Macrocyclic drug conjugate as well as preparation method and application thereof

Abstract

The invention provides a macrocyclic drug conjugate as well as a preparation method and application thereof. The macrocyclic drug conjugate has a structure as shown in a formula Ab-[M-L-E-D] x. The antibody drug conjugate has an excellent targeted killing effect on solid tumors such as gastric cancer, breast cancer, lung cancer and urothelial cancer.

Description

Macrocyclic drug conjugate 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, and the application with CN application number 202311329994.7 and application date October 13, 2023, and claims the priority of the aforementioned applications. All contents of the said CN applications are hereby introduced as a whole into this application. Technical Field

[0002] The present application relates to the field of targeted therapy, and in particular to a macrocyclic drug conjugate. Background Art

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

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

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

[0006] Specifically, the linker portion of both Disitamab vedotin and Trastuzumab deruxtecan uses a maleimide linker (MC). Literature reports that MC linkers are prone to reverse Michael reactions and sulfhydryl exchange under physiological conditions, resulting in reduced efficacy and increased toxicity (Nat Biotechnol. 2012, 32, 184-189; Bioconjugate Chem. 2015, 26, 145-152). 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 tubulin 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 antibody-drug conjugate of the structure shown in FIG. Results showed that the conjugate had excellent binding activity and proliferation inhibition against Her2-positive cells, and had a good targeted killing effect on Her2-positive tumors (e.g., breast cancer, lung cancer, or gastric cancer). Therefore, the present application provides an antibody-drug conjugate for treating Her2-expressing cancers, a pharmaceutical composition containing the antibody-drug conjugate, and their use in treating Her2-expressing cancers.

[0010] Antibody Drug Conjugates

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

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

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

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

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

[0016] D is the cytotoxic drug fragment;

[0017] X is 1 to 10.

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

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

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

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

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

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

[0024] In some embodiments, 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), Ala-Ala-Glu), Wherein R' is composed of one or more of the following groups, including but not limited to hydrogen, C 1-6 Alkyl, C 1-6Alkylene, 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 1-6 Alkyl, polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH2CH2O) 1-10 -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 (e.g., an integer of 1-15, such as 1-12, 3-12, 1-10, 1-8, 3-8, 1-6, 1-4, an integer of 1-2, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); s is selected from an integer of 1-20 (for example, an integer of 1-15, for example, an integer of 1-12, 3-12, 5-10, an integer of 8-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).

[0025] In some embodiments, s is represented by n.

[0026] In some embodiments, "-NOTA" refers to

[0027] In some embodiments, "-DOTA" refers to

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

[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, -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,G ly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Ly s-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), Where R' represents hydrogen, C 1-6 Alkyl, glucose, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl, or polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH2CH2O) r -C 1-6 alkyl), wherein r is selected from an integer of 1-20; s is selected from an integer of 1-20.

[0030] 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.

[0031] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following:

[0032] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following:

[0033] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following: 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, 5-10, 8-10, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0034] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following:

[0035] 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, 5-10, 8-10, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; R' is composed of one or more of the following groups: C 1-6 Alkyl, C 1-6Alkylene, amino, acyl, -O-, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue).

[0036] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following: 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, 5-10, 8-10, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0037] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following: 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, 5-10, 8-10, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0038] In some embodiments, L is selected from the following substituted or unsubstituted structural fragments:

[0039] In some embodiments, L is selected from the following substituted or unsubstituted structural fragments:

[0040] 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, 5-10, 8-10, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0041] In some embodiments, L is selected from the following substituted or unsubstituted structural fragments:

[0042] 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, 5-10, 8-10, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0043] In some embodiments, L is selected from the following substituted or unsubstituted structural fragments:

[0044] 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, 5-10, 8-10, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0045] 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:

[0046] In some embodiments, E is a single bond, substituted or unsubstituted -NH-CH2- or

[0047] In some embodiments, E is substituted or unsubstituted -NH-CH2- or

[0048] In some embodiments, E is a single bond or

[0049] In some embodiments, Selected from the following substituted or unsubstituted structures: s is an integer selected from 1-20.

[0050] In some embodiments, Selected from the following substituted or unsubstituted structures: s is an integer selected from 1-20.

[0051] In some embodiments, Selected from the following substituted or unsubstituted structures:

[0052] In some embodiments, Selected from the following substituted or unsubstituted structures:

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

[0054] In some embodiments, Selected from the following substituted or unsubstituted structures:

[0055] In some embodiments, Selected from the following substituted or unsubstituted structures: 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, 5-10, 8-10, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0056] In some embodiments, Selected from the following substituted or unsubstituted structures:

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

[0058] 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, 5-10, 8-10, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

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

[0060] 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.

[0061] 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.

[0062] In some embodiments, the cytotoxic drug is selected from the class of compounds of the eribulin class.

[0063] In some embodiments, the cytotoxic drug is selected from the following substituted or unsubstituted compounds or isotopically labeled compounds thereof:

[0064] In some embodiments, D is selected from the following substituted or unsubstituted structures:

[0065] In some embodiments, D is the following structure:

[0066] 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.

[0067] In one aspect, the present application provides a compound or a pharmaceutically acceptable salt thereof having a structure shown in formula DEL-M', wherein:

[0068] M' is -M-Lg, wherein Lg is a leaving group of the nucleophilic substitution reaction and M is a structural fragment that binds to the targeting moiety;

[0069] L is the structural fragment connecting M and E;

[0070] E is a structural fragment connecting L and D;

[0071] D is a cytotoxic drug fragment.

[0072] 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 C1-6 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.

[0073] 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.

[0074] In some embodiments, Lg is selected from halogen, substituted or unsubstituted C 1-6 Alkylsulfonyl (C 1-6 alkyl-SO2-), halogenated phenoxy, hydroxyl (-OH), mercapto (-SH) or amino (-NH2).

[0075] In some embodiments, Lg is selected from halogen, substituted or unsubstituted methylsulfonyl, halophenoxy, hydroxyl (-OH), thiol (-SH), or amino (-NH2).

[0076] In some embodiments, Lg is selected from C 1-6 Alkylsulfonyl or halogenated phenoxy.

[0077] In some embodiments, Lg is selected from methylsulfonyl or pentafluorophenoxy.

[0078] In some embodiments, in the compound having the structure shown in DEL-M' or a pharmaceutically acceptable salt thereof, M' is -M-Lg,

[0079] Lg is selected from halogen, substituted or unsubstituted C 1-6 Alkylsulfonyl (C 1-6 alkyl-SO2-), halogenated phenoxy, hydroxyl (-OH), mercapto (-SH) or amino (-NH2), preferably Lg is selected from methylsulfonyl or pentafluorophenoxy;

[0080] M is selected from the following substituted or unsubstituted structural fragments:

[0081] L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following: 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, 5-10, 8-10, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0082] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following: 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, 5-10, 8-10, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

[0083] E is a single bond, -NH-CH2- or Preferably a single bond or

[0084] D is selected from the following substituted or unsubstituted structures:

[0085] Preferably, D is the following structure:

[0086] In some embodiments, in the compound having the structure shown in DEL-M' or a pharmaceutically acceptable salt thereof, M' is -M-Lg,

[0087] Lg is selected from C 1-6 Alkylsulfonyl or halogenated phenoxy, preferably methylsulfonyl or pentafluorophenoxy;

[0088] M is selected from the following substituted or unsubstituted structural fragments:

[0089] L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following: 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, 5-10, 8-10, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

[0090] Preferably, L is selected from the following substituted or unsubstituted structural fragments:

[0091] 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, 5-10, 8-10, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

[0092] E is a single bond or

[0093] D is the following structure:

[0094] In some embodiments, the free form of the "drug-linker" is selected from J-1 to J-9 and H-1 to H-15 shown below:

[0095] In some embodiments, the present application provides the following compounds or pharmaceutically acceptable salts thereof:

[0096] wherein n is selected from an integer of 1-20, preferably an integer of 1-15, such as an integer of 1-12, 3-12, 5-10, 8-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10.

[0097] In some embodiments, the present application provides compounds shown in J-1 to J-9, H-1 to H-15, H'-1 to H'-12 or pharmaceutically acceptable salts thereof, wherein n is selected from an integer of 1-20, preferably an integer of 1-15, for example, an integer of 1-12, 3-12, 5-10, 8-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10.

[0098] In some embodiments, the present application provides compounds shown in J-1 to J-9, H'-1 to H'-12, H-13 to H-16 or pharmaceutically acceptable salts thereof, wherein n is selected from an integer of 1-20, preferably an integer of 1-15, for example, an integer of 1-12, 3-12, 5-10, 8-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10.

[0099] In some embodiments, the present application provides compounds shown in J-1 to J-9, H-1 to H-15 or pharmaceutically acceptable salts thereof.

[0100] In some embodiments, the present application provides compounds shown in J-1 to J-9 or pharmaceutically acceptable salts thereof.

[0101] In some embodiments, the aforementioned compounds or pharmaceutically acceptable salts thereof may be optionally substituted with one or more suitable substituents.

[0102] Conjugate

[0103] The second aspect of the present invention provides a conjugate as shown in formula (II), wherein: Ab-[MLED]x

[0104] Formula (II)

[0105] Ab is the targeting moiety; M, L, E, and D are as described above;

[0106] x is 1 to 10.

[0107] In some embodiments, Ab is an antibody or an antigen-binding fragment thereof. In some embodiments, Ab is an antibody or an antigen-binding fragment thereof that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases.

[0108] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0109] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL):

[0110] (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

[0111] (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;

[0112] 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;

[0113] or,

[0114] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0115] (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

[0116] (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;

[0117] 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;

[0118] or,

[0119] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0120] (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

[0121] (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;

[0122] wherein the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0123] or,

[0124] (4) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0125] (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

[0126] (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;

[0127] 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.

[0128] In some embodiments, the antibody or antigen-binding fragment thereof comprises

[0129] (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:

[0130] (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

[0131] (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;

[0132] 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;

[0133] or,

[0134] (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:

[0135] (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

[0136] (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;

[0137] 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;

[0138] or,

[0139] (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:

[0140] (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

[0141] (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;

[0142] wherein the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0143] or,

[0144] (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:

[0145] (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

[0146] (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;

[0147] 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.

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

[0149] (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:

[0150] (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

[0151] (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;

[0152] or,

[0153] (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:

[0154] (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

[0155] (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;

[0156] or,

[0157] (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:

[0158] (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

[0159] (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;

[0160] or,

[0161] (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:

[0162] (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

[0163] (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.

[0164] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0165] (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

[0166] (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;

[0167] 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.

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

[0169] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0170] (b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4.

[0171] In some embodiments, the antibody or antigen-binding fragment thereof further comprises:

[0172] (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

[0173] (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.

[0174] 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.

[0175] 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).

[0176] 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).

[0177] 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.

[0178] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0179] (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

[0180] (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.

[0181] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0182] (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

[0183] (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.

[0184] 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.

[0185] As known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] In some embodiments, M is linked to a sulfhydryl (—SH) or amino (—NH 2 ) group on Ab.

[0192] In some embodiments, M is linked to a sulfhydryl (—SH) group on Ab.

[0193] 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 J-1 to J-9, H-1 to H-15 or H'-1 to H'-12, preferably by removing the -SO2Me or pentafluorophenoxy group of the compound, Ab is as defined above, and x is 1 to 10.

[0194] In some embodiments, in the presence of Ab-[MLED] x In the antibody-drug conjugates of the structures shown, MLED is formed by the compounds shown in J-1 to J-9 or H-1 to H-15, preferably by removing the -SO2Me or pentafluorophenoxy group of the compounds, Ab is as defined above, and x is 1 to 10.

[0195] In some embodiments, in the presence of Ab-[MLED] x In the antibody-drug conjugates of the structures shown, MLED is formed by removing the -SO2Me or pentafluorophenoxy group from the compound shown in J-3, J-6, J-8 or H-8, Ab is as defined above, and x is 1 to 10.

[0196] In some embodiments, in the presence of Ab-[MLED] x In the antibody drug conjugate of the structure shown,

[0197] M is selected from the following substituted or unsubstituted structural fragments:

[0198] L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following: 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, 5-10, 8-10, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

[0199] Preferably, L is selected from the following substituted or unsubstituted structural fragments:

[0200] 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, 5-10, 8-10, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

[0201] E is a single bond or

[0202] D is the following structure:

[0203] Ab is an antibody or its antigen-binding fragment that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases.

[0204] The antibody or antigen-binding fragment thereof is as defined above,

[0205] Preferably, the antibody or antigen-binding fragment thereof comprises:

[0206] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL):

[0207] (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

[0208] (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;

[0209] 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;

[0210] or,

[0211] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0212] (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

[0213] (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;

[0214] 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;

[0215] or,

[0216] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0217] (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

[0218] (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;

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

[0220] or,

[0221] (4) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0222] (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

[0223] (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;

[0224] wherein the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0225] Preferably, the antibody or antigen-binding fragment thereof comprises:

[0226] (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:

[0227] (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

[0228] (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;

[0229] 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;

[0230] or,

[0231] (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:

[0232] (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

[0233] (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;

[0234] 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;

[0235] or,

[0236] (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:

[0237] (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

[0238] (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;

[0239] wherein the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0240] or,

[0241] (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:

[0242] (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

[0243] (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;

[0244] wherein the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0245] Preferably, the antibody or antigen-binding fragment thereof comprises:

[0246] (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:

[0247] (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

[0248] (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;

[0249] or,

[0250] (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:

[0251] (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

[0252] (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;

[0253] or,

[0254] (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:

[0255] (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

[0256] (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;

[0257] or,

[0258] (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:

[0259] (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

[0260] (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;

[0261] Preferably, the antibody or antigen-binding fragment thereof comprises:

[0262] (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

[0263] (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;

[0264] 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;

[0265] Preferably, the antibody or antigen-binding fragment thereof comprises:

[0266] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0267] (b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4;

[0268] Preferably, the antibody or antigen-binding fragment thereof is selected from Trastuzumab or Pertuzumab or an antigen-binding fragment thereof.

[0269] In some embodiments, the antibody drug conjugate is selected from ADC J-1 to ADC J-9 and ADC H-1 to ADC H-15 shown below:

[0270] Wherein, HA in each antibody-drug conjugate represents an antibody or antigen-binding fragment thereof that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases; n is selected from an integer of 1-20, preferably an integer of 1-15, such as an integer of 1-12, 3-12, 5-10, or 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, preferably 5, 8, or 10;

[0271] in, Or it indicates the specific connection mode between the sulfhydryl group in the antibody or antigen-binding fragment thereof and the M fragment; In some embodiments, the antibody drug conjugate is selected from ADC H'-1 to ADC H'-12 shown below:

[0272] Wherein, HA in each antibody-drug conjugate represents an antibody or antigen-binding fragment thereof that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases; n is selected from an integer of 1-20, preferably an integer of 1-15, such as an integer of 1-12, 3-12, 5-10, or 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, preferably 5, 8, or 10;

[0273] Or it indicates the specific connection mode between the sulfhydryl group in the antibody or antigen-binding fragment thereof and the M fragment;

[0274] Indicates the specific connection method between the amino group in the antibody or its antigen-binding fragment and the M fragment.

[0275] In some embodiments, the antibody or antigen-binding fragment thereof in each antibody drug conjugate is as defined above.

[0276] In some embodiments, the HA comprises:

[0277] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL):

[0278] (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

[0279] (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;

[0280] 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;

[0281] or,

[0282] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0283] (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

[0284] (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;

[0285] 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;

[0286] or,

[0287] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0288] (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

[0289] (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;

[0290] wherein the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0291] or,

[0292] (4) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0293] (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

[0294] (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;

[0295] 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.

[0296] In some embodiments, the HA comprises:

[0297] (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:

[0298] (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

[0299] (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;

[0300] 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;

[0301] or,

[0302] (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:

[0303] (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

[0304] (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;

[0305] 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;

[0306] or,

[0307] (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:

[0308] (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

[0309] (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;

[0310] wherein the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0311] or,

[0312] (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:

[0313] (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

[0314] (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;

[0315] 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.

[0316] In some embodiments, the HA comprises:

[0317] (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:

[0318] (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

[0319] (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;

[0320] or,

[0321] (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:

[0322] (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

[0323] (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;

[0324] or,

[0325] (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:

[0326] (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

[0327] (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;

[0328] or,

[0329] (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:

[0330] (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

[0331] (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.

[0332] In some embodiments, the HA comprises:

[0333] (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

[0334] (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;

[0335] 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.

[0336] In some embodiments, the HA comprises:

[0337] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0338] (b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4.

[0339] In some embodiments, the HA further comprises:

[0340] (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

[0341] (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.

[0342] 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.

[0343] In some embodiments, the HA comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 35, or a variant thereof, which has up to 20 conservative amino acid substitutions compared to SEQ ID NO: 35 (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; for example, 1, 2, 3, 4 or 5 conservative amino acid substitutions).

[0344] In some embodiments, the HA comprises a light chain constant region (CL) as set forth in SEQ ID NO: 36, or a variant thereof, which has up to 20 conservative amino acid substitutions compared to SEQ ID NO: 36 (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; for example, 1, 2, 3, 4 or 5 conservative amino acid substitutions).

[0345] In some embodiments, the HA comprises a heavy chain constant region (CH) as set forth in SEQ ID NO:35 and a light chain constant region (CL) as set forth in SEQ ID NO:36.

[0346] In some embodiments, the HA comprises:

[0347] (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

[0348] (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.

[0349] In some embodiments, the HA comprises:

[0350] (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

[0351] (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.

[0352] In some embodiments, HA is selected from Trastuzumab or Pertuzumab, the amino acid sequence of Trastuzumab has an IMGT database accession number (IMGT / mAb-DB ID): 97, and the amino acid sequence of Pertuzumab has an IMGT database accession number (IMGT / mAb-DB ID): 80.

[0353] In some embodiments, HA in each antibody drug conjugate represents trastuzumab, pertuzumab, or an antigen-binding fragment thereof.

[0354] In some embodiments, HA in each antibody drug conjugate represents the following antibody or antigen-binding fragment:

[0355] (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

[0356] (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.

[0357] 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.

[0358] As known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0359] 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.

[0360] 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.

[0361] 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.

[0362] 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.

[0363] In some embodiments, x in the conjugate represented by Ab-[MLED]x is 1-10, e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, or 4-10.

[0364] In some embodiments, x in the conjugate represented by Ab-[MLED]x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0365] In some embodiments, x in the conjugate represented by Ab-[MLED]x is 1, 2, 3, or 4.

[0366] In some embodiments, x in the conjugate represented by Ab-[MLED]x is 3, 4, or 5.

[0367] In some embodiments, x in the conjugate represented by Ab-[MLED]x is 2 or 4.

[0368] In some embodiments, the conjugate of the present invention is an antibody drug conjugate (ADC).

[0369] In some embodiments, the conjugates described herein are optionally substituted with one or more suitable substituents.

[0370] The present application further provides a formula Ab-[L'-D] x The antibody-drug conjugate shown, wherein:

[0371] Ab is an antibody or an antigen-binding fragment thereof;

[0372] D is a cytotoxic drug fragment;

[0373] L' is a linker connecting Ab and D;

[0374] x is an integer from 1 to 10;

[0375] The antibody or antigen-binding fragment thereof is coupled to a drug-linker selected from the group consisting of:

[0376] In certain embodiments, the antibody comprises the heavy chain amino acid sequence of SEQ ID NO:37, and the light chain amino acid sequence of SEQ ID NO:38.

[0377] In certain embodiments, the antibody is Trastuzumab.

[0378] 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:

[0379] Where * is the attachment point of the lysine amino group of the conjugated antibody.

[0380] 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:

[0381] Where * is the attachment point of the lysine amino group of the conjugated antibody.

[0382] 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:

[0383] Where * is the attachment point of the lysine amino group of the conjugated antibody.

[0384] intermediates

[0385] 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:

[0386] 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:

[0387] in

[0388] PG1 is each independently H or a carboxyl protecting group, such as C 1-6 Alkyl, allyl, benzyl, 2,4-dimethoxybenzyl, p-methoxybenzyl, methoxyethoxymethyl, pentafluorophenyl, 4-p-methylbenzyloxybenzyl;

[0389] PG2 is independently H or an amino protecting group, such as an alkoxycarbonyl amino protecting group, for example, benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), methyloxycarbonyl (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).

[0390] 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.

[0391] Composition

[0392] 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.

[0393] For example, in any embodiment, the ADC compositions described herein have a DAR of about 1 to about 10, or any subrange therebetween, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to 10, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7, about 2 to 8, about 2 to 9, about 1 to 10. or about 9 to 10.

[0394] In certain embodiments, the DAR of the ADC compositions described herein is about 1 to 8, e.g., about 1.0 to 1.5, about 1.5 to 2.0, about 2.0 to 2.5, about 2.5 to 3.0, about 3.0 to 3.5, about 3.5 to 4.0, about 4.0 to 4.5, about about 4.5 to 5.0, about 5.0 to 5.5, about 5.5 to 6.0, about 5.5 to 6.5, about 5.5 to 7.0, about 5.5 to 7.5, about 5.5 to 8.0, about 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5, about 6.0 to 8.5, about 6.5 to 7.0, about 6.5 to 7.5, about 6.5 to 8.0, about 6.5 to 8.5, about 7.0 to 7.5, about 7.0 to 8.0.

[0395] In certain embodiments, the DAR of the ADC compositions described herein is about 1 to 5, e.g., about 1.0 to 1.5, about 1.5 to 2.0, about 2.0 to 2.5, about 2.5 to 3.0, about 3.0 to 3.5, about 3.5 to 4.0, about 3.5 to 4.5, about 4.0 to 4.5, about 4.5 to 5.0.

[0396] In certain embodiments, the DAR of the ADC compositions described herein is about 1 to 3, e.g., about 1.0 to 1.5, about 1.0 to 2.0, about 1.0 to 2.5, about 1.0 to 3.0, about 1.5 to 2.0, about 1.5 to 2.5, about 1.5 to 3.0, about 2.0 to 2.5, about 2.0 to 3.0, about 2.5 to 3.0.

[0397] In certain embodiments, the DAR of the ADC compositions described herein is from about 1.0 to 6.0, e.g., from about 1.0 to 5.5, from about 1.0 to 5.0, from about 1.5 to 6.0, from about 1.5 to about 5.5, from about 1.5 to 5.0, 2.0 to 5.5, from about 2.0 to about 5.0, e.g., 1.0, about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.1, about 1.11, about 1.12, about 1.13, about 1.14, about 1.15, about 1.16, about 1.17, about 1.18, about 1.19, about 1.2, about 1.21, about 1.22, about 1.23, about 1.24, about 1.25, about 1.26, about 1.27, about 1.28, about 1.29, about 1.30, about 1.31, about 1.32, about 1.33, about 1.34, about 1.35 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 1.8 6, about 1.87, about 1.88, about 1.89, about 1.9, about 1.91, about 1.92, about 1.93, about 1.94, about 1.95, about 1.96, about 1.97, about 1.98, about 1.99, about 2.0, about 2.01, about 2.02, about 2.03, about 2.04, about 2.05, about 2.06, about 2. 07, about 2.08, about 2.09, about 2.1, about 2.11, about 2.12, about 2.13, about 2.14, about 2.15, about 2.16, about 2.17, about 2.18, about 2.19, about 2.2, about 2.21, about 2.22, about 2.23, about 2.24, about 2.25, about 2.26, about 2.27, about 2.28, about 2.29, about 2.3, about 2.31, about 2.32, about 2.33, about 2.34, about 2.35, about 2.36, about 2.37, about 2.38, about 2.39, about 2.4, about 2.41, about 2.42, about 2.43, about 2.44, about 2.45, about 2.46, about 2.47, about 2.48, about 2.49, about 2.5, about 2.51, about 2.52, about 2.53, about 2.54, about 2.55, about 2.56, about 2.57, about 2.58, about 2.59, about 2.6, about 2.61, about 2.62, about 2.63, about 2.64, about 2.65, about 2.66, about 2.67, about 2.68, about 2.69, about 2.7, about 2.71, about 2.72, about 2. 73, about 2.74, about 2.75, about 2.76, about 2.77, about 2.78, about 2.79, about 2.8, about 2.81, about 2.82, about 2.83, about 2.84, about 2.85, about 2.86, about 2.87, about 2.88, about 2.89, about 2.9, about 2.91, about 2.92, about 2.93, about 2.9 4, about 2.95, about 2.96, about 2.97, about 2.98, about 2.99, about 3.0, about 3.01, about 3.02, about 3.03, about 3.04, about 3.05, about 3.06, about 3.07, about 3.08, about 3.09, about 3.1, about 3.11, about 3.12, about 3.13, about 3.14, about 3.1 5, about 3.16, about 3.17, about 3.18, about 3.19, about 3.2, about 3.21, about 3.22, about 3.23, about 3.24, about 3.25, about 3.26, about 3.27, about 3.28, about 3.29, about 3.3, about 3.31, about 3.32, about 3.33, about 3.34, about 3.35, about 3.36 , about 3.37, about 3.38, about 3.39, about 3.4, about 3.41, about 3.42, about 3.43, about 3.44, about 3.45, about 3.46, about 3.47, about 3.48, about 3.49, about 3.5, about 3.51, about 3.52, about 3.53, about 3.54, about 3.55, about 3.56, about 3.57 , about 3.58, about 3.59, about 3.6, about 3.61, about 3.62, about 3.63, about 3.64, about 3.65, about 3.66, about 3.67, about 3.68, about 3.69, about 3.7, about 3.71, about 3.72, about 3.73, about 3.74, about 3.75, about 3.76, about 3.77, about 3.78, about 3.79, about 3.8, about 3.81, about 3.82, about 3.83, about 3.84, about 3.85, about 3.86, about 3.87, about 3.88, about 3.89, about 3.9, about 3.91, about 3.92, about 3.93, about 3.94, about 3.95, about 3.96, about 3.97, about 3.98, about 3.99, about 4.0, about 4.01, about 4.02, about 4.03, about 4.04, about 4.05, about 4.06, about 4.07, about 4.08, about 4.09, about 4.1, about 4.11, about 4.12, about 4.13, about 4.14, about 4.15, about 4.16, about 4.17, about 4.18, about 4.19, about 4.2, about 4.21, about 4.22, about 4.23, about 4.24, about 4.25, about 4.26, about 4.27, about 4.28, about 4.29, about 4.3, about 4.31, about 4.32, about 4.33, about 4.34, about 4.35, about 4.36, about 4.37, about 4.38, about 4.39, about 4.4, about 4.41, about 4.42, about 4.43, about 4.44, about 4.45, about 4.46, about 4.47, about 4.48, about 4.49, about 4.5, about 4.51, about 4.52, about 4.53, about 4.54, about 4.55, about 4.56, about 4.57, about 4.58, about 4.59, about 4.6 4.61, about 4.62, about 4.63, about 4.64, about 4.65, about 4.66, about 4.67, about 4.68, about 4.69, about 4.7, about 4.71, about 4.72, about 4.73, about 4.74, about 4.75, about 4.76, about 4.77, about 4.78, about 4.79, about 4.8, about 4.81, about 4.82, about 4.83, about 4.84, about 4.85, about 4.86, about 4.87, about 4.88, about 4.89, about 4.9, about 4.91, about 4.92, about 4.93, about 4.94, about 4.95, about 4.96, about 4.97, about 4.98, about 4.99, about 5.0.

[0398] In certain embodiments, the DAR of the ADC compositions described herein is about 2.28, about 2.70, about 2.14, about 2.09, about 4.17, about 4.06, about 3.89, or about 4.02.

[0399] In certain embodiments, the DAR of the ADC compositions described herein is 2.28, 2.70, 2.14, 2.09, 4.17, 4.06, 3.89, or 4.02.

[0400] In certain embodiments, the composition is composed of Ab-[L'-D] x In certain embodiments, the composition comprises an antibody drug conjugate. In certain embodiments, the DAR of the composition is 1-3, for example, about 2. In certain embodiments, the DAR of the composition is 2.28. In certain embodiments, the DAR of the composition is 2.14. In certain embodiments, the DAR of the composition is 2.09. In certain embodiments, the DAR of the composition is 2.28, and 50-90% (e.g., 60%-90%, such as 60%-70%, and another example is 67.43%) of the antibody drug conjugate with x being 2. In certain embodiments, the DAR of the composition is 2.14, and 50-90% (e.g., 55%-70%, such as 55%-65%, and another example is 60.52% or 62.70%) of the antibody drug conjugate with x being 2.

[0401] Pharmaceutical composition

[0402] In another aspect, the present application provides a pharmaceutical composition comprising the antibody-drug conjugate described in any one of the foregoing items, or the drug-linker described in any one of the foregoing items, and one or more pharmaceutical excipients.

[0403] The antibody drug conjugates, compounds or drug-linkers described herein can be formulated in a unit injectable form together with a pharmaceutically acceptable parenteral vehicle for parenteral use, such as bolus injection, intravenous injection, intratumoral injection, etc. Optionally, the antibody drug conjugate having the desired purity is mixed with a pharmaceutically acceptable diluent, carrier, excipient or stabilizer in the form of a lyophilized agent or solution (Remington's Pharmaceutical Sciences (1980) 16 th The antibody drug conjugates described herein or pharmaceutical compositions containing the same can be administered by any route appropriate to the subject to be treated.

[0404] application

[0405] The antibody drug conjugates, drug-linkers, compositions, or pharmaceutical compositions thereof described herein can be used to treat a variety of diseases or conditions, such as Her2-expressing cancers, including solid tumors or hematological malignancies, such as urothelial carcinoma, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, particularly lung adenocarcinoma), or lymphoma.

[0406] Therefore, the present application provides use of any of the above-mentioned antibody-drug conjugates, drug-linkers, compositions, or pharmaceutical compositions containing the same in the preparation of drugs for preventing or treating Her2-expressing cancers.

[0407] At the same time, the present application provides any of the above-mentioned antibody-drug conjugates, drug-linkers, compositions, or pharmaceutical compositions containing the same, and their use in drugs for preventing or treating Her2-expressing cancers.

[0408] At the same time, the present application also provides a method for preventing or treating Her2-expressing cancer, which comprises the step of administering an effective amount of any of the above-mentioned antibody-drug conjugates, drug-linkers, compositions, or pharmaceutical compositions containing the same to a subject in need thereof.

[0409] In some embodiments, the antibody drug conjugate, drug-linker, combination thereof, or pharmaceutical composition is sufficient (e.g., in a subject):

[0410] (1) Inhibit the proliferation of cells (such as tumor cells);

[0411] (2) inhibit tumor growth;

[0412] (3) induce and / or increase antibody-dependent cellular cytotoxicity activity;

[0413] (4) inhibiting HER2-mediated signal transduction;

[0414] (5) preventing and / or treating HER2-mediated diseases / disorders; or

[0415] (6) Any combination of (1) to (5) above.

[0416] In some embodiments, the cancer is selected from a solid tumor or a hematological malignancy; for example, selected from gastric cancer, breast cancer, lung cancer (eg, non-small cell lung cancer, particularly lung adenocarcinoma), and urothelial carcinoma.

[0417] 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.

[0418] definition

[0419] 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.

[0420] 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.

[0421] 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).

[0422] 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.

[0423] 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.

[0424] 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.

[0425] 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.

[0426] The term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0427] 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.

[0428] 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.

[0429] 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.

[0430] 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.

[0431] 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.

[0432] 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.

[0433] 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.

[0434] 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.

[0435] 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.

[0436] 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.

[0437] 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.

[0438] 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).

[0439] 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.

[0440] The term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which amino acid residues are substituted with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0441] 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.

[0442] 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.

[0443] 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.

[0444] 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).

[0445] 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.

[0446] As used herein, Indicates the site where the structural fragment is connected to the rest of the molecule.

[0447] 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.

[0448] The term "alkenyl" refers to a straight or branched chain hydrocarbon group containing at least one carbon-carbon double bond, including, for example, "C 2-6 Alkenyl", "C 2-4 Examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, and the like.

[0449] The term "alkynyl" refers to a straight or branched chain hydrocarbon group containing at least one carbon-carbon triple bond. 2-6 Alkynyl", "C 4-6 Examples include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1,3-butadiynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,4-hexadiynyl, and the like.

[0450] The term "cycloalkyl" refers to a saturated cyclic hydrocarbon group, including but not limited to monocyclic alkyl and bicyclic alkyl (such as spirocyclic alkyl, bicyclic alkyl and bridged cycloalkyl). 3-6 "Cycloalkyl" refers to a cycloalkyl group having 3 to 6 ring carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc., which may be optionally substituted with 1 or more (such as 1, 2 or 3) suitable substituents, for example methyl substituted cyclopropyl.

[0451] The term "heterocyclyl" or "heterocycle" refers to a saturated or partially saturated, monocyclic or polycyclic (such as a bicyclic) non-aromatic cyclic structure, the ring atoms of which are composed of carbon atoms and at least one (e.g., 1, 2, or 3) heteroatoms selected from nitrogen, oxygen, and sulfur. If the valence bond requirements are met, the heterocyclyl can be connected to the rest of the molecule via any one of the ring atoms. The heterocyclyl in the present invention is preferably a 3-6 membered heterocyclyl. The term "3-6 membered heterocyclyl" as used in the present invention refers to a heterocyclyl having 3 to 6 ring atoms, including 3-membered heterocyclyl, 4-membered heterocyclyl, 5-membered heterocyclyl, and 6-membered heterocyclyl, including nitrogen-containing heterocyclyl, oxygen-containing heterocyclyl, such as 4-6 membered heterocyclyl, such as 4-6 membered nitrogen-containing heterocyclyl, 4-6 membered oxygen-containing heterocyclyl. Common heterocyclic groups include, but are not limited to, azetidinyl, oxetanyl, tetrahydrofuryl, pyrrolidinyl, pyrrolidinonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, and morpholinyl. The heterocyclic groups of the present invention may be optionally substituted with one or more substituents described herein. The heterocyclic groups of the present invention may be optionally fused to one or more aromatic or non-aromatic rings.

[0452] The term "oxygen-containing heterocycle" refers to a heterocycle as described above in which one or more (e.g., 1, 2, or 3) ring atoms are oxygen atoms, such as a 5-6 membered oxygen-containing heterocycle, and specific examples include but are not limited to an oxirane ring, a tetrahydrofuran ring, a furan ring, a tetrahydropyran ring, a pyran ring, and the like. The term "nitrogen-containing heterocycle" as used herein refers to a heterocycle as described above in which one or more (e.g., 1, 2, or 3) ring atoms are nitrogen atoms.

[0453] The term "alkoxy" refers to a group having the structure "alkyl-O-", wherein alkyl is as defined above. 1-6 Alkoxy, C 1-4 Alkoxy, C 1-3 Alkoxy or C 1-2 Alkoxy, etc. Common alkoxy groups include (but are not limited to) methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy, etc. The alkoxy group in the present invention is optionally substituted with one or more substituents described herein.

[0454] The term "halo" or "halogen" group is defined to include F, Cl, Br, or I.

[0455] 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).

[0456] 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 1-6Haloalkoxy, halogen, hydroxy, carboxyl and ester groups (e.g. -C 1-6 Alkyl-C(=O)-OC 1-6 alkyl).

[0457] The term "substituted" refers to the replacement of one or more (e.g., 1, 2, 3, 4, or 5) hydrogens on the specified compound or structural fragment by a substituent, provided that the normal valence of the specified atom in the current situation is not exceeded and the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form stable compounds. In some embodiments, the substituents are each independently composed of one or more of the following structures: -O-, -S-, -NR'-, halogen, -CN, -OH, -NH2, -NO2, -CN, =O, C1-C6 (alkylene), C1-C6 haloalkylene, C1-C6 alkoxy, C2-C6 (alkenyl), C2-C6 (alkynyl), C3-C8 (cycloalkyl), 3-8 membered (heterocyclyl), C6-C 10 (Ethyl)aryl and 5-10 membered (Ethyl)heteroaryl, etc. In some embodiments, the substituents are each independently composed of one or more of the following structures: NR 8 R 9 , -O-, -S-, -NR'-, halogen, -CN, -OH, -SH, -NH2, -NO2, -C(O)-, -CN, =O, C1-C6 (alkylene), C1-C6 haloalkylene, C1-C6 alkoxy, C2-C6 (alkenyl), C2-C6 (alkynyl), C3-C8 (cycloalkyl), 3-10 membered (heterocyclyl), C6-C 10 (Ethyl)aryl and 5-10 membered (Ethyl)heteroaryl, etc., wherein R 8 、R 9 and R' are as defined above. For example, the substituent may be a suitable substituent as described above.

[0458] 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.

[0459] As used herein, the term "one or more" means 1 or more than 1, such as 2, 3, 4, 5 or 10, where reasonable.

[0460] Unless otherwise indicated, as used herein, the point of attachment of a substituent may be from any suitable position of the substituent.

[0461] Pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts and base addition salts thereof. Suitable acid addition salts are formed from acids that form pharmaceutically acceptable salts, including aspartate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, etc. Suitable base addition salts are formed from bases that form pharmaceutically acceptable salts, including aluminum salts, arginine salts, choline salts, diethylamine salts, etc. Suitable salts are reviewed in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0462] 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

[0463] Figure 1: Efficacy of anti-human HER2 antibody-drug conjugate in the NCI-N87 cell subcutaneous tumor-bearing mouse model

[0464] Figure 2 Changes in body weight of mice in each group in the human gastric cancer cell NCI-N87 CDX model

[0465] Figure 3: Efficacy of anti-human HER2 antibody-drug conjugate in JIMT-1 breast cancer xenograft model

[0466] Figure 4. Changes in body weight of mice in each group in the JIMT-1 breast cancer transplant model DETAILED DESCRIPTION

[0467] 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.

[0468] The information of the sequences involved in the present invention is described in the following table:

[0469] The abbreviations used in this document have the following meanings:

[0470] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0471] 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).

[0472] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the examples are shown below.

[0473] 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.

[0474] Mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0475] Intermediate Preparation Example 1: 2-((((4-(Methoxycarbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (INT-1)

[0476] Step 1: Preparation of 1-(N-((2-(benzyloxy)-2-oxoethoxy)carbonyl)sulfamoyl)piperidine-4-carboxylic acid methyl ester (INT-1-2)

[0477] Dissolve chlorosulfonyl isocyanate (500 mg, 3.53 mmol, 307 μL) in dichloromethane (10.0 mL) at 0°C. Add benzyl glycolate (533 mg, 3.21 mmol, 455 μL) and continue stirring for 1 hour. Then, add a solution of methyl 4-piperidinylcarboxylate (459 mg, 3.21 mmol) and triethylamine (974 mg, 9.63 mmol, 1.34 mL) in dichloromethane (5.00 mL) to the reaction mixture. Warm to 25°C and continue stirring for 1 hour. Add water (100 mL) to the reaction mixture, and extract with dichloromethane three times (50.0 mL x 3). The combined organic phases were washed with cooled 1N dilute hydrochloric acid (20.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (1.30 g, 3.14 mmol), which was used directly in the next step without purification.

[0478] The structural characterization data are as follows:

[0479] ESI-MS (m / z): 414.9 [M+H] +

[0480] Step 2: Preparation of 2-((((4-(methoxycarbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (INT-1)

[0481] Under a nitrogen atmosphere, 10% Pd / C (0.70 g) was added to a solution of compound INT-1-2 (700 mg, 1.69 mmol) in methanol (20.0 mL). The atmosphere was replaced with hydrogen three times and then reacted at 25°C for 3 hours (15 PSI). The reaction mixture was filtered, and the filter cake was rinsed with methanol three times (100 mL x 3). The filtrate was concentrated to obtain the crude title compound (590 mg).

[0482] The structural characterization data are as follows:

[0483] ESI-MS (m / z): 325.1 [M+H] +

[0484] Intermediate Preparation Example 2: 2-((((4-((allyloxy)carbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (INT-2)

[0485] Step 1: Preparation of allyl 1-(N-((2-(tert-butoxy)-2-oxoethoxy)carbonyl)sulfamoyl)piperidine-4-carboxylate (INT-2-2)

[0486] Dissolve chlorosulfonyl isocyanate (1.76 g, 12.41 mmol) in acetonitrile (80.0 mL) at 0°C, add tert-butyl glycolate (1.56 g, 11.82 mmol, 455 μL), and continue stirring for 1 hour. Then, add allyl 4-piperidinylcarboxylate (2 g, 11.82 mmol) and pyridine (1.12 g, 14.18 mmol) to the reaction mixture, warm to 25°C, and continue stirring for 1 hour. Water (100 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (25.0 mL x 3). The combined organic phases were washed with cooled 1N dilute hydrochloric acid (20.0 mL) and saturated sodium chloride solution (30.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (2.3 g, 5.66 mmol), which was used directly in the next step without purification.

[0487] Its structural characterization data are as follows:

[0488] ESI-MS (m / z): 407.1 [M+H] +

[0489] Step 2: Preparation of 2-((((4-((allyloxy)carbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (INT-2)

[0490] Compound INT-2-2 (2.3 g, 5.66 mmol) was dissolved in dichloromethane (10 mL), TFA (2 mL) was added, and the mixture was reacted at 25°C for 2 hours. The reaction solution was concentrated, and the pH was adjusted to 8 by adding 1N aqueous sodium bicarbonate solution. The mixture was extracted twice with ethyl acetate (25.0 mL x 2). The combined aqueous phases were adjusted to pH 3 with 1N dilute hydrochloric acid, and then extracted three times with ethyl acetate (25.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (1.6 g, 4.57 mmol).

[0491] Its structural characterization data are as follows:

[0492] ESI-MS (m / z): 351.1 [M+H] +

[0493] Intermediate Preparation Example 3: 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-3)

[0494] Step 1: Preparation of 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid methyl ester (INT-3-2)

[0495] 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.

[0496] Its structural characterization data are as follows:

[0497] ESI-MS (m / z): 385.1 [M+H] + .

[0498] Step 2: Preparation of 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid (INT-3-3)

[0499] 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.

[0500] Its structural characterization data are as follows:

[0501] ESI-MS (m / z): 371.1 [M+H] + .

[0502] 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-3-4)

[0503] 3,5-Bis(2-(methylthio)pyrimidin-5-yl)benzoic acid (3.00 g, 8.10 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptane-27-ate (4.03 g, 8.10 mmol) were added to DMF (40 mL), and HOBt (3.28 g, 24.3 mmol), EDCI (4.66 g, 24.3 mmol) and DIPEA (4.19 g, 32.4 mmol, 5.64 mL) were added in sequence, and the reaction system was stirred at 60 ° C for 2 hours. Water (100 mL) and ethyl acetate (60 mL x 3) were added to the reaction solution, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give tert-butyl 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oate (4.20 g, 4.14 mmol), which was used in the next step without purification.

[0504] 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-3-5)

[0505] Dissolve tert-butyl 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosadecane-29-oate (3.60 g, 4.24 mmol) in dichloromethane (30 mL). Add TFA (15.3 g, 134 mmol, 10 mL). Stir the reaction system at 25°C for 6 hours. Add water (60 mL) and extract with ethyl acetate (40 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by preparative high performance liquid chromatography and freeze-dried to give 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid (2.93 g, 3.63 mmol).

[0506] Its structural characterization data are as follows:

[0507] ESI-MS (m / z): 794.3 [M+H] + .

[0508] The purification method is as follows:

[0509] Chromatographic column: Phenomenex luna C18 (250mm*70mm*10μm)

[0510] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0511] 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-3)

[0512] 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonaconicoside-29-oic acid (148 mg, 0.186 mmol) 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 minutes. The reaction system was extracted with water and ethyl acetate and concentrated to obtain the title compound (155 mg).

[0513] Its structural characterization data are as follows:

[0514] ESI-MS (m / z): 858.3 [M+H] + .

[0515] Intermediate Preparation Example 4: 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-4)

[0516] 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-4-2)

[0517] Use standard solid phase synthesis methods:

[0518] 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.

[0519] 2) Coupling: A solution of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methylglycine (5.60 g, 6.00 equiv) and HATU (6.58 g, 5.70 equiv) in DMF (10.0 mL) was added to the resin under nitrogen bubbling. DIPEA (6.00 equiv) was added dropwise, and nitrogen was bubbled at 20°C for 30 minutes. The resin was washed with DMF (30.0 mL x 5) before proceeding to the next step.

[0520] 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.

[0521] 4) Repeat steps 2 and 3 using the amino acids in Table 1: numbers 2-10 in Table 1.

[0522] 5) The resin was then washed with DMF (30.0 mL x 5) and MeOH (30.0 mL x 5), and then dried under vacuum.

[0523] Table 1:

[0524] Peptide cleavage and purification:

[0525] 1) Add cleavage solution (TFA / DCM, 1 / 100, v / v, 200.0 mL) to the flask containing the side-chain protected peptide at room temperature and stir for 3 minutes twice.

[0526] 2) After filtration, the filtrates were combined and concentrated.

[0527] 3) The crude product was purified by HPLC and freeze-dried to obtain the title compound (1117.1 mg, TFA salt).

[0528] Its structural characterization data are as follows:

[0529] ESI-MS (m / z): 1010.4 (M+H) + .

[0530] The purification method is as follows:

[0531] 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-4)

[0532] 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26-nonamethyl-1,4,7,10,13,16,19,22,25-nonaoxo-2,5,81,14,17,0,23,26-nonaazaoctacosane-28-ynoic 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).

[0533] Its structural characterization data are as follows:

[0534] ESI-MS (m / z): 1074.4 (M+H) + .

[0535] Intermediate Preparation Example 5: 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 (INT-5)

[0536] Step 1: Preparation of 2,5-dioxopyrrolidin-1-yl 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoate (INT-5-1)

[0537] 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), then add dicyclohexylcarbodiimide (0.4 g, 1.94 mmol), and stir at room temperature for 2 hours. After completion of the reaction, filter, 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.

[0538] 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 (INT-5-2)

[0539] 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).

[0540] Its structural characterization data are as follows:

[0541] MS m / z(ESI):662.2[M+H] +

[0542] 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 (INT-5)

[0543] Dissolve 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosane-20-oleic 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).

[0544] Its structural characterization data are as follows:

[0545] MS m / z(ESI):726.1[M+H] +

[0546] Intermediate Preparation Example 6: Preparation of Allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6)

[0547] Step 1: Preparation of allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6-2)

[0548] Allyl (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (150.0 mg, 0.31 mmol) and (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine (115.1 mg, 0.37 mmol) were dissolved in DCM (8 mL) and MeOH (2 mL). 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (229.7 mg, 0.93 mmol) was added to the mixture. 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).

[0549] Its structural characterization data are as follows:

[0550] MS m / z(ESI):782.2[M+H] +

[0551] Step 2: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (INT-6-3)

[0552] 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).

[0553] Its structural characterization data are as follows:

[0554] MS m / z(ESI):566.1[M+Na] +

[0555] Step 3: Preparation of allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6)

[0556] (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).

[0557] Its structural characterization data are as follows:

[0558] MS m / z(ESI):731.2[M+Na] +

[0559] Example 1: 1-(N-((9S,13S)-13-hydroxy-6-isopropyl-14-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctacoshydro-2 Pentafluorophenyl 5H-2,27:5,8:11,15-triepoxy-23,25-ethano-2,28-furo[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)-9-methyl-4,7,10-trioxo-2-oxa-5,8,11-triazatetradecanoyl)sulfamoyl)piperidine-4-carboxylate (J-1)

[0560] Step 1: Preparation of 2,5-dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)-D-valyl-L-alaninate (J-1-2)

[0561] (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valyl-L-alanine (300 mg, 730.88 μmol), N-hydroxysuccinimide (84.12 mg, 730.88 μmol) and DCC (165.88 mg, 803.97 μmol) were added to THF (20 mL) and reacted at 25°C for 18 h. A large amount of white solid precipitated during the reaction. The solid was removed by filtration, and the filtrate was collected and concentrated to dryness to obtain 515 mg of the title compound with a purity of 70%, which was used directly in the next reaction without purification.

[0562] Step 2: (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21- Preparation of 2,27:5,8:11,15-triepoxy-2,3,25-ethano-2,28-methylfuro[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxolan-18-yl)propyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (J-1-3)

[0563] J-1-2 (198.67 mg, 274.01 μmol, 70%), eribulin (50 mg, 68.50 μmol), and DIPEA (35.41 mg, 274.01 μmol) were added to DMF (10 mL) and stirred at 25°C for 3 h. The reaction was monitored by LC-MS. The reaction solution was concentrated to obtain the crude product, which was purified by preparative HPLC and freeze-dried to afford 47 mg of the title compound.

[0564] Its structural characterization data are as follows:

[0565] ESI-MS (m / z): 1122.7 [M+H] + .

[0566] The preparation method is as follows:

[0567] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0568] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0569] Step 3: (S)-2-amino-N-((S)-1-(((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene Preparation of -21-oxooctahydro-25H-2,27:5,8:11,15-triepoxy-23,25-ethano-2,28-methylfuro[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxolane-18-yl)propyl)amino)-1-oxopropane-2-yl)-3-methylbutanamide (J-1-4)

[0570] J-1-3 (47 mg, 41.88 μmol) and DBU (12.75 mg, 83.75 μmol) were added to DMF (5 mL) and reacted at 25 °C for 2 h. Formic acid (7.71 mg, 167.51 μmol) was added to the reaction system and stirred for 20 min. Water and MTBE were added to the reaction system, and some impurities were extracted and removed. The aqueous phase was freeze-dried to obtain 55 mg (crude) of the title compound, which was used directly in the next reaction without further purification.

[0571] Its structural characterization data are as follows:

[0572] ESI-MS (m / z): 900.6 [M+H]+ .

[0573] Step 4: 1-(N-((9S,13S)-13-hydroxy-6-isopropyl-14-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctacosyl-25H Preparation of methyl-2,27:5,8:11,15-triepoxy-23,25-ethano-2,28-furo[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)-9-methyl-4,7,10-trioxo-2-oxa-5,8,11-triazatetradecanoyl)sulfamoyl)piperidine-4-carboxylate (J-1-5)

[0574] J-1-4 (53.85 mg, 38.88 μmol, crude) and DIPEA (25.13 mg, 194.42 μmol) were added to DMF (10 mL) and stirred for 5 minutes. 2-[(4-methoxycarbonyl-1-piperidinyl)sulfonylaminoformyloxy]acetic acid (25.22 mg, 77.77 μmol) and HATU (29.57 mg, 77.77 μmol) were then added to the reaction system. The reaction was allowed to react at 25°C for 18 hours. The reaction was monitored by LC-MS. The reaction solution was concentrated to obtain the crude product, which was purified by column chromatography (MeOH / EA = 0-15%) to afford 28 mg of the title compound.

[0575] Its structural characterization data are as follows:

[0576] ESI-MS (m / z): 1206.6 [M+H] + .

[0577] Step 5: 1-(N-((9S,13S)-13-hydroxy-6-isopropyl-14-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctacoshydro-25 Preparation of H-2,27:5,8:11,15-triepoxy-23,25-ethano-2,28-furoxane [3,2-i] furo [2',3':5,6] pyrano [4,3-b] [1,4] dioxol-18-yl) -9-methyl-4,7,10-trioxo-2-oxa-5,8,11-triazatetradecanoyl) sulfamoyl) piperidine-4-carboxylic acid (J-1-6)

[0578] J-1-5 (30 mg, 24.87 μmol) and lithium hydroxide monohydrate (40.74 mg, 994.70 μmol) were added to MeOH (12 mL) and water (6 mL), and the mixture was reacted at 35 °C for 4 h. The reaction was monitored by LC-MS. The pH of the system was adjusted to about 3 with 0.5 N HCl. The mixture was extracted with ethyl acetate and concentrated to dryness to obtain 28 mg of the title compound, which was used directly in the next step without further purification.

[0579] Its structural characterization data are as follows:

[0580] ESI-MS (m / z): 1193.5 [M+H] + .

[0581] Step 6: 1-(N-((9S,13S)-13-hydroxy-6-isopropyl-14-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctacoshydro-25H Preparation of pentafluorophenyl-2,27:5,8:11,15-triepoxy-2,3,25-ethano-2,28-furo[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)-9-methyl-4,7,10-trioxo-2-oxa-5,8,11-triazatetradecanoyl)sulfamoyl)piperidine-4-carboxylate (J-1)

[0582] Compound J-1-6 (28 mg, 23.48 μmol) and EDCI (45.02 mg, 234.83 μmol) were added to DMF (5 mL) and reacted at 25°C for 16 h. The reaction was monitored by LC-MS. The reaction solution was concentrated to obtain a crude product, which was purified by preparative HPLC and freeze-dried to obtain 0.50 mg of the title compound.

[0583] Its structural characterization data are as follows:

[0584] ESI-MS (m / z): 1358.6 [M+H] + .

[0585] The preparation method is as follows:

[0586] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0587] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0588] Example 2: 1-(N-((6S,9S,12S,16S)-16-hydroxy-17-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctadecanoic acid-25H-2, Pentafluorophenyl 2,28-furofuryl[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)-6,9,12-trimethyl-4,7,10,13-tetraoxo-2-oxa-5,8,11,14-tetraazaheptadecanoyl)sulfamoyl)piperidine-4-carboxylate (J-3)

[0589] Step 1: Preparation of 2,5-dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanyl-L-alaninate (J-3-2)

[0590] (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanyl-L-alanine (200.00 mg, 441.03 μmol), N-hydroxysuccinimide (101.51 mg, 882.05 μmol, FR), and EDCI (169.09 mg, 882.05 μmol) were added to DMF (10 mL) and reacted at 25°C for 1 hour. Water and ethyl acetate were added to the reaction solution, extracted, dried over sodium sulfate, and concentrated to obtain 206 mg of the title compound with a purity of 90%. It was used directly in the next reaction without purification.

[0591] Its structural characterization data are as follows:

[0592] ESI-MS (m / z): 551.3 [M+H] + .

[0593] Step 2: (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxo Preparation of octadisohydro-25H-2,27:5,8:11,15-triepoxy-23,25-ethano-2,28-furo[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)propyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)carbamate (J-3-3)

[0594] Compound J-3-2 (178.65 mg, 308.26 μmol, 90% purity), eribulin (90 mg, 123.31 μmol), and DIPEA (47.81 mg, 369.92 μmol) were added to DMF (10 mL) and stirred at 25°C for 18 hours. The reaction mixture was directly concentrated to obtain a crude product, which was purified by preparative HPLC and freeze-dried to yield 67 mg of the title compound.

[0595] Its structural characterization data are as follows:

[0596] ESI-MS (m / z): 1165.7 [M+H] + .

[0597] The preparation method is as follows:

[0598] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0599] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0600] Step 3: (S)-2-amino-N-((S)-1-(((S)-1-(((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene- Preparation of 21-oxooctacoshydro-25H-2,27:5,8:11,15-triepoxy-23,25-ethano-2,28-furo[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)propyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propionamide (J-3-4)

[0601] Compound J-3-3 (66 mg, 56.63 μmol) and DBU (17.24 mg, 113.27 μmol) were added to DMF (10 mL) and reacted at 25 ° C for 2 hours. Formic acid (10.43 mg, 226.54 μmol) was added to the reaction system and stirred for 20 minutes. Water and MTBE were added to the reaction system, and some impurities were extracted and removed. The aqueous phase was freeze-dried to obtain 73 mg (crude) of the title compound, which was used directly in the next reaction without further purification.

[0602] Its structural characterization data are as follows:

[0603] ESI-MS (m / z): 943.6 [M+H] + .

[0604] Step 4: 1-(N-((6S,9S,12S,16S)-16-hydroxy-17-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctacosyl-25H-2,27 Preparation of methyl (5,8:11,15-triepoxy-23,25-ethano-2,28-furoxamethylene[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)-6,9,12-trimethyl-4,7,10,13-tetraoxo-2-oxa-5,8,11,14-tetraazaheptadecanoyl)sulfamoyl)piperidine-4-carboxylate (J-3-5)

[0605] Compound J-3-4 (65 mg, 48.24 μmol, crude) and DIPEA (31.18 mg, 241.22 μmol) were added to DMF (10 mL) and stirred for 10 min. 2-[(4-methoxycarbonyl-1-piperidinyl)sulfonylaminoformyloxy]acetic acid (31.29 mg, 96.49 μmol) and HATU (36.69 mg, 96.49 μmol) were then added to the reaction system and allowed to react at 25°C for 16 hours. The reaction solution was directly concentrated to obtain the crude product, which was purified by column chromatography (MeOH / DCM = 0-10%) to obtain 32 mg of the title compound.

[0606] Its structural characterization data are as follows:

[0607] ESI-MS (m / z): 1250.6 [M+H] + .

[0608] Step 5: 1-(N-((6S,9S,12S,16S)-16-hydroxy-17-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctacoshydro-25H-2,2 Preparation of 7:5,8:11,15-triepoxy-23,25-ethano-2,28-furoxamethylene[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)-6,9,12-trimethyl-4,7,10,13-tetraoxo-2-oxa-5,8,11,14-tetraazaheptadecanoyl)sulfamoyl)piperidine-4-carboxylic acid (J-3-6)

[0609] Compound J-3-5 (39 mg, 31.21 μmol, FR) and lithium hydroxide monohydrate (38.85 mg, 936.44 μmol) were added to MeOH (15 mL) and water (7.5 mL), reacted at 35 ° C for 4 h, and the pH of the system was adjusted to about 3 with 0.5N HCl. The mixture was extracted with ethyl acetate and concentrated to dryness to obtain 34 mg of the title compound, which was used directly in the next reaction without further purification.

[0610] Step 6: 1-(N-((6S,9S,12S,16S)-16-hydroxy-17-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctacoshydro-25H-2,27 Preparation of pentafluorophenyl (5,8:11,15-triepoxy-23,25-ethano-2,28-furoxamethylene[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)-6,9,12-trimethyl-4,7,10,13-tetraoxo-2-oxa-5,8,11,14-tetraazaheptadecanoyl)sulfamoyl)piperidine-4-carboxylate (J-3)

[0611] Compound J-3-6 (34 mg, 27.52 μmol), 2,3,4,5,6-pentafluorophenol (75.99 mg, 412.82 μmol), and EDCI (94.97 mg, 495.39 μmol) were added to DMF (10 mL) and reacted at 25°C for 5 hours. The reaction solution was directly concentrated to obtain a crude product, which was purified by preparative HPLC and freeze-dried to obtain 3 mg of the title compound.

[0612] Its structural characterization data are as follows:

[0613] ESI-MS (m / z): 1401.6 [M+H] + .

[0614] The preparation method is as follows:

[0615] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0616] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0617] Example 3: 1-(N-((12S,19S)-12-benzyl-19-hydroxy-20-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctadecanoyl-25H- Pentafluorophenyl 2,27:5,8:11,15-triepoxy-2,3,25-ethano-2,28-furofuryl[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)-4,7,10,13,16-pentaoxo-2-oxa-5,8,11,14,17-pentaazaeicosanoyl)sulfamoyl)piperidine-4-carboxylate (J-6)

[0618] Step 1: Preparation of (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanylglycine 2,5-dioxopyrrolidin-1-yl ester (J-6-2)

[0619] (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanylglycine (500.00 mg, 895.13 μmol), N-hydroxysuccinimide (206.04 mg, 1.79 mmol) and EDCI (343.19 mg, 1.79 mmol) were added to DMF (10 mL) and reacted at 25°C for 2 hours. Water and ethyl acetate were added, extracted, dried over sodium sulfate, and concentrated to give 640 mg of the title compound with a purity of 90%. It was used directly in the next reaction without purification.

[0620] Its structural characterization data are as follows:

[0621] ESI-MS (m / z): 673.3 [M+18] + .

[0622] Step 2: (9H-fluoren-9-yl)methyl((7S,14S)-7-benzyl-14-hydroxy-15-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21 Preparation of 2,5,8,11-tetrahydro-2,6,9,12-tetraazapentadecyl-2,2,8-furylmethane-2,2,7:5,8:11,15-triepoxy-2,3,25-ethano-2,28-furylmethane [3,2-i] furo [2',3':5,6] pyrano [4,3-b] [1,4] dioxol-18-yl]-2,5,8,11-tetrahydro-3,6,9,12-tetraazapentadecyl] carbamate (J-6-3)

[0623] Compound J-6-2 (349.33 mg, 479.52 μmol, 90% purity), eribulin (100 mg, 137.01 μmol), and DIPEA (70.83 mg, 548.02 μmol) were added to DMF (10 mL) and stirred at 25°C for 18 hours. The reaction mixture was directly concentrated to obtain a crude product, which was purified by preparative HPLC and freeze-dried to yield 125 mg of the title compound.

[0624] Its structural characterization data are as follows:

[0625] ESI-MS (m / z): 1270.7 [M+H] + .

[0626] The preparation method is as follows:

[0627] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0628] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0629] Step 3: (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7, Preparation of 14-dimethylene-21-oxooctacoshydro-25H-2,27:5,8:11,15-triepoxy-23,25-ethano-2,28-furoxamethylene[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)propyl)amino)-2-oxoethyl)-3-phenylpropionamide (J-6-4)

[0630] Compound J-6-3 (125 mg, 98.39 μmol) and DBU (44.94 mg, 295.17 μmol) were added to DMF (8 mL) and reacted at 25°C for 2 hours. The reaction solution was directly concentrated to obtain a crude product, which was purified by preparative HPLC and freeze-dried to obtain 64 mg of the title compound.

[0631] Its structural characterization data are as follows:

[0632] ESI-MS (m / z): 1048.6 [M+H] + .

[0633] The preparation method is as follows:

[0634] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0635] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0636] Step 4: 1-(N-((12S,19S)-12-benzyl-19-hydroxy-20-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctahydro-25H-2,2 Preparation of 7:5,8:11,15-triepoxy-23,25-ethano-2,28-furomethane[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)-4,7,10,13,16-pentaoxo-2-oxa-5,8,11,14,17-pentaazaeicosanoyl)sulfamoyl)piperidine-4-carboxylic acid allyl ester (J-6-5)

[0637] Compound J-6-4 (53 mg, 48.44 μmol) and DIPEA (31.30 mg, 242.18 μmol) were added to DMF (10 mL) and stirred for 5 minutes. 2-[(4-allyloxycarbonyl-1-piperidinyl)sulfonylcarbamoyloxy]acetic acid (37.71 mg, 96.87 μmol, 90% purity) and HATU (36.83 mg, 96.87 μmol) were then added to the reaction system and allowed to react at 25°C for 3 hours. The reaction system was directly concentrated to obtain a crude product, which was purified by preparative HPLC and freeze-dried to obtain 33 mg of the title compound.

[0638] Its structural characterization data are as follows:

[0639] ESI-MS (m / z): 1381.6 [M+H] + .

[0640] The preparation method is as follows:

[0641] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0642] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0643] Step 5: 11-(N-((12S,19S)-12-benzyl-19-hydroxy-20-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctahydro-25H-2 Preparation of 2,28-furoxyl-2,3,25-ethanoyl-2,28-furoxyl-2,27:5,8:11,15-triepoxy ...

[0644] Compound J-6-5 (30 mg, 21.73 μmol), morpholine (18.93 mg, 217.30 μmol), and tetrakistriphenylphosphine palladium (15.07 mg, 13.04 μmol) were dissolved in THF (20 mL). The mixture was evacuated and replaced with nitrogen three times. The reaction was allowed to proceed at 25°C for 16 hours. The reaction solution was directly concentrated to obtain a crude product, which was purified by preparative HPLC and freeze-dried to obtain 11 mg of the title compound.

[0645] Its structural characterization data are as follows:

[0646] ESI-MS (m / z): 1341.5 [M+H] + .

[0647] The preparation method is as follows:

[0648] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0649] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0650] Step 6: 1-(N-((12S,19S)-12-benzyl-19-hydroxy-20-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctacoshydro-25H-2, Preparation of Pentafluorophenyl (27:5,8:11,15-triepoxy-23,25-ethano-2,28-furo[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)-4,7,10,13,16-pentaoxo-2-oxa-5,8,11,14,17-pentaazaeicosanoyl)sulfamoyl)piperidine-4-carboxylate (J-6)

[0651] Compound J-6-6 (16 mg, 11.94 μmol), 2,3,4,5,6-pentafluorophenol (32.95 mg, 179.04 μmol), and EDCI (41.19 mg, 214.85 μmol) were added to DMF (5 mL) and reacted at 25°C for 4 hours. The reaction solution was directly concentrated to obtain a crude product, which was purified by preparative HPLC and freeze-dried to obtain 2.60 mg of the title compound.

[0652] Its structural characterization data are as follows:

[0653] ESI-MS (m / z): 1506.6 [M+H] + .

[0654] The preparation method is as follows:

[0655] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0656] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0657] Example 4: 1-(N-((6S,9S)-14-amino-9-((4-(((((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctahydro-25H-2,27: (5,8:11,15-Triepoxy-23,25-ethano-2,28-furo[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)propyl)carbamoyl)oxy)methyl)phenyl)carbamoyl)-6-isopropyl-4,7,14-trioxo-2-oxa-5,8,13-triazaester)tetradecanoyl)sulfamoyl)piperidine-4-carboxylic acid pentafluorophenyl ester (J-8)

[0658] Step 1: 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19- Preparation of methoxy-13-methyl-7,14-dimethylene-21-oxooctacoshydro-25H-2,27:5,8:11,15-triepoxy-23,25-ethano-2,28-furofurylmethane[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)propyl)carbamate (J-8-2)

[0659] Eribulin (90 mg, 123.31 μmol), (9H-fluoren-9-yl)methyl ((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino))-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate (199.05 mg, 246.61 μmol, purity 95%), HOBT (33.32 mg, 246.61 μmol) and pyridine (48.77 mg, 616.53 μmol) were added to DMF (10 mL), and the reaction was stirred at 25° C. for 18 hours. The reaction solution was directly concentrated to obtain a crude product, which was purified by preparative HPLC and freeze-dried to obtain 100 mg of the title compound.

[0660] Its structural characterization data are as follows:

[0661] ESI-MS (m / z): 1357.8 [M+H] + .

[0662] The preparation method is as follows:

[0663] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0664] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0665] Step 2: 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19- Preparation of methoxy-13-methyl-7,14-dimethylene-21-oxooctacosahydro-25H-2,27:5,8:11,15-triepoxy-23,25-ethano-2,28-furofurylmethane[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)propyl)carbamate (J-8-3)

[0666] Compound J-8-2 (100 mg, 73.66 μmol) was added to DMF (10 mL) and diethylamine (2 mL), stirred at 25°C for 50 minutes, and the reaction solution was directly concentrated to obtain a crude product, which was purified by preparative HPLC and freeze-dried to obtain 64 mg of the title compound.

[0667] Its structural characterization data are as follows:

[0668] ESI-MS (m / z): 1135.6 [M+H] + .

[0669] The preparation method is as follows:

[0670] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0671] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0672] Step 3: 1-(N-((6S,9S)-14-amino-9-((4-(((((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctahydro-25H-2,27:5, Preparation of 8:11,15-triepoxy-23,25-ethano-2,28-furomethane[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)propyl)carbamoyl)oxy)methyl)phenyl)carbamoyl)-6-isopropyl-4,7,14-trioxo-2-oxa-5,8,13-triazaester (tetradecanoyl)sulfamoyl)piperidine-4-carboxylic acid allyl ester (J-8-4)

[0673] Compound J-8-3 (27 mg, 22.85 μmol, crude) and DIPEA (14.77 mg, 114.77 μmol) were added to DMF (5 mL) and stirred for 5 minutes. 2-[(4-allyloxycarbonyl-1-piperidinyl)sulfonylcarbamoyloxy]acetic acid (17.79 mg, 45.71 μmol, 90% purity) and HATU (17.38 mg, 45.71 μmol) were then added to the reaction system. The reaction was allowed to proceed at 25°C for 16 hours. The reaction solution was concentrated to obtain the crude product, which was purified by preparative HPLC and freeze-dried to afford 19 mg of the title compound.

[0674] Its structural characterization data are as follows:

[0675] ESI-MS (m / z): 1467.7 [M+H] + .

[0676] The preparation method is as follows:

[0677] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0678] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0679] Step 4: 1-(N-((6S,9S)-14-amino-9-((4-(((((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctahydro-25H-2,27:5 Preparation of 1,8:11,15-triepoxy-2,3,25-ethano-2,28-furoxamethylene[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)propyl)carbamoyl)oxy)methyl)phenyl)carbamoyl)-6-isopropyl-4,7,14-trioxo-2-oxa-5,8,13-triazaester tetradecanoyl)sulfamoyl)piperidine-4-carboxylic acid (J-8-5)

[0680] Compound J-8-4 (35 mg, 23.85 μmol), morpholine (20.78 mg, 238.47 μmol), and tetrakistriphenylphosphine palladium (16.53 mg, 14.31 μmol) were dissolved in THF (25 mL). The mixture was evacuated and replaced with nitrogen three times. The reaction was allowed to react at 25°C for 2 hours. The reaction solution was directly concentrated to obtain a crude product, which was purified by preparative HPLC and freeze-dried to obtain 18 mg of the title compound.

[0681] Its structural characterization data are as follows:

[0682] ESI-MS (m / z): 1427.7 [M+H] + .

[0683] The preparation method is as follows:

[0684] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0685] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0686] Step 5: 1-(N-((6S,9S)-14-amino-9-((4-(((((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctahydro-25H-2,27:5, Preparation of 8:11,15-triepoxy-23,25-ethano-2,28-furo[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)propyl)carbamoyl)oxy)methyl)phenyl)carbamoyl)-6-isopropyl-4,7,14-trioxo-2-oxa-5,8,13-triazaester (tetradecanoyl)sulfamoyl)piperidine-4-carboxylic acid pentafluorophenyl ester (J-8)

[0687] Compound 5 (18 mg, 12.61 μmol), 2,3,4,5,6-pentafluorophenol (34.81 mg, 189.13 μmol), and EDCI (43.51 mg, 226.95 μmol) were added to DMF (5 mL) and reacted at 25°C for 3 hours. The reaction solution was directly concentrated to obtain a crude product, which was purified by preparative HPLC and freeze-dried to obtain 6 mg of the title compound.

[0688] Its structural characterization data are as follows:

[0689] ESI-MS (m / z): 1594.7 [M+H] + .

[0690] The preparation method is as follows:

[0691] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0692] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0693] Example 5: 4-((31S,34S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-31-isopropyl-1,29,32-trioxo-34-(3-ureidopropyl))-5,8,11,14,17,20,23,26-octaoxa-2,30,33-triazapentatriacontane-35-amido)benzyl((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R, (19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctacoshydro-25H-2,27:5,8:11,15-triepoxy-23,25-ethano-2,28-furofurylmethane[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)propyl)carbamate (H-1)

[0694] Compound J-8-3 (10 mg, 8.46 μmol) and DIPEA (5.47 mg, 42.32 μmol) were added to DMF (3 mL) and stirred for 10 min. 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 (14.52 mg, 16.93 μmol) and HATU (6.44 mg, 16.93 μmol) were then added to the reaction system and reacted at 25°C for 3.5 hours. The reaction solution was directly purified by preparative HPLC and freeze-dried to obtain 10.70 mg of the title compound.

[0695] Its structural characterization data are as follows:

[0696] ESI-MS (m / z): 1997.1 [M+Na] + .

[0697] The preparation method is as follows:

[0698] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0699] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0700] Example 6: 4-((33S,36S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-33-isopropyl-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28,31,34-dodecaoxo-36-(3-ureidopropyl)-2,5, 8,11,14,17,20,23,26,29,32,35-Dodecaazaheptatriacontanoyl-37-amido)benzyl((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19- Methoxy-13-methyl-7,14-dimethylene-21-oxooctacoshydro-25H-2,27:5,8:11,15-triepoxy-23,25-ethano-2,28-furofurylmethane[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)propyl)carbamate (H-6)

[0701] Compound J-8-3 (10 mg, 8.46 μmol), 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatriacontane-31-carboxylic acid (19.39 mg, 16.93 μmol), HATU (6.44 mg, 16.93 μmol), and DIPEA (5.47 mg, 42.32 μmol) were added to DMF (3 mL) and reacted at 25°C for 3 hours. The reaction solution was directly purified by preparative HPLC and freeze-dried to obtain 16 mg of the title compound.

[0702] Its structural characterization data are as follows:

[0703] ESI-MS (m / z): 1132.3 [1 / 2M+H] + .

[0704] The preparation method is as follows:

[0705] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0706] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0707] Example 7: N-((31S,34S,37S,41S)-41-hydroxy-42-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctahydro-25H-2,27:5,8:11,15-triepoxy-23,25-ethano-2,28-furomethane[3,2-i]furan[2 ',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)-3,6,9,12,15,18,21,24,27,31,34,37-dodecamethyl-2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaoxo-3,6,9,12,15,18,21,24,27,30,33,36,39-trideazatetradecanoyl)-N-methyl-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (H-8)

[0708] Compound J-3-4 (12 mg, 12.13 μmol), 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatriacontane-31- Carboxylic acid (27.79 mg, 24.26 μmol), HATU (9.23 mg, 24.26 μmol), and DIPEA (7.84 mg, 60.66 μmol) were added to DMF (3 mL). After reacting at 25°C for one hour, some starting material remained. HATU (9.23 mg, 24.26 μmol) and DIPEA (7.84 mg, 60.66 μmol) were added and the reaction continued for another hour. The reaction solution was directly concentrated to obtain a crude product, which was purified by preparative HPLC and freeze-dried to yield 14 mg of the title compound.

[0709] Its structural characterization data are as follows:

[0710] ESI-MS (m / z): 1044.6 [1 / 2 (M+18)] + .

[0711] The preparation method is as follows:

[0712] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0713] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0714] Example 8: N-((37S,44S)-37-benzyl-44-hydroxy-45-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctacoshydro-25H-2,27:5,8:11,15-triepoxy-23,25-ethano-2,28-furoxamethylene[3,2-i]furan [2',3':5,6]pyrano[4,3-b][1,4]dioxol-18-yl)-3,6,9,12,15,18,21,24,27-nonamethyl-2,5,8,11,14,17,20,23,26,29,32,35,38,41-tetradecano-3,6,9,12,15,18,21,24,27,30,33,36,39,42-tetradecazapentatetradecane)-N-methyl-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (H-10)

[0715] 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 (52.33 mg, 45.69 μmol l) was dissolved in DMF (5 mL), and HATU (17.37 mg, 45.69 μmol), DIPEA (14.76 mg, 114.23 μmol) and compound J-6-4 (25.0 mg, 22.85 μmol, formate) were added, and the mixture was reacted at 25°C for 2 h. The reaction solution was directly purified by HPLC and lyophilized to obtain the title compound (18.0 mg, 8.19 μmol).

[0716] Its structural characterization data are as follows:

[0717] ESI-MS (m / z): 1096.6 (1 / 2M+H) + .

[0718] The purification method is as follows:

[0719] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0720] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0721] Example 9: 1-(N-((12S,15S,19S)-19-hydroxy-12-isopropyl-20-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctadecanoyl-25H-2,2 Preparation of Pentafluorophenyl (7:5,8:11,15-triepoxy-23,25-ethano-2,28-furo[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxacyclopentacosan-18-yl)-15-methyl-4,7,10,13,16-pentaoxo-2-oxa-5,8,11,14,17-pentaazaeicosanoyl)sulfamoyl)piperidine-4-carboxylate (J-5)

[0722] Step 1: Preparation of 2,5-dioxypyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine ester (J-5-2)

[0723] (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine (1.50 g, 4.23 mmol), N-hydroxysuccinimide (974.34 mg, 8.47 mmol) and EDCI (1.62 g, 8.47 mmol) were added to DMF (20 mL) and reacted at 25 ° C for 2 h. Water and ethyl acetate were added for extraction and concentrated to dryness to obtain 1.9 g of the title compound, which was used directly in the next reaction without purification.

[0724] Its structural characterization data are as follows:

[0725] MS m / z(ESI):474.1[M+Na]+

[0726] Step 2: (9H-fluoren-9-yl)methyl((7S,10S,14S)-14-hydroxy-7-isopropyl-15-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21- Preparation of 2,27:5,8:11,15-triepoxy-2,3,25-ethano-2,28-methylfuro[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxacyclopentacosan-18-yl)-10-methyl-2,5,8,11-tetraoxo-3,6,9,12-tetraazapentadecyl)carbamate (J-5-3)

[0727] J-5-2 (127.87 mg, 283.26 μmol), J-1-4 (67 mg, 70.81 μmol, formate salt), and DIPEA (27.46 mg, 212.44 μmol) were added to DMF (5 mL) and stirred at 25°C for 6 h. The reaction solution was concentrated to obtain a crude product, which was purified by preparative HPLC and freeze-dried to obtain 49 mg of the title compound.

[0728] Its structural characterization data are as follows:

[0729] ESI-MS (m / z): 1237.8 [M+H] + .

[0730] The preparation method is as follows:

[0731] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0732] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0733] Step 3: (S)-2-(2-(2-aminoacetamido)acetamido)-N-((S)-1-(((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7 ... Preparation of 1-oxopropyl-2-(1,4-dimethyl-21-oxooctahydro-25H-2,27:5,8:11,15-triepoxy-23,25-ethano-2,28-methylfuro[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxacyclopentacosan-18-yl)propyl)amino)-1-oxopropyl-2-yl)-3-methylbutanamide (J-5-4)

[0734] J-5-3 (49 mg, 39.63 μmol) and DBU (18.10 mg, 118.89 μmol) were added to DMF (3 mL) and reacted at 25°C for 1 h. The reaction solution was concentrated to obtain a crude product, which was purified by reverse phase purification (ACN / 0.05% aqueous ammonium bicarbonate solution = 0-60%) and freeze-dried to obtain 40 mg of the title compound.

[0735] Its structural characterization data are as follows:

[0736] ESI-MS (m / z): 1014.6 [M+H] + .

[0737] Step 4: 1-(N-((12S,15S,19S)-19-hydroxy-12-isopropyl-20-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctacoshydro-25H-2,27:5,8:11,15-triepoxy-23,25 Preparation of 1,2-dioxo-2,28-furoxano[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxacyclopentacosan-18-yl)-15-methyl-4,7,10,13,16-pentaoxo-2-oxa-5,8,11,14,17-pentaazaeicosanoyl)sulfamoyl)piperidine-4-carboxylic acid allyl ester (J-5-5)

[0738] J-5-4 (40 mg, 39.44 μmol), INT2 (41.45 mg, 118.32 μmol), HATU (15.00 mg, 78.88 μmol), and DIPEA (25.49 mg, 197.20 μmol) were added to DMF (8 mL) and reacted at 25°C for 5 h. The reaction solution was concentrated to obtain a crude product, which was purified by reverse phase chromatography (ACN / H2O = 0-50%, 0.05% formic acid) and lyophilized to obtain 48 mg of the title compound.

[0739] Its structural characterization data are as follows:

[0740] MS m / z(ESI):1346.6[M+H] +

[0741] Step 5: 1-(N-((12S,15S,19S)-19-hydroxy-12-isopropyl-20-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctacoshydro-25H-2,2 Preparation of 7:5,8:11,15-triepoxy-23,25-ethano-2,28-furomethane [3,2-i] furo [2',3':5,6] pyrano [4,3-b] [1,4] dioxacyclopentacosan-18-yl)-15-methyl-4,7,10,13,16-pentaoxo-2-oxa-5,8,11,14,17-pentaazaeicosanoyl)sulfamoyl)piperidine-4-carboxylic acid (J-5-6)

[0742] Dissolve J-5-5 (48 mg, 35.65 μmol), morpholine (31.06 mg, 356.47 μmol), and tetrakistriphenylphosphine palladium (24.72 mg, 21.39 μmol) in THF (10 mL). Vacuum and replace with nitrogen three times. Allow to react at 25°C for 2 hours. Concentrate the reaction mixture to obtain the crude product, which is then purified by reverse phase chromatography (ACN / H2O = 0-50%, 0.05% formic acid) and lyophilized to afford 16 mg of the title compound.

[0743] Its structural characterization data are as follows:

[0744] MS m / z(ESI):1306.5[M+H] +

[0745] Step 6: 1-(N-((12S,15S,19S)-19-hydroxy-12-isopropyl-20-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctacosyl-25H-2,27 Preparation of pentafluorophenyl (5,8:11,15-triepoxy-23,25-ethano-2,28-furo[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxacyclopentacosan-18-yl)-15-methyl-4,7,10,13,16-pentaoxo-2-oxa-5,8,11,14,17-pentaazaeicosanoyl)sulfamoyl)piperidine-4-carboxylate (J-5)

[0746] Compound J-5-5 (16 mg, 12.25 μmol), 2,3,4,5,6-pentafluorophenol (33.81 mg, 183.70 μmol), and EDCI (42.26 mg, 220.44 μmol) were added to DMF (5 mL) and reacted at 25°C for 4 hours. The reaction solution was directly concentrated to obtain a crude product, which was purified by preparative HPLC and freeze-dried to obtain 6.00 mg of the title compound.

[0747] Its structural characterization data are as follows:

[0748] ESI-MS (m / z): 1472.6 [M+H] + .

[0749] The preparation method is as follows:

[0750] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

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

[0752] 2. Antibody Preparation and Binding Activity Assay

[0753] 1. Antibody acquisition and purification

[0754] Based on the amino acid sequences of trastuzumab (IMGT / mAb-DB ID: 97) and pertuzumab (IMGT / mAb-DB ID: 80) in the IMGT database, codon optimization was performed and the encoding genes were synthesized. These genes were then constructed into expression vectors, transfected into CHO cells, and subjected to pressure screening to establish stable expressing cell lines. Expression was performed, the supernatant was collected, and the corresponding antibodies, trastuzumab and pertuzumab, were purified using a Protein A affinity medium. The amino acid sequences of trastuzumab and pertuzumab are shown above.

[0755] 3. Conjugation of Compounds Containing Cellular Bioactive Molecules and Linkers to Antibodies

[0756] 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.

[0757] The conjugate preparation of the antibody drug conjugate sample is as follows:

[0758] Example Preparation of Trastuzumab-J-3

[0759] 0.926 mL of trastuzumab antibody (16.2 mg / mL) was taken and the pH was adjusted to 7.40 with 1 M Na2HPO4 solution. A solution of J-3 (54.39 μL, 10 mM, equivalent to 5 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 (ADC, trastuzumab-J-3). The DAR value determined by mass spectrometry was 2.28.

[0760] Table 1: Trastuzumab-J-3 Measured Molecular Weight and Calculated DAR

[0761] Example Preparation of Trastuzumab-J-5

[0762] 0.27 mL of trastuzumab antibody (14.8 mg / mL) was taken and the pH was adjusted to 7.40 with 1M Na2HPO4 solution. A 5-fold amount of J-5 (14.5 μL, 10 mM) dissolved in dimethyl sulfoxide was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 4 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 (ADC, trastuzumab-J-5). The DAR value determined by mass spectrometry was 2.70.

[0763] Table 2: Measured molecular weight and DAR calculation of KB106-Her2-1089

[0764] Example Preparation of Trastuzumab-J-6

[0765] 0.926 mL of trastuzumab antibody (16.2 mg / mL) was adjusted to pH 7.40 with 1 M Na₂HPO₄ solution. A solution of J-6 (50.11 μL, 10 mM, equivalent to 4.8 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 at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (ADC, trastuzumab-J-6). The DAR value, determined by mass spectrometry, was 2.14.

[0766] Table 3: Measured molecular weight and DAR calculation of Trastuzumab-J-6

[0767] Example Preparation of Trastuzumab-J-8

[0768] 0.926 mL of trastuzumab antibody (16.2 mg / mL) was adjusted to pH 7.40 with 1 M Na₂HPO₄ solution. A solution of J-8 (53.27 μL, 10 mM, equivalent to 5 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 at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (ADC, trastuzumab-J-8). The DAR value, determined by mass spectrometry, was 2.09.

[0769] Table 4: Trastuzumab-J-8 Measured Molecular Weight and Calculated DAR

[0770] Example Preparation of Trastuzumab-H-1

[0771] 0.75mL of trastuzumab antibody (16.2mg / mL) was diluted with 37.5µL of 20mM PB + 0.1M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1M Na2HPO4 solution. 46µL of 10mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A solution of H-1 dissolved in dimethyl sulfoxide (DMSO) (50.73µL, 10mM, equivalent to 6 times the amount of antibody) was then added, mixed, and allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (ADC, trastuzumab-H-1). The DAR value, determined by mass spectrometry, was 4.17.

[0772] Table 5: Trastuzumab-H-1 Measured Molecular Weight and DAR Calculation

[0773] Example Preparation of Trastuzumab-H-6

[0774] 0.185mL of trastuzumab antibody (16.2mg / mL) was diluted with 19.3µL of 20mM PB + 0.1M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1M Na2HPO4 solution. 30mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60, 3.79µL) was added, mixed, and allowed to stand at room temperature for 1.5 hours. H-6 dissolved in dimethyl sulfoxide (12.54µL, 10mM, equivalent to 6 times the amount of antibody) was then added, mixed, and allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20mM histidine buffer at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (ADC, trastuzumab-H-6). The DAR value, determined by mass spectrometry, was 4.06.

[0775] Table 6: Trastuzumab-H-6 Measured Molecular Weight and Calculated DAR

[0776] Example Preparation of Trastuzumab-H-8

[0777] 0.926 mL of trastuzumab antibody (16.2 mg / mL) was diluted with 46.3 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. A 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.84 μL, pH 7.60) solution was added, mixed, and allowed to stand at room temperature for 1.5 hours. H-8 dissolved in dimethyl sulfoxide (62.63 μL, 10 mM, equivalent to 6 times the amount of antibody) was then added, mixed, and allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (ADC, trastuzumab-H-8). The DAR value, determined by mass spectrometry, was 3.89.

[0778] Table 7: Trastuzumab-H-8 Measured Molecular Weight and DAR Calculation

[0779] Example Preparation of Trastuzumab-H-10

[0780] 0.926 mL of trastuzumab antibody (16.2 mg / mL) was diluted with 46.3 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. A 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.84 μL, pH 7.60) solution was added, mixed, and allowed to stand at room temperature for 1.5 hours. H-10 (62.63 μL, 10 mM, equivalent to 6 times the amount of antibody) dissolved in dimethyl sulfoxide 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-5 gel column (Cytiva) to obtain the antibody-drug conjugate (ADC, trastuzumab-H-10). The DAR value, determined by mass spectrometry, was 4.02.

[0781] Table 8: Trastuzumab-H-10 Measured Molecular Weight and DAR Calculation

[0782] IV. Evaluation of the Antibody-Drug Conjugate's Inhibitory Effect on Tumor Growth in a Mouse Subcutaneous Xenograft Tumor Model

[0783] The preparations containing the ADC of the present invention were administered via tail vein injection to CDX mouse models subcutaneously transplanted with human gastric cancer cells NCI-N87. The tumor volume and animal body weight changes were measured twice a week, and the tumor inhibition efficacy of the ADC of the present invention on tumor-bearing mice was calculated.

[0784] Test drug

[0785] Drug name, source, and preparation method: Take an appropriate amount of the ADC of the present invention, dilute the stock solution with 0.9% NaCl injection to a dosing solution at a 1 mg / kg dosing volume. Use 0.9% NaCl injection as a vehicle control.

[0786] Experimental animals and cell lines

[0787] Balb / c Nude mice (Chengdu Yaokang Biotechnology Co., Ltd.)

[0788] Human gastric cancer cells NCI-N87 (ATCC)

[0789] Experimental grouping and evaluation methods

[0790] The average tumor volume was about 150 mm. 3 Tumor-bearing mice were randomly divided into groups (the number of groups was determined based on the sample size). The groups were administered 0.9% NaCl injection (hereinafter referred to as vehicle control) and the ADC of the present invention. The dosing frequency was as described in the specific examples. The administration method was tail vein injection, and the administration volume was 10 ml / kg. Tumor diameter was measured twice weekly with a vernier caliper, and tumor volume was calculated using the following formula: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively. Animal mortality was recorded daily.

[0791] The tumor growth inhibition rate (TGI) was calculated using the following formula to evaluate the tumor inhibition efficacy of the ADC of the present invention:

[0792] 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%.

[0793] Where V T末 : Mean tumor volume of treatment group at the end of the experiment

[0794] VT0 : Mean tumor volume at the start of drug administration in the treatment group

[0795] V C末 : Mean tumor volume of negative control group at the end of the experiment

[0796] V C0 : Mean tumor volume of negative control group at the beginning of drug administration

[0797] The following formula was used to calculate the tumor relative proliferation rate T / C (%), which was used to evaluate the tumor inhibition efficacy of the ADC of the present invention:

[0798] T / C=(VTend / V T0 ) / (VC end / V C0 ).

[0799] (1) Efficacy testing of anti-human Her2 antibody-drug conjugates in the NCI-N87 model

[0800] 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. When the average tumor volume reached approximately 150 mm, the cells were cultured. 3 At about 30 minutes, the patients were randomly divided into groups according to tumor size, namely: vehicle control group (i.e. negative control, Vehicle group), Trastuzumab-J-8 1 mg / kg group of the present invention, Trastuzumab-J-6 1 mg / kg group, Trastuzumab-J-3 1 mg / kg group and Trastuzumab-H-8 0.5 mg / kg group. Each group was injected with tail vein (iv) and the drugs were given on Day 0 and Day 7, for a total of 2 doses.

[0801] The ADC of this invention demonstrated 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 1 mg / kg trastuzumab-J-8 group, the 1 mg / kg trastuzumab-J-6 group, the 1 mg / kg trastuzumab-J-3 group, and the 0.5 mg / kg trastuzumab-H-8 group were 93.82%, 90.25%, 135.66%, and 57.29%, respectively. On Day 30, there were no animal deaths or significant weight loss in any treatment group, and no significant drug toxicity was observed. The ADC of this invention was well tolerated by mice during treatment. Detailed results are shown in Table 9, Figures 1 and 2.

[0802] Table 9 Human gastric cancer cell NCI-N87 CDX model

[0803] Note: TGI is tumor growth inhibition rate, T / C is relative tumor proliferation rate, the same below.

[0804] 5. Evaluation of the Antibody-Drug Conjugate's Inhibitory Effect on Tumor Growth in a Mouse Subcutaneous Xenograft Tumor Model

[0805] The preparations containing the ADC of the present invention were administered via tail vein injection to CDX mouse models subcutaneously transplanted with human breast cancer cells JIMT-1. The tumor volume and animal body weight changes were measured twice a week, and the tumor inhibition efficacy of the ADC of the present invention on tumor-bearing mice was calculated.

[0806] Test drug

[0807] Drug name, source, and preparation method: Take an appropriate amount of the ADC of the present invention, dilute the stock solution with 0.9% NaCl injection to a dosing solution at a dose volume of 3 mg / kg. Use 0.9% NaCl injection as a vehicle control.

[0808] Experimental animals and cell lines

[0809] NOD SCID mice (Chengdu Yaokang Biotechnology Co., Ltd., production license number: SCXK (Sichuan) 2020-0034, animal certificate number: 511214900024561)

[0810] Human breast cancer cells JIMT-1 (Nanjing Kebai)

[0811] Experimental grouping and evaluation methods

[0812] The average tumor volume was about 150 mm. 3 Tumor-bearing mice were randomly divided into groups (the number of groups was determined based on the sample size). The groups were administered 0.9% NaCl injection (hereinafter referred to as vehicle control) and the ADC of the present invention. The dosing frequency was as described in the specific examples. The administration method was tail vein injection, and the administration volume was 10 ml / kg. Tumor diameter was measured twice weekly with a vernier caliper, and tumor volume was calculated using the following formula: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively. Animal mortality was recorded daily.

[0813] The tumor growth inhibition rate (TGI) was calculated using the following formula to evaluate the tumor inhibition efficacy of the ADC of the present invention: 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 V T末 : Mean tumor volume of treatment group at the end of the 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 the ADC of the present invention: T / C = (V T末 / V T0 ) / (V C末 / V C0 ).

[0819] (1) Efficacy testing of anti-human Her2 antibody-drug conjugates in the JIMT-1 model

[0820] JIMT-1 cells were cultured in DMEM containing 10% fetal bovine serum at 37°C and 5% CO2. JIMT-1 cells in the exponential growth phase were harvested, resuspended in PBS containing 50% Matrigel to an appropriate concentration, and inoculated subcutaneously into female NOD SCID mice to establish a breast cancer model. When the average tumor volume reached approximately 150 mm, the cells were inoculated with a 5% PBS solution containing 50% Matrigel. 3 At approximately 14 days, the patients were randomly divided into the following groups based on tumor size: a vehicle control group (i.e., negative control, or vehicle group), a 3 mg / kg trastuzumab-J-8 group, a 3 mg / kg trastuzumab-J-6 group, and a 3 mg / kg trastuzumab-J-3 group. Each group received a single dose of trastuzumab-J-3 via tail vein injection (iv) on Day 0.

[0821] The ADC of this invention demonstrated significant tumor growth inhibition in the JIMT-1 breast cancer xenograft model. Compared with the vehicle group, the tumor growth inhibition rates (TGI) of the 3 mg / kg trastuzumab-J-8, 3 mg / kg trastuzumab-J-6, and 3 mg / kg trastuzumab-J-3 groups were 106.33%, 78.33%, 102.14%, and 45.30%, respectively. On Day 20, there were no animal deaths or significant weight loss in any treatment group, and no significant drug toxicity was observed. The ADC of this invention was well tolerated by mice during treatment. Detailed results are shown in Table 10, Figures 3 and 4.

[0822] Table 10 Human breast cancer cell JIMT-1CDX model

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

[0824] 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, wherein: 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 a structural fragment connecting linkers 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, wherein: The structure M is substituted or unsubstituted 3. The antibody-drug conjugate according to claim 1 or 2, wherein: L is selected from a divalent substituted or unsubstituted structural fragment consisting of one or more of the following groups: C 1-6 Alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, -N(R')-, carbonyl, -O-, natural amino acids or unnatural amino acids and their analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), 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, Ala-Ala-Glu), 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-, glucose, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl, polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH2CH2O) 1-10 -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, such as an integer of 1-15, such as 1-12, 3-12, 1-10, 1-8, 3-8 , an integer selected from 1-6, 1-4, 1-2, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; s is selected from an integer selected from 1-20, for example an integer selected from 1-15, for example an integer selected from 1-12, 3-12, 5-10, 8-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

4. The antibody-drug conjugate according to any one of claims 1 to 3, wherein: L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following: 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, 5-10, 8-10, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

5. The antibody drug conjugate according to any one of claims 1 to 4, wherein L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following: 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, 5-10, 8-10, 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: 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, 5-10, 8-10, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

6. The antibody-drug conjugate according to any one of claims 1 to 5, wherein: L is selected from the following substituted or unsubstituted structural fragments: 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, 5-10, 8-10, 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 the following substituted or unsubstituted structural fragments: 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, 5-10, 8-10, 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 the following substituted or unsubstituted structural fragments: 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, 5-10, 8-10, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

7. The antibody-drug conjugate according to any one of claims 1 to 6, wherein: E is a single bond, a substituted or unsubstituted -NH-CH2-, or a substituted or unsubstituted structural fragment selected from the following:

8. The antibody-drug conjugate according to any one of claims 1 to 7, wherein: E is a single bond, substituted or unsubstituted -NH-CH2-, or For example, a single key or 9. The antibody-drug conjugate according to any one of claims 1 to 8, wherein: Selected from the following substituted or unsubstituted structures: Preferably, Selected from the following substituted or unsubstituted structures: Preferably, Selected from the following substituted or unsubstituted structures: Preferably, Selected from the following substituted or unsubstituted structures: Preferably, Selected from the following substituted or unsubstituted structures: 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, 5-10, 8-10, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

10. The antibody-drug conjugate according to any one of claims 1 to 9, wherein: The cytotoxic drug is an eribulin compound; Preferably, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof: Preferably, the cytotoxic drug is connected to E in the antibody-drug conjugate via the -OH, primary amino, secondary amine or tertiary amine group on the cytotoxic drug.

11. The antibody-drug conjugate according to any one of claims 1 to 10, wherein: -MLED can be obtained by the following compounds J-1 to J-9, H-1 to H-15, H'-1 to H'-12, preferably by compounds J-1 to J-9, H-1 to H-15 by substitution reaction (for example, removal of the methylsulfonyl structure thereon): wherein n is selected from an integer of 1-20, preferably an integer of 1-15, such as an integer of 1-12, 3-12, 5-10, 8-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10.

12. The antibody-drug conjugate according to any one of claims 1 to 11, wherein: The antibody or antigen-binding fragment thereof comprises: (1) the following heavy chain variable region (VH) and / or light chain variable region (VL): (1a) a heavy chain variable region (VH) comprising the following three CDRs: a sequence of SEQ ID NO: 5 or a variant thereof CDR-H1, CDR-H2 of SEQ ID NO: 6 or a variant thereof, 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, CDR-L3 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): (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): (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), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or 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): (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; 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, 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 / 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 having a sequence of SEQ ID NO: 33 or a variant thereof, CDR-H2 having a sequence of SEQ ID NO: 34 or a variant thereof, and CDR-H3 having a sequence of SEQ ID NO: 22 or a variant thereof. and / or, a light chain variable region (VL) comprising the following three CDRs: a CDR-L1 having a sequence of SEQ ID NO: 23 or a variant thereof, a CDR-L2 having a sequence of SEQ ID NO: 24 or a variant thereof, and a CDR-L3 having 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), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or 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 having a sequence of SEQ ID NO: 13 or a variant thereof, CDR-H2 having a sequence of SEQ ID NO: 14 or a variant thereof, and CDR-H3 having a sequence of SEQ ID NO: 15 or a variant thereof and / or, a light chain variable region (VL) comprising the following three CDRs: a CDR-L1 having a sequence of SEQ ID NO: 16 or a variant thereof, a CDR-L2 having a sequence of SEQ ID NO: 17 or a variant thereof, and a CDR-L3 having a sequence 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 having a sequence of SEQ ID NO: 33, A CDR-H2 with a sequence of SEQ ID NO: 34, a CDR-H3 with a sequence of SEQ ID NO: 22; and a light chain variable region (VL) comprising the following three CDRs: a CDR-L1 with a sequence of SEQ ID NO: 23, a CDR-L2 with a sequence of SEQ ID NO: 24, and a CDR-L3 with a sequence of SEQ ID NO: 25; or, (3) the following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system: (3a) 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 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; or, (4) the following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system: (4a) 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.

13. The antibody-drug conjugate according to any one of claims 1 to 12, wherein: 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.

14. The antibody-drug conjugate according to any one of claims 1 to 13, wherein: 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, 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; 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.

15. The antibody-drug conjugate according to any one of claims 1 to 14, wherein: 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 the VH of SEQ ID NO: 3 and the heavy chain constant region (CH) of SEQ ID NO: 35, and a light chain comprising the VL of SEQ ID NO: 4 and the light chain constant region (CL) of SEQ ID NO:

36.

16. The antibody drug conjugate according to any one of claims 1 to 15, wherein: x is 3 to 6, and more preferably, x is about 4.

17. The antibody-drug conjugate according to any one of claims 1 to 16, wherein: x is 1 to 4, and more preferably, x is about 2.

18. The antibody drug conjugate of claims 1-17, selected from: in, HA is an antibody or an antigen-binding fragment thereof that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family receptor tyrosine kinase. Preferably, the antibody or antigen-binding fragment thereof is as described in any one of claims 12 to 15; n is selected from an integer of 1 to 20, preferably an integer of 1 to 15, such as an integer of 1 to 12, 3 to 12, 5 to 10, 8 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10; 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 method between the amino group in the antibody or its antigen-binding fragment and the linker.

19. A composition comprising one or more antibody drug conjugates according to any one of claims 1 to 18, 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 ~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 to 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, 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 .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 2.2 6, 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 3.50, 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 3.60 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, 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, and about 5.

0.

20. A pharmaceutical composition comprising the antibody drug conjugate according to any one of claims 1 to 18, or the composition according to claim 19, and one or more pharmaceutical excipients.

21. Use of the antibody-drug conjugate according to any one of claims 1 to 18, or the composition according to claim 19, or the pharmaceutical composition according to claim 20 in the preparation of a drug for treating cancer.

22. The use of claim 21, 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, particularly lung adenocarcinoma) and urothelial carcinoma.

23. The antibody drug conjugate of any one of claims 1 to 18, or the composition of claim 19, or the pharmaceutical composition of claim 20, for use in treating cancer.

24. The antibody drug conjugate of any one of claims 1 to 18, or the composition of claim 19, or the pharmaceutical composition of claim 20, for use in treating solid tumors or hematological malignancies; for example, selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) and urothelial carcinoma.

25. A method for preventing or treating cancer, comprising administering to an individual in need thereof an effective amount of the antibody drug conjugate of any one of claims 1 to 18, or the composition of claim 19, or the pharmaceutical composition of claim 20.

26. The method of claim 25, wherein the cancer is selected from a solid tumor or a hematological malignancy; for example, selected from gastric cancer, breast cancer, lung cancer (eg, non-small cell lung cancer, in particular lung adenocarcinoma) and urothelial carcinoma.

27. A compound having the structure shown below, or a salt, stereoisomer, tautomer or isotope-labeled compound thereof:

28. A compound having the structure shown below, or a salt, stereoisomer, tautomer or isotope-labeled compound thereof: in PG1 is each independently H or a carboxyl protecting group, such as C 1-6 Alkyl, allyl, benzyl, 2,4-dimethoxybenzyl, p-methoxybenzyl, methoxyethoxymethyl, pentafluorophenyl, 4-p-methylbenzyloxybenzyl; PG2 is independently H or an amino protecting group, wherein the amino protecting group is an alkoxycarbonyl amino protecting group, such as benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), methyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methyl (or ethyl)oxycarbonyl; an acyl amino protecting group, such as phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), o-butyloxycarbonyl (O-butyloxycarbonyl), tert ... (p-)nitrobenzenesulfonyl (Ns), pivaloyl, benzoyl, tert-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, benzyloxycarbonyl, p-toluenesulfonyl, p-nitrobenzenesulfonyl, trifluoroacetyl, methoxycarbonyl, or ethoxycarbonyl; alkyl amino protecting groups, such as trityl (Trt), 2,4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), benzyl (Bn).

29. Use of the compound according to claim 27 or 28 or a salt, stereoisomer, tautomer or isotope-labeled compound thereof in the preparation of an antibody drug conjugate according to any one of claims 1 to 18.

Images