality or significant weight loss occurred in the Day 27 treatment group, and no significant drug toxicity was observed. The mice tolerated the ADC of the present invention well during treatment. Specific results are shown in Table 2, Figures 3, 4, and 5.

[0843] Table 2 Human gastric cancer cell NCI-N87 CDX model

[0844] Note: TGI is tumor growth inhibition rate, T / C is relative tumor proliferation rate.

[0845] 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 shown in the formula Ab-[MLED]x, in: 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: M is a linker site with an antibody or antigen-binding fragment thereof; L is the structural fragment connecting M and E; E is a structural fragment connecting L and D; D is the cytotoxic drug fragment; X is 1 to 10.

2. The antibody-drug conjugate according to claim 1, in, M is selected from the following substituted or unsubstituted structural fragments: Preferably, M is selected from the following substituted or unsubstituted structural fragments: Preferably, M is selected from the following substituted or unsubstituted structural fragments:

3. The antibody-drug conjugate according to claim 1, in, M is selected from the following substituted or unsubstituted structural fragments: Preferably, M is selected from the following substituted or unsubstituted structural fragments: Preferably, M is selected from the following substituted or unsubstituted structural fragments: Alternatively, M is selected from the following substituted or unsubstituted structural fragments: Alternatively, M is selected from the following substituted or unsubstituted structural fragments:

4. The antibody-drug conjugate according to any one of claims 1 to 3, in, L is selected from substituted or unsubstituted structural fragments 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 , -NHCH(R')-C(=O)-, 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 (COCH 2 CH 2 (OCH 2 CH 2 )rOCH 3 )), Glu(R'), 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, G ly-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-Ly s-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' is composed of one or more of the following groups, including but not limited to hydrogen, C 1-6 Alkyl, C 1-6 Alkylene, amine, hydroxyl, carboxyl, acyl, -O-, -C 1-6 AlkyleneCO 2 H, -C 1-6 Alkylene SO 3 H, -SO 3 H, -PO 3 H 2 , -C 1-6 Alkylene-NHC 1-6 Alkyl, -C 1-6 Alkylene-N(C 1-6 alkyl) 2 , -C 1-6 Alkylene-heterocycle, -CH 2 N(C 1-6 alkyl)-C(=O)C 1- 6 Alkylene-heterocycle, -CH 2 NH-SO 3 H, -CH 2 N(C 1-6 Alkyl)-SO 3 H, -CH 2 NHC 1-6 Alkylene-SO 3 H, -CH 2 N(C 1-6 Alkyl)C 1-6 Alkylene-SO 3 H, -CH 2 N(C 1-6 Alkylene-SO 3 H) 2 、-CH 2 N + (C 1-6 Alkylene-SO 3 H) 3 、-CH 2 N + (C 1-6 alkyl) 2 -C 1-6 Alkylene-SO 3 H, -CH 2 N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1- 6 Alkylene-SO 3 H) 3 、-CH 2 NH-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene-SO 3 H) 3 、-CH 2 N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 alkyl) 3 、-CH 2 NH-C(=O)C 1-6 Alkylene-N + (C 1-6 alkyl) 3 、-CH 2 N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 alkyl) 3 、-CH 2 N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 alkyl) 2 -CH 2 CO 2 H, -CH 2 N(C 1-6 Alkyl)-C 1-6 Alkylene-CO 2 H, -CH 2 N + (C 1-6 alkyl) 2 -C 1-6 Alkylene-CO 2 H, glucose, galactosyl, glucuronic acid, galacturonic acid, -CH 2 N(C 1-6 alkyl)-C(=O)-(CH 2 CH 2 O) r -C 1-6 Alkyl, -CH 2 N(C 1-6 alkyl)-C(=O)-(OCH 2 CH 2 ) r -OC 1-6 Alkyl, -(CH 2 N(Me)-C(=O)) r -C 1-6 Alkyl, polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH 2 CH 2 O) 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), NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue), -C 1-6 Alkylene-N(C 1-6 Alkyl)-DOTA, -C 1-6 Alkyl-N(C 1-6 Alkyl)-DOTAGA, or -C 1-6 Alkyl-N(C 1-6 alkyl)-NOTA, wherein r is selected from an integer of 1-20, preferably 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, for example r is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; 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, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; Preferably, L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following groups: 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, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; Preferably, L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following groups: wherein 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, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; Preferably, L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following groups: wherein 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, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably, s is selected from 5, 8, 10; Preferably, L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following groups: wherein 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, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably, s is selected from 5, 8, 10; Preferably, L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following groups: wherein 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, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably, s is selected from 5, 8, 10; Preferably, L contains the following structural fragment Preferably, L is formed by connecting a structural fragment of group I with one or more substituted or unsubstituted structural fragments selected from group II: Group I Group II consists of Composed of, preferably Group II consists of Composed of, preferably Group II consists of wherein 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, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably, s is selected from 5, 8, 10; Alternatively, 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 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 (COCH 2 CH 2 (OCH 2 CH 2 )rOCH 3 )), 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-A rg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Al a. 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 AlkyleneCO 2 H, -C 1- 6 Alkylene SO 3 H, -SO 3 H, -PO 3 H 2 , -C 1-6 Alkylene-NHC 1-6 Alkyl, -C 1-6 Alkylene-N(C 1-6 alkyl) 2 、-CH 2 N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-heterocycle, -CH 2 NH-SO 3 H, -CH 2 N(C 1-6 Alkyl)-SO 3 H, -CH 2 NHC 1-6 Alkylene-SO 3 H, -CH 2 N(C 1-6 Alkyl)C 1-6 Alkylene-SO 3 H, -CH 2 N(C 1-6 Alkylene-SO 3 H) 2 、-CH 2 N + (C 1-6 Alkylene-SO 3 H) 3 、-CH 2 N + (C 1-6 alkyl) 2 -C 1-6 Alkylene-SO 3 H, -CH 2 N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene-SO 3 H) 3 、-CH 2 NH-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene-SO 3 H) 3 、-CH 2 N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 alkyl) 3 、-CH 2 NH-C(=O)C 1-6 Alkylene-N + (C 1-6 alkyl) 3 、-CH 2 N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 alkyl) 3 、-CH 2 N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 alkyl) 2 -CH 2 CO 2 H, -CH 2 N(C 1-6 Alkyl)-C 1-6 Alkylene-CO 2 H, -CH 2 N + (C 1-6 alkyl) 2 -C 1-6 Alkylene-CO 2 H, glucose, galactosyl, glucuronic acid, galacturonic acid, -CH 2 N(C 1-6 alkyl)-C(=O)-(CH 2 CH 2 O) r -C 1-6 Alkyl, -CH 2 N(C 1-6 alkyl)-C(=O)-(OCH 2 CH 2 ) r -OC 1-6 Alkyl, -(CH 2 N(Me)-C(=O)) r -C 1-6 Alkyl, or a polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH 2 CH 2 O) r -C 1-6 Alkyl), wherein r is selected from an integer of 1-20; s is selected from an integer of 1-20; Preferably, L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following groups: s is an integer selected from 1 to 20; Alternatively, L is selected from a divalent substituted or unsubstituted structural fragment consisting of one or more of the following groups: 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 (COCH 2 CH 2 (OCH 2 CH 2 )rOCH 3 )), 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-A rg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Al a. 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, -CH 2 N(C 1-6 alkyl)-C(=O)-(CH 2 CH 2 O) r -C 1-6 Alkyl, -(CH 2 N(Me)-C(=O)) r -C 1-6 Alkyl, or a polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH 2 CH 2 O) r -C 1-6 Alkyl), wherein r is selected from an integer of 1-20; s is selected from an integer of 1-20; Preferably, L is selected from a divalent substituted or unsubstituted structural fragment consisting of one or more of the following groups: s is an integer selected from 1-20.

5. The antibody-drug conjugate according to any one of claims 1 to 4, in, E is a single bond, substituted or unsubstituted -NH-CH 2 -, or selected from the following substituted or unsubstituted structural fragments: Preferably, E is a single bond, substituted or unsubstituted -NH-CH 2 -,or 6. The antibody-drug conjugate according to any one of claims 1 to 5, in, The cytotoxic drug is selected from anti-tubulin agents, DNA intercalators, DNA topoisomerase inhibitors, RNA polymerase inhibitors and gene transcription inhibitors; Preferably, the cytotoxic drug is a topoisomerase I inhibitor (e.g., Camptothecin, Hydroxycamptothecin, 9-aminocamptothecin, SN-38, Irinotecan, Ixinotecan, Topotecan, Belotecan, Rubitecan, Diflomotecan, Lurtotecan, Karenitecin, Gimatecan, Namitecan, Simmitecan, Chimmitecan, Silatecan or Elomotecan); Preferably, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof: Preferably, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof: Preferably, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof: Preferably, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof: Preferably, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof: Preferably, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof: Alternatively, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof: 1-1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-26, 1-27, 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34, 1-35, 1-36, 1-37, 1-38, 1-39, 2-1, 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 2-24, 2-25, 2-26, 2-27, 2-28, 2-29, 2-30, 2-31, 2-32, 2-33, 2 -34, 2-35, 2-36, 2-37, 2-38, 2-39, 2-40, 2-41, 2-42, 2-43, 2-44, 2-45, 2-46, 2-47, 2-48, 2-49, 2-50, 2-51, 2-52, 2-53, 2-54, 2-55, 2-56, 2-57, 2-58, 2-59, 2-60, 2-61, 2-62, 2-63, 2-64, 2-65, 2-66, 2-67, 2-68, 2-69, 2-70, 2-71 71, 2-72, 2-73, 2-74, 2-75, 2-76, 2-77, 2-78, 2-79, 2-80, 2-81, 2-82, 2-83, 2-84, 2-85, 2-86, 2-87, 2-88, 2-89, 2-90, 2-91, 2-92, 2-93, 2-94, 2-95, 2-96, 2-97, 2-98, 2-99, 2-100, 2-101, 2-102, 2-103, 3-1, 3-2, 3-3, 3-4, 3-5, Preferably, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof: Alternatively, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof: Preferably, the cytotoxic drug is connected to the The E in the compound is connected.

7. The antibody-drug conjugate according to any one of claims 1 to 6, in, -MLED can be formed by the following structures: E'-1 to E'-50, F'-1 to F'-3, F'-7 to F'-12, G'-1 to G'-4, I'-10 to I'-49, E-1 to E-50, F-1 to F-14, G-1 to G-26, I-1 to I-50, for example, by a substitution reaction (for example, removing the methylsulfonyl structure thereon): wherein n is selected from an integer of 1-20, such as an integer of 1-12, 3-12, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; Alternatively, -MLED can be obtained by the following structures: E-1 to E-50, F-1 to F-14, G-1 to G-5 by substitution reaction (for example, removing the methylsulfonyl structure thereon); Alternatively, -MLED can be obtained by the following structures: E-1 to E-50, F-1 to F-12 through substitution reaction (e.g., removal of The method of obtaining the methylsulfonyl structure on the surface of the substrate is as follows; Alternatively, -MLED can be obtained through the following structures: E-1 to E-50, I-1 to I-49 through substitution reaction (for example, removing the methylsulfonyl structure thereon).

8. The antibody-drug conjugate according to any one of claims 1 to 7, in, The antibody or antigen-binding fragment thereof 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) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 5 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 6 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 8 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 9 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 10 or a variant thereof; or, (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 20 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 21 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 23 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 24 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 25 or a variant thereof; 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 more 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 substitutions are conservative substitutions; 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) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 18 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 19 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 8 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 9 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 10 or a variant thereof; or, (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 33 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 34 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 23 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 24 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 25 or a variant thereof; 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 more 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 substitutions are conservative substitutions; 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) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 11 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 12 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 8 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 9 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 10 or a variant thereof; or, (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; wherein the variant described in any one of (3a) and (3b) 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 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; 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 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, (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; 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 more 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 substitutions are conservative substitutions; Preferably, the antibody or antigen-binding fragment thereof 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) 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, (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 20, CDR-H2 with a sequence of SEQ ID NO: 21, and CDR-H3 with a sequence of SEQ ID NO: 22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 23, CDR-L2 with a sequence of SEQ ID NO: 24, and CDR-L3 with a 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) 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, (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; 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) 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, (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 26, CDR-H2 with a sequence of SEQ ID NO: 27, and CDR-H3 with a sequence of SEQ ID NO: 22; and, comprising the following A light chain variable region (VL) having 3 CDRs: CDR-L1 having a sequence of SEQ ID NO: 23, CDR-L2 having a sequence of SEQ ID NO: 24, and CDR-L3 having a sequence of SEQ ID NO: 25; or, (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: (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, (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO:28, CDR-H2 with a sequence of SEQ ID NO:29, and CDR-H3 with a sequence of SEQ ID NO:30; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO:31, CDR-L2 with a sequence of SEQ ID NO:32, and CDR-L3 with a sequence of SEQ ID NO:

25.

9. The antibody-drug conjugate according to claim 1 or 8, in, The antibody or antigen-binding fragment thereof comprises: (a) VH or a variant thereof as shown in SEQ ID NO: 1, and / or VL or a variant thereof as shown in SEQ ID NO: 2; or (b) VH or a variant thereof as shown in SEQ ID NO: 3, and / or VL or a variant thereof as shown in SEQ ID NO: 4; 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 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; Preferably, the antibody or antigen-binding fragment thereof 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, in, The antibody or antigen-binding fragment thereof further comprises: (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., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids) compared to the wild-type sequence from which it is derived; and (b) a light chain constant region (CL) of a human immunoglobulin or a variant thereof, wherein the variant is identical to the wild type from which it is derived One or more amino acid substitutions, deletions or additions compared to the sequence (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids; such as 1, 2, 3, 4 or 5 amino acids); Preferably, 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; Preferably, 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 substitutions of amino acids 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); Preferably, 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 substitutions of amino acids 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); Preferably, 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.

11. The antibody-drug conjugate according to any one of claims 1, 8 to 10, in, The antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising a VH sequence set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) set forth in SEQ ID NO: 35, and a light chain comprising a VL sequence set forth in SEQ ID NO: 2 and a light chain constant region (CL) set forth in SEQ ID NO: 36; or (2) a heavy chain comprising a VH sequence set forth in SEQ ID NO: 3 and a heavy chain constant region (CH) set forth in SEQ ID NO: 35, and a light chain comprising a VL sequence set forth in SEQ ID NO: 4 and a light chain constant region (CL) set forth in SEQ ID NO: 36; Optionally, the N-terminal glutamine of the VH of the sequence as shown in SEQ ID NO: 1 or 3 or its variants or the heavy chain of the sequence as shown in SEQ ID NO: 37 or 39 or its variants undergoes cyclization to form pyroglutamate or pyroglutamate salt; Optionally, 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.

12. The antibody-drug conjugate according to any one of claims 1 to 11, wherein M binds to a sulfhydryl (-SH) or amino (-NH 2 )connect; Preferably, M is linked to a sulfhydryl (-SH) group on Ab.

13. The antibody-drug conjugate according to any one of claims 1 to 12, in, The antibody or antigen-binding fragment thereof is selected from the antibody or antigen-binding fragment thereof described in claim 11; -MLED is selected from the structural compound shown in claim 7; x is 1 to 10; preferably, x is 1 to 6, or x is 3 to 6, and further preferably, x is about 2, or x is about 4.

14. The antibody drug conjugate according to any one of claims 8 to 13, which is selected from: in, HA in each antibody drug conjugate represents an antibody or an antigen-binding fragment, preferably the antibody or antigen-binding fragment is as defined in any one of claims 8 to 11; n is selected from an integer of 1-20, such as an integer of 1-12, 3-12, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; in, It indicates the specific connection mode between the thiol group in the antibody or antigen-binding fragment thereof and the linker; It indicates the specific connection mode between the amino group in the antibody or its antigen-binding fragment and the M fragment.

15. A composition comprising one or more antibody drug conjugates according to any one of claims 1 to 14, wherein the DAR value (drug antibody conjugate ratio) of the composition is 1-10, for example: 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, for example, 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; preferably 3.5-5.0 (for example, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0); Alternatively, the composition has a DAR value of about 1.0 to 6.0, such as about 1.0 to 5.5, about 1.0 to 5.0, about 1.5 to 6.0, about 1.5 to about 5.5, about 1.5 to 5.0, 2.0 to 5.5, 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, 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 2.94, 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 3.15, 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 3.8 3, 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, 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 to 14, the composition according to claim 15, and one or more pharmaceutical excipients.

17. Use of the antibody drug conjugate of any one of claims 1 to 14, the composition of claim 15, or the pharmaceutical composition of claim 16 in the preparation of a medicament for treating Her2-expressing cancer.

18. The use of claim 17, wherein the cancer is selected from solid tumors or hematological malignancies; for example, selected from gastric cancer, breast cancer, lung cancer (eg, non-small cell lung cancer, in particular lung adenocarcinoma) and urothelial carcinoma.

19. The antibody drug conjugate of any one of claims 1 to 14, the composition of claim 15, or the pharmaceutical composition of claim 16, for use in treating Her2-expressing cancer.

20. The antibody drug conjugate of any one of claims 1 to 14, the composition of claim 15, or the pharmaceutical composition of claim 16, for use in treating solid tumors or hematological malignancies; for example, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) or urothelial carcinoma.

21. A method for treating Her2-expressing cancer, the method comprising administering to an individual in need thereof a therapeutically effective amount of the antibody drug conjugate of any one of claims 1 to 14, the composition of claim 15, or the pharmaceutical composition of claim 16.

22. The method of treatment of claim 21, wherein the cancer is selected from a solid tumor or a hematological malignancy; for example, selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, particularly lung adenocarcinoma) and urothelial carcinoma.

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