Antibody drug conjugate as well as preparation method and application thereof

Abstract

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

Description

Antibody drug conjugates and preparation methods and uses thereof

[0001] This application is based on the application with CN application number 202310538792.7 and application date May 12, 2023, and claims its priority. The disclosed content of the CN application is hereby introduced as a whole into this application. Technical Field

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

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

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

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

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

[0007] Summary of the Invention

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

[0009] The present application relates to an antibody drug conjugate, and exemplarily discloses an antibody drug conjugate having the general formula Ab-[MLED] with trastuzumab as the targeting moiety. x The antibody-drug conjugate of the structure shown. The results showed that the conjugate had a relatively good drug-antibody coupling ratio (e.g., 6.5-8.5), and the conjugate had excellent binding activity and proliferation inhibition on Her2-positive cells, and had a good targeted killing effect on Her2-positive tumors (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 the structural fragment connecting the 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 structural fragments:

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

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

[0022] In some embodiments, M is the following structural fragment:

[0023] In some embodiments, M is the following structural fragment:

[0024] In some embodiments, L is selected from a divalent structural fragment consisting of one or more of the following groups: 1-6 an alkylene group, a 6-10 membered arylene group, a 5-6 membered heteroarylene group, -N(R')-, a carbonyl group, -O-, a residue of an amino acid or an analog thereof (the amino acid is a natural amino acid or a non-natural amino acid, 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) r OCH3)), Lys(R'), a fragment of a short peptide consisting of two or more amino acids (the short peptide is, for example, selected from 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, A la-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 (SEQ ID NO:41), Gly-Gly-Val-Ala (SEQ ID NO:42), Gly-Phe-Leu-Gly (SEQ ID NO:43), Glu-Ala-Ala-Ala (SEQ ID NO:44) and Gly-Gly-Gly-Gly-Gly (SEQ ID NO:45)), wherein R' and R" each independently represent hydrogen, C 1-6Alkyl, glucose, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl, -CH2N(C 1-6 alkyl)-C(=O)-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) r - C 1-6 alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), or NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue), wherein r is selected from an integer of 1 to 20, such as 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; s is selected from an integer of 1-20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, preferably, s is 5, 8 or 9; t is selected from an integer of 1-5, for example, 1, 2, 3, 4, 5; preferably, t is 1.

[0025] In some embodiments, "-NOTA" refers to

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

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

[0028] In some embodiments, r is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 1-10, 1-8, 3-8, 1-6, 1-4, 1-2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0029] In some embodiments, s is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 5-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0030] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following: C 1-6 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) r OCH3)), 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-A la, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), Where R' represents hydrogen, C 1-6 Alkyl, glucose, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl, or polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH2CH2O) r -C 1-6 alkyl), wherein r is selected from an integer of 1-20; s is selected from an integer of 1-20.

[0031] In some embodiments, L is selected from a divalent structural fragment consisting of one or more of the following groups: 1-6 Alkylene, Carbonyl, -NH-, Lys(R'), Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Cit, Val -Lys, Ala-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly- Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-G ly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, wherein R' and R" each independently represent 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(C 1-6 alkyl)-C(=O)-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) r -C 1-6 alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), or NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue), wherein r is selected from an integer of 1-20; s is selected from an integer of 1-20, preferably, s is 5, 8 or 9; t is selected from an integer of 1-5; preferably, t is 1.

[0032] 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.

[0033] In some embodiments, L is selected from a divalent structural fragment consisting of one or more of the following groups: carbonyl, Lys(R'), Val-Ala, Val-Cit, Ala-Ala-Ala, D-Leu-Ala-Glu, Glu-Val-Ala, Ser-D-Ala-Pro, Val-Lys-Gly, Gly-Gly-Val-Ala, wherein R' and R" each independently represent 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(C 1-6 alkyl)-C(=O)-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 1-6alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), or NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue), wherein r is selected from an integer of 1-20; s is selected from an integer of 1-20; t is selected from an integer of 1-5; preferably, t is 1.

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

[0035] In some embodiments, L is selected from a structural fragment consisting of one or more of the following groups:

[0036] In some embodiments, L is selected from a divalent structural segment consisting of one or more of the following groups:

[0037] s is selected from an integer from 1 to 20. In some embodiments, L is selected from the following substituted or unsubstituted structural fragments:

[0038] In some embodiments, L is selected from:

[0039] s is an integer selected from 1-20.

[0040] In any of the above embodiments, s is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0041] In any of the above embodiments, s is selected from an integer of 5-15, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0042] In some embodiments, L is selected from:

[0043] s is selected from an integer of 5-15, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0044] In some embodiments, E is a single bond, -NH-CH2-, or is selected from the following structural fragments:

[0045] In some embodiments, E is a single bond, -NH-CH2- or

[0046] In some embodiments, E is -NH-CH2- or

[0047] In some embodiments, E is

[0048] In some embodiments, Selected from the following structures:

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

[0050] In some embodiments, Selected from the following substituted or unsubstituted structures:

[0051] n is an integer selected from 1-20; preferably, n is an integer selected from 5-15, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In some embodiments, Selected from the following substituted or unsubstituted structures:

[0052] n is selected from an integer of 5-15, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0053] In some embodiments, the cytotoxic drug is selected from an anti-tubulin agent. In some embodiments, the tubulin inhibitor is an auristatin compound.

[0054] In some embodiments, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0055] 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.

[0056] 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.

[0057] In some embodiments, the cytotoxic drug fragment D is selected from:

[0058] 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.

[0059] In one aspect, the present application provides a compound or a pharmaceutically acceptable salt thereof having a structure shown in formula DEL-M', wherein:

[0060] M' is -M-Lg, wherein Lg is a leaving group for a nucleophilic substitution reaction, and M is a structural fragment that binds to a targeting moiety (e.g., an antibody or an antigen-binding fragment thereof);

[0061] L is the structural fragment connecting M and E;

[0062] E is a structural fragment connecting L and D;

[0063] D is a cytotoxic drug fragment.

[0064] 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 1-6Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 Alkyl sulfoxide, halogenated phenoxy, hydroxyl (-OH), thiol (-SH), amino (-NH2), nitro, azido, cyano, alkenyl, alkynyl and alkynyl-containing structural fragments, the halogenated C 1-6 Alkyl, C 1-6 Alkylsulfonyl, halo C 1-6 Alkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 The alkylsulfoxide group, halophenoxy group, alkenyl group, alkynyl group and alkynyl group-containing structural fragments are optionally substituted with one or more suitable substituents.

[0065] 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.

[0066] 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).

[0067] In some embodiments, Lg is selected from halogen, substituted or unsubstituted methylsulfonyl, halophenoxy, hydroxyl (-OH), thiol (-SH), or amino (-NH2).

[0068] In some embodiments, Lg is selected from C 1-6 Alkylsulfonyl or halogenated phenoxy.

[0069] In some embodiments, Lg is selected from methylsulfonyl or pentafluorophenoxy.

[0070] In some embodiments, M, L, E, and D are as defined in any of the preceding clauses.

[0071] In some embodiments, the free form of the "drug-linker" is selected from F-1 to F-20, L-1 to L-11 shown below:

[0072] In some embodiments, the free form of the "drug-linker" is selected from F'-1 to F'-12, F'-16 to F'-18, and L'-6 to L'-11 shown below:

[0073] wherein n is selected from an integer of 1-20, preferably n is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 5-15, 5-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 9 or 10.

[0074] In some embodiments, the drug-linker described above may be optionally substituted with one or more suitable substituents.

[0075] In some embodiments, the antibody drug conjugate of the present application has a structure shown in the formula Ab-[MLED]x, wherein:

[0076] 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;

[0077] x is 1-10, preferably, x is 1-4, more preferably, x is 4;

[0078] -MLED is formed from compounds F'-1-F'-12, F'-16-F'-18, L'-6-L'-11, F-1 to F-20, or L-1 to L-11 (preferably F-1 to F-20 or L-1 to L-11) described above.

[0079] In some embodiments, the antibody drug conjugate of the present application has a structure shown in the formula Ab-[MLED]x, wherein:

[0080] 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;

[0081] x is 1-10, preferably, x is 1-4, more preferably, x is 4;

[0082] -MLED is formed by removing the methanesulfonyl group from the above-mentioned compounds F-1 to F-20.

[0083] In some embodiments, the antibody drug conjugate of the present application has a structure shown in the formula Ab-[MLED]x, wherein:

[0084] 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;

[0085] x is 1-10, preferably, x is 1-4, more preferably, x is 2;

[0086] -MLED is formed by removing the pentafluorophenoxy group from the compounds L-1 to L-11 described above.

[0087] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0088] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL):

[0089] (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

[0090] (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;

[0091] 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;

[0092] or,

[0093] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0094] (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

[0095] (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;

[0096] 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;

[0097] or,

[0098] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0099] (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

[0100] (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;

[0101] wherein the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0102] or,

[0103] (4) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0104] (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

[0105] (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;

[0106] 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.

[0107] In some embodiments, the antibody or antigen-binding fragment thereof comprises

[0108] (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:

[0109] (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

[0110] (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;

[0111] 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;

[0112] or,

[0113] (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:

[0114] (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

[0115] (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;

[0116] 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;

[0117] or,

[0118] (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:

[0119] (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

[0120] (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;

[0121] 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., 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;

[0122] or,

[0123] (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:

[0124] (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

[0125] (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;

[0126] 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.

[0127] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0128] (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:

[0129] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6, and CDR-H3 of SEQ ID NO: 7; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0130] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20, CDR-H2 of SEQ ID NO: 21, and CDR-H3 of SEQ ID NO: 22; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0131] or,

[0132] (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:

[0133] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18, CDR-H2 of SEQ ID NO: 19, and CDR-H3 of SEQ ID NO: 7; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0134] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33, CDR-H2 of SEQ ID NO: 34, and CDR-H3 of SEQ ID NO: 22; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0135] or,

[0136] (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:

[0137] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11, CDR-H2 of SEQ ID NO: 12, and CDR-H3 of SEQ ID NO: 7; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0138] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26, CDR-H2 of SEQ ID NO: 27, and CDR-H3 of SEQ ID NO: 22; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0139] or,

[0140] (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:

[0141] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13, CDR-H2 of SEQ ID NO: 14, and CDR-H3 of SEQ ID NO: 15; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16, CDR-L2 of SEQ ID NO: 17, and CDR-L3 of SEQ ID NO: 10; or

[0142] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:28, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:30; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:31, CDR-L2 of SEQ ID NO:32, and CDR-L3 of SEQ ID NO:25.

[0143] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0144] (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:

[0145] (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

[0146] (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;

[0147] or,

[0148] (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:

[0149] (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

[0150] (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;

[0151] or,

[0152] (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:

[0153] (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

[0154] (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;

[0155] or,

[0156] (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:

[0157] (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

[0158] (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.

[0159] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0160] (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

[0161] (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;

[0162] 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.

[0163] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0164] (a) VH shown in SEQ ID NO: 1, and / or VL shown in SEQ ID NO: 2; or

[0165] (b) VH represented by SEQ ID NO: 3, and / or VL represented by SEQ ID NO: 4.

[0166] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0167] (a) VH or a variant thereof shown in SEQ ID NO: 1, and VL or a variant thereof shown in SEQ ID NO: 2; or

[0168] (b) VH shown in SEQ ID NO: 3 or a variant thereof, and VL shown in SEQ ID NO: 4 or a variant thereof.

[0169] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0170] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0171] (b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4.

[0172] In some embodiments, the antibody or antigen-binding fragment thereof further comprises:

[0173] (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

[0174] (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.

[0175] 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.

[0176] 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).

[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.

[0178] 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).

[0179] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO:36.

[0180] 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.

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

[0182] (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

[0183] (2) A heavy chain comprising the VH sequence of SEQ ID NO: 3 and the heavy chain constant region (CH) of SEQ ID NO: 35, and a light chain comprising the VL sequence of SEQ ID NO: 4 and the light chain constant region (CL) of SEQ ID NO: 36.

[0184] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0185] (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

[0186] (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.

[0187] 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.

[0188] 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.

[0189] As known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0190] 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.

[0191] 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.

[0192] In certain embodiments, the N-terminal glutamine of the VH or variant thereof shown in SEQ ID NO: 1 or 3 or the heavy chain or variant thereof shown in SEQ ID NO: 37 or 39 undergoes cyclization to form pyroglutamate or pyroglutamate salt.

[0193] In certain embodiments, the heavy chain constant region (CH) of SEQ ID NO: 35 or a variant thereof, or the heavy chain of SEQ ID NO: 37 or 39 or a variant thereof lacks a C-terminal lysine.

[0194] In some embodiments, the antibody drug conjugate is selected from ADC F-1 to ADC F-20, ADC L-1 to ADC L-11 shown below:

[0195] 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 n is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 5-15, or 5-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, preferably 5, 8, 9, or 10;

[0196] in, Or it indicates the specific connection mode between the sulfhydryl group in the antibody or antigen-binding fragment thereof and the M fragment;

[0197] Indicates the specific connection method between the amino group in the antibody or its antigen-binding fragment and the M fragment.

[0198] In some embodiments, the antibody drug conjugate is selected from the group consisting of ADC F'-1 to ADC F'-12, ADC F'-16 to ADC F'-18, and ADC L'-6 to ADC L'-11 shown below:

[0199] Wherein, HA in each antibody drug conjugate represents an antibody or an antigen-binding fragment thereof; n is an integer selected from 1-20, preferably n is an integer selected from 1-15, such as 1-12, 3-12, 5-15, 5-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 9 or 10;

[0200] in, Or it indicates the specific connection mode between the sulfhydryl group in the antibody or antigen-binding fragment thereof and the M fragment;

[0201] Indicates the specific connection method between the amino group in the antibody or its antigen-binding fragment and the M fragment.

[0202] In some embodiments, the HA in each antibody drug conjugate comprises:

[0203] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL):

[0204] (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

[0205] (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;

[0206] 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;

[0207] or,

[0208] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0209] (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

[0210] (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;

[0211] 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;

[0212] or,

[0213] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0214] (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

[0215] (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;

[0216] wherein the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0217] or,

[0218] (4) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0219] (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

[0220] (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;

[0221] 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.

[0222] In some embodiments, the HA in each antibody drug conjugate comprises:

[0223] (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:

[0224] (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

[0225] (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;

[0226] 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;

[0227] or,

[0228] (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:

[0229] (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

[0230] (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;

[0231] 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;

[0232] or,

[0233] (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:

[0234] (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

[0235] (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;

[0236] wherein the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0237] or,

[0238] (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:

[0239] (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

[0240] (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;

[0241] 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.

[0242] In some embodiments, the HA in each antibody drug conjugate comprises:

[0243] (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:

[0244] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6, and CDR-H3 of SEQ ID NO: 7; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0245] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20, CDR-H2 of SEQ ID NO: 21, and CDR-H3 of SEQ ID NO: 22; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0246] or,

[0247] (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:

[0248] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18, CDR-H2 of SEQ ID NO: 19, and CDR-H3 of SEQ ID NO: 7; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0249] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33, CDR-H2 of SEQ ID NO: 34, and CDR-H3 of SEQ ID NO: 22; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0250] or,

[0251] (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:

[0252] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11, CDR-H2 of SEQ ID NO: 12, and CDR-H3 of SEQ ID NO: 7; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0253] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26, CDR-H2 of SEQ ID NO: 27, and CDR-H3 of SEQ ID NO: 22; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0254] or,

[0255] (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:

[0256] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13, CDR-H2 of SEQ ID NO: 14, and CDR-H3 of SEQ ID NO: 15; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16, CDR-L2 of SEQ ID NO: 17, and CDR-L3 of SEQ ID NO: 10; or

[0257] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:28, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:30; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:31, CDR-L2 of SEQ ID NO:32, and CDR-L3 of SEQ ID NO:25.

[0258] In some embodiments, the HA in each antibody drug conjugate comprises:

[0259] (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:

[0260] (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

[0261] (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;

[0262] or,

[0263] (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:

[0264] (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

[0265] (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;

[0266] or,

[0267] (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:

[0268] (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

[0269] (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;

[0270] or,

[0271] (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:

[0272] (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

[0273] (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.

[0274] In some embodiments, the HA in each antibody drug conjugate comprises:

[0275] (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

[0276] (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;

[0277] 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.

[0278] In some embodiments, the HA in each antibody drug conjugate comprises:

[0279] (a) VH shown in SEQ ID NO: 1, and / or VL shown in SEQ ID NO: 2; or

[0280] (b) VH represented by SEQ ID NO: 3, and / or VL represented by SEQ ID NO: 4.

[0281] In some embodiments, the HA in each antibody drug conjugate comprises:

[0282] (a) VH or a variant thereof shown in SEQ ID NO: 1, and VL or a variant thereof shown in SEQ ID NO: 2; or

[0283] (b) VH shown in SEQ ID NO: 3 or a variant thereof, and VL shown in SEQ ID NO: 4 or a variant thereof.

[0284] In some embodiments, the HA in each antibody drug conjugate comprises:

[0285] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0286] (b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4.

[0287] In some embodiments, the HA in each antibody drug conjugate further comprises:

[0288] (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

[0289] (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.

[0290] 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.

[0291] In some embodiments, the HA in each antibody drug conjugate comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 35, or a variant thereof having 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; e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions).

[0292] In some embodiments, the HA in each antibody drug conjugate comprises a heavy chain constant region (CH) as set forth in SEQ ID NO:35.

[0293] In some embodiments, the HA in each antibody drug conjugate comprises a light chain constant region (CL) as set forth in SEQ ID NO: 36, or a variant thereof having 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; e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions).

[0294] In some embodiments, the HA in each antibody drug conjugate comprises a light chain constant region (CL) as set forth in SEQ ID NO:36.

[0295] In some embodiments, the HA in each antibody drug conjugate 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.

[0296] In some embodiments, the HA in each antibody drug conjugate comprises:

[0297] (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

[0298] (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.

[0299] In some embodiments, HA in each antibody drug conjugate represents the following antibody or antigen-binding fragment:

[0300] (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

[0301] (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.

[0302] In some embodiments, the HA in each antibody drug conjugate is selected from trastuzumab or pertuzumab.

[0303] In some embodiments, the query accession number of the amino acid sequence of Trastuzumab in the IMGT database (IMGT / mAb-DB ID) is: 97, and the query accession number of the amino acid sequence of Pertuzumab in the IMGT database (IMGT / mAb-DB ID) is: 80.

[0304] In certain embodiments, the HA in each antibody drug conjugate can 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.

[0305] In certain embodiments, the HA in each antibody drug conjugate may include post-translational modifications thereof (e.g., cleavage of a C-terminal lysine in the heavy chain, conversion of an N-terminal glutamine or glutamic acid in the heavy or light chain to pyroglutamic acid or pyroglutamate), which may occur upon recombinant expression in a host cell (e.g., CHO cells) or during purification / storage.

[0306] In certain embodiments, the N-terminal glutamine of the VH or variant thereof shown in SEQ ID NO: 1 or 3 or the heavy chain or variant thereof shown in SEQ ID NO: 37 or 39 undergoes cyclization to form pyroglutamate or pyroglutamate salt.

[0307] In certain embodiments, the heavy chain constant region (CH) of SEQ ID NO: 35 or a variant thereof, or the heavy chain of SEQ ID NO: 37 or 39 or a variant thereof lacks a C-terminal lysine.

[0308] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-4 of the following structures:

[0309] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0310] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-4 of the following structures:

[0311] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0312] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-4 of the following structures:

[0313] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0314] In some embodiments, x in the antibody drug conjugate represented by Ab-[MLED]x is 1-10, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10.

[0315] In some embodiments, x in the antibody drug conjugate represented by Ab-[MLED]x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0316] The antibody-drug conjugates of the present invention are optionally substituted with one or more suitable substituents.

[0317] The present application further provides a formula Ab-[L'-D] x The antibody-drug conjugate shown, wherein:

[0318] Ab is an antibody or an antigen-binding fragment thereof;

[0319] D is a cytotoxic drug fragment;

[0320] L' is a linker connecting Ab and D;

[0321] x is an integer from 1 to 10;

[0322] The antibody or antigen-binding fragment thereof is coupled to a drug-linker selected from the group consisting of:

[0323] 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.

[0324] In certain embodiments, the antibody is Trastuzumab.

[0325] 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:

[0326] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0327] 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:

[0328] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0329] 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:

[0330] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0331] Composition

[0332] 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.

[0333] 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 or about 9 to 10.

[0334] In certain embodiments, the DAR of the ADC compositions described herein is about 3 to 9, e.g., about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 3.0 to 6.0, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5, about 4.0 to 4.5, about 4.0 to 5.0, about 4.0 to 5.5, about 4.0 to 6.0, about 4.0 to 6.5, about 4.0 to 7.0, about 4.0 to 8.0, about 4.5 to 5.0, about 4.5 to 5.5, about 4.5 to 6.0, about 4.5 to 6.5, about 4.5 to 7.0, about 4.5 to 7.5, about 5.0 to 8.0, about 5.5 to 6.0, about 5.5 to 6.5, about 5.5 to 7.0, about 5.5 to 7.5, about 5.5 to 8.0, about 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5, about 6.0 to 8.5, about 6.5 to 7.0, about 6.5 to 7.5, about 6.5 to 8.5, about 7.0 to 7.5.

[0335] In some embodiments, the DAR of the ADC compositions described herein is between about 1.0 and 3.0, or between about 3.0 and 5.0; e.g., about 1.0, about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.1, about 1.11, about 1.12, about 1.13, about 1.14, about 1.15, about 1.16, about 1.17, about 1.18, about 1.19, about 1.2, about 1.21, about 1.22, about 1.23, about 1.24, about 1.25, about 1.26, about 1.27, about 1.28, about 1.29, about 1.3, about 1.31, about 1.32, about 1.33, about 1.34, about 1.35 4, 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 1.5 5, about 1.56, about 1.57, about 1.58, about 1.59, about 1.6, about 1.61, about 1.62, about 1.63, about 1.64, about 1.65, about 1.66, about 1.67, about 1.68, about 1.69, about 1.7, about 1.71, about 1.72, about 1.73, about 1.74, about 1.75, about 1.76 , about 1.77, about 1.78, about 1.79, about 1.8, about 1.81, about 1.82, about 1.83, about 1.84, about 1.85, about 1.86, about 1.87, about 1.88, about 1.89, about 1.9, about 1.91, about 1.92, about 1.93, about 1.94, about 1.95, about 1.96, about 1.97 , about 1.98, about 1.99, about 2.0, about 2.01, about 2.02, about 2.03, about 2.04, about 2.05, about 2.06, about 2.07, about 2.08, about 2.09, about 2.1, about 2.11, about 2.12, about 2.13, about 2.14, about 2.15, about 2.16, about 2.17, about 2.18, about 2.19, about 2.2, about 2.21, about 2.22, about 2.23, about 2.24, about 2.25, about 2.26, about 2.27, about 2.28, about 2.29, about 2.3, about 2.31, about 2.32, about 2.33, about 2.34, about 2.35, about 2.36, about 2.37, about 2.38, about 2.39, about 2.4, about 2.41, about 2.42, about 2.43, about 2.44, about 2.45, about 2.46, about 2.47, about 2.48, about 2.49, about 2.5, about 2.51, about 2.52, about 2.53, about 2.54, about 2.55, about 2.56, about 2.57, about 2.58, about 2.59, about 2.6, about 2.61, about 2.62, about 2.63, about 2.64, about 2.65, about 2.66, about 2.67, about 2.68, about 2.69, about 2.7, about 2.71, about 2.72, about 2.73, about 2.74, about 2.75, about 2.76, about 2.77, about 2.78, about 2.79, about 2.8, about 2.81, about 2. 82, about 2.83, about 2.84, about 2.85, about 2.86, about 2.87, about 2.88, about 2.89, about 2.9, about 2.91, about 2.92, about 2.93, about 2.94, about 2.95, about 2.96, about 2.97, about 2.98, about 2.99, about 3.0, about 3.01, about 3.02, about 3.0 3, about 3.04, about 3.05, about 3.06, about 3.07, about 3.08, about 3.09, about 3.1, about 3.11, about 3.12, about 3.13, about 3.14, about 3.15, about 3.16, about 3.17, about 3.18, about 3.19, about 3.2, about 3.21, about 3.22, about 3.23, about 3.24 , about 3.25, about 3.26, about 3.27, about 3.28, about 3.29, about 3.3, about 3.31, about 3.32, about 3.33, about 3.34, about 3.35, about 3.36, about 3.37, about 3.38, about 3.39, about 3.4, about 3.41, about 3.42, about 3.43, about 3.44, about 3.45, about 3.46, about 3.47, about 3.48, about 3.49, about 3.5, about 3.51, about 3.52, about 3.53, about 3.54, about 3.55, about 3.56, about 3.57, about 3.58, about 3.59, about 3.6, about 3.61, about 3.62, about 3.63, about 3.64, about 3.65, about 3.66, about 3.67, about 3.68, about 3.69, about 3.7, about 3.71, about 3.72, about 3.73, about 3.74, about 3.75, about 3.76, about 3.77, about 3.78, about 3.79, about 3.8, about 3.81, about 3.82, about 3.83, 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, about 5.0.

[0336] In certain embodiments, the DAR of the ADC compositions described herein is about 2.28, about 2.35, about 4.12, about 3.6, or about 4.08.

[0337] In certain embodiments, the DAR of the ADC compositions described herein is 2.28, 2.35, 4.12, 3.6, or 4.08.

[0338] In certain embodiments, the composition is composed of Ab-[L'-D] x The composition of the antibody drug conjugate shown. In certain embodiments, the DAR of the composition is 2-6, such as 3-5, such as about 4. In certain embodiments, the DAR of the composition is 4.12. In certain embodiments, the DAR of the composition is 3.6. In certain embodiments, the DAR of the composition is 4.08. In certain embodiments, the DAR of the composition is 4.12, and the antibody drug conjugates with x being 4 account for 80-90% (e.g., 85%-90%, and another example, 88.43%). In certain embodiments, the DAR of the composition is 3.6, and the antibody drug conjugates with x being 4 account for 70-80% (e.g., 70%-75%, and another example, 72.01%). In certain embodiments, the DAR of the composition is 4.08, and the antibody drug conjugates with x being 4 account for 85-95% (e.g., 90%-95%, and another example, 92.32%).

[0339] Pharmaceutical composition

[0340] In another aspect, the present application provides a pharmaceutical composition comprising the antibody-drug conjugate or compound described in any of the foregoing items, and one or more pharmaceutical excipients.

[0341] The antibody drug conjugates, compounds or drug-linkers described herein are generally 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.

[0342] application

[0343] The antibody drug conjugates, compositions, or pharmaceutical compositions 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.

[0344] Therefore, the present application provides the use of any of the above-mentioned antibody-drug conjugates, compositions or pharmaceutical compositions in the preparation of drugs for treating Her2-expressing cancers.

[0345] At the same time, the present application provides the antibody-drug conjugate, composition or pharmaceutical composition described in any of the above items, and its use in a drug for treating Her2-expressing cancer.

[0346] At the same time, the present application also provides a method for treating Her2-expressing cancer, which comprises the step of administering an effective amount of any of the above-described antibody-drug conjugates, compositions, or pharmaceutical compositions to a subject in need thereof.

[0347] In some embodiments, the antibody drug conjugate, composition, or pharmaceutical composition is sufficient (e.g., in a subject):

[0348] (1) Inhibit cell (such as tumor cell) proliferation;

[0349] (2) inhibit tumor growth;

[0350] (3) induce and / or increase antibody-dependent cellular cytotoxicity activity;

[0351] (4) inhibiting HER2-mediated signal transduction;

[0352] (5) preventing and / or treating HER2-mediated diseases / disorders; or

[0353] (6) Any combination of (1) to (5) above.

[0354] In some embodiments, the cancer disease is selected from solid tumors or hematological malignancies; for example, selected from gastric cancer, breast cancer, lung cancer (eg, non-small cell lung cancer, in particular lung adenocarcinoma) and urothelial carcinoma.

[0355] intermediates

[0356] In some embodiments, the present invention provides the following synthetic intermediate compound or a salt, stereoisomer, tautomer or isotope-labeled compound thereof:

[0357] in:

[0358] 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;

[0359] PG2 is each independently H or a hydroxyl protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), methyl, tert-butyl, allyl, benzyl, methoxymethyl (MOM), ethoxyethyl, 2-tetrahydropyranyl (THP), formyl, acetyl, benzoyl, or p-nitrobenzoyl;

[0360] PG3 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);

[0361] Lg is a leaving group, as defined above;

[0362] s1 is selected from integers of 1-20, preferably from integers of 1-15, such as integers of 1-12, 3-12, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10.

[0363] In some embodiments, the present invention provides the following synthetic intermediate compound or a salt, stereoisomer, tautomer or isotope-labeled compound thereof:

[0364] Synthesis method

[0365] In some embodiments, the present application provides a method 1 for synthesizing the following synthetic intermediate compound:

[0366] The method comprises the following steps:

[0367] Step 1: Compound Int-2-3 is subjected to coupling reaction to synthesize compound Int-5-1;

[0368] Step 2: Compound Int-5-1 is subjected to substitution reaction to synthesize compound Int-5-2;

[0369] Step 3: Compound Int-5-2' is subjected to oxidation reaction to synthesize compound Int-5';

[0370] In some embodiments, step 1 is performed in the presence of N-hydroxysuccinimide;

[0371] In some embodiments, step 1 is performed in the presence of dicyclohexylcarbodiimide.

[0372] In some embodiments, step 2 is performed in the presence of 1-amino-3,6,9,12,15-pentaoxaoctadecane-18-carboxylic acid.

[0373] In some embodiments, step three is performed in the presence of an oxidizing agent.

[0374] In some embodiments, the oxidizing agent is sodium periodate.

[0375] In some embodiments, step three is performed in the presence of ruthenium trichloride monohydrate.

[0376] In some embodiments, the present application provides a method 1 for synthesizing the following synthetic intermediate compound:

[0377] The method comprises the following steps:

[0378] Step 1: Compound Int-2-3 is subjected to coupling reaction to synthesize compound Int-2-4';

[0379] Step 2: removing the protecting group of compound Int-2-4' to form compound Int-2-5';

[0380] Step 3: Compound Int-2-5' undergoes a substitution reaction to form compound Int-5';

[0381] In some embodiments, step 1 is performed in the presence of the following compound:

[0382] In some embodiments, step 1 is performed in the presence of HOBt, EDCI and / or DIPEA.

[0383] In some embodiments, step 2 is performed in the presence of an organic acid.

[0384] In some embodiments, the organic acid is TFA.

[0385] In some embodiments, step three is performed in the presence of an oxidizing agent.

[0386] In some embodiments, the oxidizing agent is sodium periodate.

[0387] In some embodiments, step three is performed in the presence of ruthenium trichloride monohydrate.

[0388] In some embodiments, the present application provides a method for synthesizing the following synthetic intermediate compound:

[0389] The method comprises the following steps:

[0390] Step 1: Compound Int-3' undergoes coupling reaction to synthesize compound Int-4-1';

[0391] Step 2: removing the protecting group of compound Int-4-1' to form compound Int-4-2';

[0392] Step 3: Compound Int-4-2' forms compound Int-4';

[0393] In some embodiments, step 1 is performed in the presence of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline.

[0394] In some embodiments, step 2 is performed in the presence of pyridine hydrofluoride.

[0395] In some embodiments, step three is performed in the presence of p-nitrophenyl chloroformate.

[0396] In some embodiments, step three is performed in the presence of DIPEA.

[0397] 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.

[0398] definition

[0399] 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.

[0400] 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.

[0401] 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).

[0402] 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.

[0403] 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.

[0404] 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.

[0405] 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.

[0406] The term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0407] 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.

[0408] 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.

[0409] 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.

[0410] 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.

[0411] 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.

[0412] 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.

[0413] 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.

[0414] 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.

[0415] 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.

[0416] 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.

[0417] 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.

[0418] 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).

[0419] 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.

[0420] 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).

[0421] 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.

[0422] 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.

[0423] 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.

[0424] 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).

[0425] 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.

[0426] 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.

[0427] 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).

[0428] As used herein, the term "suitable substituent" refers to modifications that can be made to a compound by one skilled in the art according to the needs of the compound substituent. "Suitable substituents" include oxo (=O), halogen, cyano, NR 8 R 9 , carboxyl, thiol, hydroxyl, ester (e.g. -C 1- 6-alkyl-C(=O)-OC 1-6 Alkyl), C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkyl-OC 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 3-10 membered heterocyclic group, 5-10 membered heteroaryl, C 6-10Aryl, benzyl, hydroxy substituted benzyl, indolylmethylene and C 1-6 Haloalkoxy, R 8 、R 9 Each independently selected from H, C 1-6 Alkyl, C 3-6 Cycloalkyl, 3-10 membered heterocyclic group, 5-10 membered heteroaryl, C 6-10 Aryl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 Haloalkoxy, halogen, hydroxy, carboxyl and ester groups (e.g. -C 1-6 Alkyl-C(=O)-OC 1- 6 alkyl).

[0429] 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.

[0430] 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.

[0431] As used herein, the term "one or more" means 1 or more than 1, such as 2, 3, 4, 5 or 10, where reasonable.

[0432] Unless otherwise indicated, as used herein, the point of attachment of a substituent may be from any suitable position of the substituent.

[0433] 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

[0434] Figure 1: Efficacy of anti-human HER2 antibody-drug conjugate ADC in the NCI-N87 cell subcutaneous tumor-bearing mouse model

[0435] Figure 2: Efficacy of anti-human HER2 antibody-drug conjugate ADC in the NCI-N87 cell subcutaneous tumor-bearing mouse model

[0436] Figure 3: Efficacy of anti-human HER2 antibody-drug conjugate ADC in NCI-N87 cell subcutaneous tumor-bearing mouse model

[0437] Figure 4 Changes in body weight of mice in each group in the human gastric cancer cell NCI-N87 CDX model DETAILED DESCRIPTION

[0438] 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.

[0439] The information of the sequences involved in the present invention is described in the following table:

[0440] The abbreviations used in this document have the following meanings:

[0441] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0442] 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).

[0443] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the examples are shown below.

[0444] 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.

[0445] Mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0446] Intermediate Preparation Example 1: Preparation of 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonane-29-oic acid (INT-2)

[0447] Step 1: Preparation of 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid methyl ester (INT-2-2)

[0448] 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.

[0449] Its structural characterization data are as follows:

[0450] ESI-MS (m / z): 385.1 [M+H] + .

[0451] Step 2: Preparation of 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid (INT-2-3)

[0452] 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.

[0453] Its structural characterization data are as follows:

[0454] ESI-MS (m / z): 371.1 [M+H] + .

[0455] Step 3: Preparation of 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid tert-butyl ester (INT-2-4)

[0456] 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.

[0457] Step 4: Preparation of 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid (INT-2-5)

[0458] 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).

[0459] Its structural characterization data are as follows:

[0460] ESI-MS (m / z): 794.3 [M+H] + .

[0461] The purification method is as follows:

[0462] Chromatographic column: Phenomenex luna C18 (250mm*70mm*10μm)

[0463] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0464] Step 5: Preparation of 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid (INT-2)

[0465] 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosanetan-29-oic acid (148 mg, 0.186 mmol) was added to acetonitrile (15 mL) and water (7.5 mL), and sodium periodate (398.71 mg, 1.86 mmol) and ruthenium trichloride were added. The hydrate (15.47 mg, 74.56 μmol) was added to the reaction system, stirred at 25 ° C for 30 minutes, and the reaction system was extracted with water and ethyl acetate and concentrated to give 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid (155 mg).

[0466] Its structural characterization data are as follows:

[0467] ESI-MS (m / z): 858.3 [M+H] + .

[0468] Intermediate Preparation Example 2: Preparation of Allyl (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-3)

[0469] Step 1: Preparation of 2-hydroxymethyl-N-methyl-5-nitrobenzamide (INT-3-2)

[0470] Add 6-nitroisobenzofuran-1(3H)-one (50.0 g, 279 mmol) to a 2M solution of methylamine in tetrahydrofuran (500 mL), heat to 75°C, and stir for 5 hours. The reaction mixture was concentrated to obtain the crude title compound (58.0 g), which was used in the next step without purification.

[0471] Its structural characterization data are as follows:

[0472] ESI-MS (m / z): 211.0 [M+H] + .

[0473] Step 2: Preparation of (2-((methylamino)methyl)-4-nitrophenyl)methanol (INT-3-3)

[0474] Dissolve 2-hydroxymethyl-N-methyl-5-nitrobenzamide (25.0 g, 119 mmol) in tetrahydrofuran (500 mL). Cool the reaction mixture to 0°C, then add a 10M borane-dimethyl sulfide solution (89.3 mL) dropwise to the mixture. After the addition is complete, heat the mixture to 70°C and stir for 5 hours. Cool the mixture again to 0°C, then add a 2M hydrogen chloride-methanol solution (100 mL) dropwise to the reaction mixture. Heat the mixture to 65°C and stir for 12 hours. Filter the reaction mixture, and concentrate the filtrate to obtain the crude title compound (44.3 g), which is used in the next step without purification.

[0475] Its structural characterization data are as follows:

[0476] ESI-MS (m / z): 197.0 [M+H] + .

[0477] Step 3: Preparation of 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethanamine (INT-3-4)

[0478] Dissolve (2-((methylamino)methyl)-4-nitrophenyl)methanol (40.3 g, 205 mmol) in dichloromethane (800 mL), cool to 0°C, add imidazole (55.9 g, 822 mmol) and tert-butyldiphenylsilyl chloride (84.0 g, 308 mmol, 78.8 mL), return to 25°C, and continue stirring for 1 hour. Quench the reaction by adding water (200 mL) and extract with dichloromethane three times (300 mL x 3). The combined organic phases are washed with saturated sodium chloride (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purify on a silica gel column (dichloromethane / methanol = 20 / 1 to 5 / 1) and reconcentrate to obtain the title compound (59.7 g, 137 mmol).

[0479] Its structural characterization data are as follows:

[0480] ESI-MS (m / z): 435.1 [M+H] + .

[0481] Step 4: Preparation of allyl (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate (INT-3-5)

[0482] Dissolve 1-(2-(((tert-Butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethanamine (54.9 g, 126 mmol) in dichloromethane (550 mL). Cool to 0°C, add DIPEA (48.9 g, 379 mmol, 66.0 mL) and allyl chloroformate (30.5 g, 253 mmol, 26.8 mL), and return to 25°C. Stirring is continued for 1 hour. Quench the reaction system with water (300 mL). Extract with dichloromethane three times (200 mL x 3). Combine the organic phases, wash with saturated sodium chloride (300 mL), dry over anhydrous sodium sulfate, and filter and concentrate to obtain the crude product. Purify on a silica gel column (petroleum ether / ethyl acetate = 20 / 1 to 3 / 1) and concentrate again to obtain the title compound (65.5 g, 126 mmol).

[0483] Its structural characterization data are as follows:

[0484] ESI-MS (m / z): 519.1 [M+H] + .

[0485] Step 5: Preparation of allyl (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-3)

[0486] Allyl (2-(((tert-Butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate (62.8 g, 121 mmol) was dissolved in a mixed solvent of ethanol (300 mL) and water (300 mL). Iron powder (33.8 g, 605 mmol) and ammonium chloride (64.8 g, 1.21 mol) were added, and the mixture was heated to 80°C and stirred for 2 hours. The reaction solution was filtered, and the filtrate was extracted with dichloromethane three times (100 mL x 3). The combined organic phases were washed with saturated aqueous sodium chloride (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (49.3 g).

[0487] Its structural characterization data are as follows:

[0488] ESI-MS (m / z): 511.7 [M+H] + .

[0489] Intermediate Preparation Example 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-4)

[0490] 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-4-1)

[0491] 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).

[0492] Its structural characterization data are as follows:

[0493] MS m / z(ESI):782.2[M+H] +

[0494] Step 2: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (INT-4-2)

[0495] 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).

[0496] Its structural characterization data are as follows:

[0497] MS m / z(ESI):566.1[M+Na] +

[0498] 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-4) (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), DIPEA (81.3 mg, 0.63 mmol) was added, p-nitrophenyl chloroformate (50.8 mg, 0.25 mmol) was added, and the mixture was stirred at room temperature for 2 hours. After the reaction was completed, the residue 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).

[0499] Its structural characterization data are as follows:

[0500] MS m / z(ESI):731.2[M+Na] +

[0501] Intermediate Preparation Example 4: 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)

[0502] Step 1: Preparation of 2,5-dioxopyrrolidin-1-yl 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoate (INT-5-1)

[0503] 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.

[0504] 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): 2,5-Dioxopyrrolidin-1-yl 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoate (331.5 mg, 0.99 mmol) was dissolved in DMF (4 mL). 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) were added and stirred at room temperature for 2 hours. After completion of the reaction, the title compound (315.50 mg, 0.44 mmol) was obtained by flash column chromatography (C18, water / acetonitrile = 0.5).

[0505] Its structural characterization data are as follows:

[0506] MS m / z(ESI):662.2[M+H] +

[0507] 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) 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) was dissolved in acetonitrile (3 mL) and water (1.5 mL), and sodium periodate (470.50 mg, 2.20 mmol) and ruthenium trichloride monohydrate (9.11 mg, 0.04 mmol) were added and stirred at room temperature for 0.5 hour. After completion of the reaction, the residue was purified by flash column chromatography (C18, water / acetonitrile = 2 / 1) and freeze-dried to obtain the title compound (115.50 mg, 0.16 mmol).

[0508] Its structural characterization data are as follows:

[0509] MS m / z(ESI):726.1[M+H] +

[0510] Intermediate Preparation Example 5: Preparation of (((9H-fluoren-9-yl)methoxy)carbonyl)-D-alanyl-L-proline (INT-6)

[0511] (2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)propionic acid (2.00 g, 6.42 mmol), 1-hydroxypyrrolidine-2,5-dione (887.2 mg, 7.71 mmol), and DCC (1.59 g, 7.71 mmol) were added sequentially to DMF (30 mL) and reacted at 25°C for 2 h. L-proline (738.6 mg, 6.42 mmol) was added all at once, followed by dropwise addition of DIPEA (829.1 mg, 6.42 mmol) and the reaction was continued at 25°C for 4 h. The reaction solution was poured into water (90 mL) and the insoluble solids were filtered off. The impurities in the filtrate were extracted with ethyl acetate (50 ml + 30 ml). The aqueous phase was adjusted to pH 3-4 with hydrochloric acid and extracted twice with ethyl acetate (100 ml + 50 ml). The combined organic phases were washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound (2.40 g, 4.70 mmol).

[0512] Its structural characterization data are as follows:

[0513] MS m / z(ESI):409.2[M+H] +

[0514] Intermediate Preparation Example 6: N 2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoyl)-N 6 Preparation of -(tert-Butoxycarbonyl)-L-lysine (INT-8)

[0515] Step 1: Preparation of 2,5-dioxypyrrolidin-1-yl 3,5-bis(2-(methylthio)pyrimidin-4-yl)benzoate (INT-8-1)

[0516] 3,5-Bis(2-(methylthio)pyrimidin-4-yl)benzoic acid (5 g, 13.50 mmol) and 1-hydroxypyrrolidine-2,5-dione (1.55 g, 13.50 mmol) were added to a mixture of acetonitrile (75 mL) and DMF (75 mL). EDCI (5.17 g, 26.99 mmol) was then added and the mixture was allowed to react at 25°C for 1.5 minutes. The reaction mixture was filtered, and the filter cake was collected and dried to obtain the title compound (2.41 g, 5.15 mmol).

[0517] Its structural characterization data are as follows:

[0518] MS m / z(ESI):468.3[M+H] +

[0519] Step 2: N 2 -(3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoyl)-N 6 Preparation of -(tert-butoxycarbonyl)-L-lysine (INT-8-2)

[0520] N 6 1-(tert-Butoxycarbonyl)-L-lysine (895.61 mg, 3.64 mmol) and 2,5-dioxopyrrolidin-1-yl 3,5-bis(2-(methylthio)pyrimidin-4-yl)benzoate (1.7 g, 3.64 mmol) were added to DMSO (50 mL) and the temperature was raised to 60°C for 14 hours. The reaction solution was poured into water, whereupon a large amount of white solid precipitated. The pH of the solution was adjusted to approximately 2 with 1N dilute hydrochloric acid, and the filter cake was filtered and dried to obtain the title compound (2.16 g, 3.61 mmol).

[0521] Its structural characterization data are as follows:

[0522] MS m / z(ESI):526.2[M+H-56] +

[0523] Step 3: N 2 -(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoyl)-N 6Preparation of -(tert-Butoxycarbonyl)-L-lysine (INT-8)

[0524] N 2 -(3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoyl)-N 6 -(tert-Butoxycarbonyl)-L-lysine (1.06 g, 1.77 mmol) was dissolved in a mixture of water (25 mL) and acetonitrile (50 mL). Sodium periodate (2.46 g, 11.51 mmol) and ruthenium trichloride hydrate (36.72 mg, 177.04 μmol) were added and reacted at 25°C for 20 minutes. Water was added to the reaction system, and the pH was adjusted to approximately 2 with 1N dilute hydrochloric acid. The reaction was extracted with EA and concentrated. The crude product was dissolved in acetonitrile, purified by reverse phase chromatography (ACN / H2O = 0-45%, 0.05% formic acid), and lyophilized to obtain the title compound (825 mg, 1.24 mmol).

[0525] Its structural characterization data are as follows:

[0526] MS m / z(ESI):680.2[M+H2O] +

[0527] Intermediate Preparation Example 7: N 6 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 2 Preparation of 3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoyl)-L-lysine (INT-9)

[0528] Step 1: N 6 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 2 Preparation of -(3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoyl)-L-lysine (INT-9-1)

[0529] To DMSO (20 mL) was added N 6 2-(((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysine trifluoroacetate (2.8 g, 5.80 mmol) and DIPEA (1.50 g, 11.61 mmol) were stirred at room temperature for 10 minutes, and the reaction became clear. 2,5-Dioxopyrrolidin-1-yl 3,5-bis(2-(methylthio)pyrimidin-4-yl)benzoate (2.71 g, 5.80 mmol) was then added. The reaction was stirred at room temperature, and solids gradually precipitated during the reaction. The reaction mixture was heated to 37°C. The reaction solution was purified by reverse-phase column chromatography (ACN / H2O = 0-45%, 0.05% formic acid) and freeze-dried to obtain the title compound (2.8 g, 3.88 mmol).

[0530] Its structural characterization data are as follows:

[0531] MS m / z(ESI):721.9[M+H] +

[0532] Step 2: Preparation of N6-(((9H-fluoren-9-yl)methoxy)carbonyl)-N2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoyl)-L-lysine (INT-9)

[0533] To a mixed solvent of acetonitrile (80 mL) and water (40 mL) was added N 6 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 2 5-(3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoyl)-L-lysine (580 mg, 804.60 μmol), sodium periodate (1.38 g, 6.44 mmol), and RuCl₃·H₂O (36.28 mg, 160.92 μmol) were added and stirred at room temperature for 40 minutes. Water (150 mL) was added to the reaction solution, which was then extracted three times with ethyl acetate (100 mL*3). The mixture was filtered through celite and separated. The organic phase was washed once with water (50 mL) and once with saturated sodium chloride solution (50 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was dissolved in acetonitrile and water and freeze-dried to obtain the title compound (590 mg, 676.56 μmol).

[0534] Its structural characterization data are as follows:

[0535] MS m / z(ESI):785.9[M+H] + .

[0536] Example 1: 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-triazatriaza-35-amino)benzyl ((S)-1-(((S)-1-(((3R,4S,5S) -1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-1)

[0537] 1-(3,5-Bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octyloxy-2-azanonane-29-oic acid (Int-2, 35 mg, 40.80 μmol) was dissolved in DMF (2 mL). DIPEA (16 mg), HATU (23 mg), and VC-MMAE (CAS: 644981-35-1; 45 mg, 40.80 μmol) were added sequentially. The mixture was reacted at 25°C for 2 hours. The reaction mixture was directly purified by preparative HPLC and freeze-dried to give the title compound (31.45 mg, 15.54 μmol).

[0538] Its structural characterization data are as follows:

[0539] MS m / z(ESI):1963.1[M+H] +

[0540] Its preparation method is as follows:

[0541] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0542] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0543] Example 2: 1-(N-((6S,9S)-14-amino-9-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine (L-1)

[0544] Step 1:

[0545] Chlorosulfonyl isocyanate (500 mg, 3.53 mmol, 307 μL) was dissolved in dichloromethane (10.0 mL) at 0°C. Benzyl glycolate (533 mg, 3.21 mmol, 455 μL) was added and stirred for 1 hour. A solution of methyl 4-piperidinylcarboxylate L-1-1 (459 mg, 3.21 mmol) and triethylamine (974 mg, 9.63 mmol, 1.34 mL) in dichloromethane (5.00 mL) was then added to the reaction mixture. The temperature was raised to 25°C and stirring was continued for 1 hour. Water (100 mL) was added to the reaction mixture, and the mixture was extracted three times with dichloromethane (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 afford crude L-1-2 (1.30 g, 3.14 mmol), which was used directly in the next step without purification.

[0546] The structural characterization data are as follows:

[0547] ESI-MS (m / z): 414.9 [M+H] +

[0548] Step 2:

[0549] Under a nitrogen atmosphere, 10% Pd / C (0.70 g) was added to a solution of L-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 crude L-1-3 (590 mg).

[0550] Step 3:

[0551] VC-MMAE (CAS: 644981-35-1; 60 mg, 53.4 μmol) and L-1-3 (17.3 mg, 53.4 μmol) were dissolved in DMF (10.0 mL). HOBt (36.0 mg, 267 μmol), DIPEA (34.5 mg, 267 μmol, 46.5 μL), and EDCI (30.7 mg, 160 μmol) were added sequentially, and the mixture was reacted at 25°C for 2 hours. Water (20.0 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (20.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to afford crude L-1-4 (60.0 mg), which was used directly in the next step without purification.

[0552] The structural characterization data are as follows:

[0553] ESI-MS (m / z): 715.4 [M / 2+H] +

[0554] Step 4:

[0555] L-1-4 (50.0 mg, 34.9 μmol) was dissolved in tetrahydrofuran (2.50 mL), water (1.00 mL), and MeOH (1.00 mL). Lithium hydroxide monohydrate (2.94 mg, 69.9 μmol) was added, and the mixture was stirred at 30°C for 1 hour. The reaction solution was adjusted to pH 6 with 1N dilute hydrochloric acid and then concentrated to obtain crude L-1-5 (50.0 mg), which was used in the next step without purification.

[0556] The structural characterization data are as follows:

[0557] ESI-MS (m / z): 1416.6 [M+H] +

[0558] Step 5:

[0559] L-1-5 (50.0 mg, 35.3 μmol) and pentafluorophenol (13.0 mg, 70.6 μmol) were dissolved in DCM (10.0 mL). EDCI (30.4 mg, 158 μmol) was added and stirred at 30°C for 2 hours. The reaction solution was concentrated at 25°C to obtain the crude product, which was purified by HPLC and freeze-dried to afford L-1 (4.70 mg, 2.86 μmol).

[0560] The structural characterization data are as follows:

[0561] ESI-MS (m / z): 1582.1 [M+H] +

[0562] The separation and purification methods are as follows:

[0563] Chromatographic column: Phenomenex luna C18 (10μm*25mm*150mm)

[0564] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0565] Example 3: 1-(N-((2-(((S)-1-(((R)-1-((S)-2-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxypropyl)pyrrolidine- (1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)pyrrolidin-1-yl)-1-oxopropan-2-yl)amino)-3-hydroxy-1-oxopropan-2-yl)amino)-2-oxoethoxy)carbonyl)sulfamoyl)piperidine-4-carboxylic acid pentafluorophenol ester (L-4)

[0566] Step 1: Preparation of methyl 1-(N-((2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethoxy)carbonyl)sulfamoyl)piperidine-4-carboxylate (L-4-1)

[0567] 2-((((4-(Methoxycarbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (50 mg, 0.169 mmol), HOSU (23.3 mg, 0.202 mmol), and DCC (41.8 mg, 0.202 mmol) were added to DMF (1 mL) and reacted at 25°C for 2 hours; the reaction solution was poured into ethyl acetate (20 ml) and water (5 ml), stirred and filtered to separate the organic phase, which was washed with brine and concentrated to give the crude title compound (35 mg, 0.066 mmol), which was used directly in the next step without purification.

[0568] Step 2: Preparation of (9H-fluoren-9-yl)methyl ((R)-1-((S)-2-((4-(hydroxymethyl)phenyl)carbamoyl)pyrrolidin-1-yl)-1-oxopropan-2-yl)carbamate (L-4-2)

[0569] (((9H-fluoren-9-yl)methoxy)carbonyl)-D-alanyl-L-proline (Int-6, 400.0 mg, 0.979 mmol) and 4-aminobenzyl alcohol (144.7 mg, 1.18 mmol) were dissolved in a mixed solvent of dichloromethane (4 mL) and MeOH (2 mL), and EEDQ (484.4 mg, 1.96 mmol) was added. The temperature was raised to 40°C and the reaction was allowed to proceed for 4 hours. The crude product was concentrated to obtain a crude product, which was purified by column chromatography (first with EA / PE = 40-60%, then with MeOH / DCM = 5-15%) to obtain the title compound (225 mg, 0.394 mmol).

[0570] Its structural characterization data are as follows:

[0571] MS m / z(ESI):514[M+H] +

[0572] Step 3: (9H-fluoren-9-yl)methyl ((R)-1-((S)-2-((4-(((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamoyl)pyrrolidin-1-yl)-1-oxopropan-2-yl)carbamate (L-4-3)

[0573] (9H-fluoren-9-yl)methyl ((R)-1-((S)-2-((4-(hydroxymethyl)phenyl)carbamoyl)pyrrolidin-1-yl)-1-oxopropan-2-yl)carbamate (225 mg, 0.438 mmol) and bis(4-nitrophenyl) carbonate (266.6 mg, 0.876 mmol) were dissolved in DMF (3 mL), and DIPEA (84.9 mg, 0.657 mmol) was added. The mixture was reacted at 25°C for 2 hr. The reaction solution was added to ethyl acetate (20 ml) and water (10 ml), the organic phase was separated, and the aqueous phase was extracted once with ethyl acetate (10 ml). The organic phases were combined, washed with brine, and concentrated to give a crude product. The crude product was purified by column chromatography (MeOH / DCM = 5-10%) and concentrated again to give the title compound (190 mg, 0.280 mmol).

[0574] Its structural characterization data are as follows:

[0575] MS m / z(ESI):701.3[M+Na] +

[0576] Step 4: Preparation of 4-((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)-D-propylamino)pyrrolidine-2-carboxamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (L-4-4)

[0577] MMAE (CAS: 474645-27-7; 150 mg, 0.209 mmol) and (9H-fluoren-9-yl)methyl ((R)-1-((S)-2-((4-(((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamoyl)pyrrolidin-1-yl)-1-oxopropan-2-yl)carbamate (170.2 mg, 0.251 mmol) were dissolved in DMF (3 mL), and then HOBT (5.65 mg) was added. , 0.042 mmol) and DIPEA (27.00 mg, 0.209 mmol), react at 25 ° C for 2 hours; the reaction solution was poured into ethyl acetate (20 ml) and water (10 ml), the organic phase was separated, and the aqueous phase was extracted once with ethyl acetate (10 ml). The organic phases were combined, washed with brine, and concentrated to give a crude product, which was purified by column chromatography (MeOH / DCM = 5-10%) and concentrated again to give the title compound (60 mg, 0.048 mmol).

[0578] Its structural characterization data are as follows:

[0579] MS m / z(ESI):1257.8[M+H] +

[0580] Step 5: Preparation of 4-((S)-1-(D-propylamino)pyrrolidine-2-carboxamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (L-4-5)

[0581] 4-((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)-D-propylamino)pyrrolidine-2-carboxamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxo To a solution of (4-heptane-2-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (60 mg, 0.048 mmol) in DMF (1 mL) was added diethylamine (17.4 mg, 0.238 mmol) and the mixture was reacted at 25°C for 1 hour. The reaction solution was purified by preparative HPLC and freeze-dried to give the title compound (36 mg, 0.032 mmol, formate).

[0582] Its structural characterization data are as follows:

[0583] MS m / z(ESI):1035.7[M+H] +

[0584] Its separation and purification method is as follows:

[0585] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0586] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0587] Step 6: Preparation of 4-((S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-L-serine-D-propylamino)pyrrolidine-2-carboxamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (L-4-6)

[0588] 4-((S)-1-(D-propylamino)pyrrolidine-2-carboxamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxoheptan-2-yl To a solution of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-serine (13.1 mg, 0.040 mmol) and (((9H-fluoren-9-yl)methoxy)carbonyl)-L-serine) in DMF (1 mL) were added HATU (25.32 mg, 0.066 mmol) and DIPEA (12.9 mg, 0.10 mmol). The mixture was reacted at 25°C for 1 hour. The reaction solution was purified by preparative HPLC and freeze-dried to give the title compound (19 mg, 0.013 mmol).

[0589] Its structural characterization data are as follows:

[0590] MS m / z(ESI):1344.8[M+H] +

[0591] Its preparation method is as follows:

[0592] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0593] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0594] Step 7: Preparation of 4-((S)-1-(L-serine-D-propylamino)pyrrolidine-2-carboxamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (L-4-7)

[0595] 4-((S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-L-serine-D-propylamino)pyrrolidine-2-carboxamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1 -oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (19 mg, 0.013 mmol) was dissolved in DMF (1 mL), diethylamine (5.2 mg, 0.071 mmol) was added, and the reaction was carried out at 25°C for 1 hour; the reaction solution was purified by preparative HPLC and freeze-dried to give the title compound (14 mg, 0.011 mmol, formate).

[0596] Its structural characterization data are as follows:

[0597] MS m / z(ESI):1122.7[M+H] +

[0598] Its preparation method is as follows:

[0599] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0600] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0601] Step 8: 1-(N-((2-(((S)-1-(((R)-1-((S)-2-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1- Preparation of methyl piperidine-4-carboxylate (L-4-8)

[0602] 4-((S)-1-(L-serine-D-propylamino)pyrrolidine-2-carboxamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1 -oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (8.0 mg, 0.0071 mmol), compound L-4-1 (6.01 mg, 0.014 mmol), and DIPEA (4.6 mg, 0.036 mmol) were added to DMF (0.5 mL) and reacted at 25 ° C for 1 hour; the reaction solution was purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (8.0 mg, 0.0056 mmol).

[0603] Its structural characterization data are as follows:

[0604] MS m / z(ESI):1428[M+H] +

[0605] Its preparation method is as follows:

[0606] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0607] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0608] Step 9: 1-(N-((2-(((S)-1-(((R)-1-((S)-2-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3- Preparation of (((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)pyrrolidin-1-yl)-1-oxopropan-2-yl)amino)-3-hydroxy-1-oxopropan-2-yl)amino]-2-oxoethoxy)carbonyl)sulfamoyl)piperidine-4-carboxylic acid (L-4-9)

[0609] 1-(N-((2-(((S)-1-(((R)-1-((S)-2-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl) )phenyl)carbamoyl)pyrrolidin-1-yl)-1-oxopropyl-2-yl)amino)-3-hydroxy-1-oxopropyl-2-yl)amino]-2-oxoethoxy)carbonyl)sulfamoyl)piperidine-4-carboxylic acid methyl ester (8.0 mg, 0.0056 mmol) was dissolved in a mixed solvent of MeOH (1 mL) and H2O (0.5 mL), and LiOH.H2O (2.4 mg, 0.056 mmol) was added, and the reaction was carried out at 25°C for 2 hours; the reaction solution was purified by preparative HPLC and freeze-dried to give the title compound (5.0 mg, 0.0035 mmol).

[0610] Its structural characterization data are as follows:

[0611] MS m / z(ESI):1414[M+H] +

[0612] Its preparation method is as follows:

[0613] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0614] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0615] Step 10: 1-(N-((2-(((S)-1-(((R)-1-((S)-2-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1- Preparation of pentafluorophenol (L-4) containing 1,2-difluoro-2-( ...

[0616] 1-(N-((2-(((S)-1-(((R)-1-((S)-2-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)aminomethyl To the mixture of 2,3,4,5,6-pentafluorophenol (5.0 mg, 0.0035 mmol) and 2,3,4,5,6-pentafluorophenol (2.0 mg, 0.011 mmol) was dissolved in DMF (0.5 mL), and EDCI (3.4 mg, 0.018 mmol) was added. The mixture was reacted at 25°C for 1 hour. The reaction solution was purified by preparative HPLC and freeze-dried to give the title compound (2.1 mg, 0.0013 mmol).

[0617] Its structural characterization data are as follows:

[0618] MS m / z(ESI):1580[M+H] +

[0619] Its preparation method is as follows:

[0620] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0621] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0622] Example 4: 2,2',2"-(10-(2-((5-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3-methylbutanamido)propionamido)-2-((12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2 Preparation of triacetic acid (F-13)

[0623] Step 1: Preparation of 4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl))amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-13-1)

[0624] Allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (1.0 g, 1.41 mmol) and MMAE (CAS: 474645-27-7; 1.52 g, 2.12 mmol) were dissolved in DMF (6 mL). DIPEA (820.6 mg) and 1-hydroxybenzotriazole (190.6 mg, 1.41 mmol) were added, and the mixture was reacted at 25°C for 2 hours. After completion of the reaction, the product was purified by flash column chromatography (C18, water / acetonitrile = 0.6) and freeze-dried to obtain the title compound (1.18 g, 0.92 mmol).

[0625] Its structural characterization data are as follows:

[0626] MS m / z(ESI):1287.3[M+H] +

[0627] Step 2: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-aminopropionamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-13-2)

[0628] 4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl (1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl))amino)-1-methoxy-2-methyl-3- Carbamate (1.18 g, 91.65 μmol) was dissolved in DMF (10 mL) and diethylamine (201.6 mg) was added. The mixture was reacted at 25°C for 0.5 h. After the reaction was complete, the solvent was drained and a mixed solution of ethyl acetate and petroleum ether (100 mL, ethyl acetate / petroleum ether = 1 / 3) was added. The solution was stirred for 2 h and filtered to obtain the title compound (878.5 mg, 0.83 mmol).

[0629] Its structural characterization data are as follows:

[0630] MS m / z(ESI):1065.3[M+H] +

[0631] Step 3: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propionamido)-2-((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R))-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-13-3)

[0632] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-aminopropionamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine (308.1 mg, 90.75 μmol) were dissolved in DMF (5 mL), and DIPEA (320 mg) and HATU (470 mg) were added sequentially. The mixture was reacted at 25°C for 2 hours. The product was purified by flash column chromatography (C18, water / acetonitrile = 0.7) and freeze-dried to obtain the title compound (800.2 mg, 0.58 mmol).

[0633] Its structural characterization data are as follows:

[0634] MS m / z(ESI):1386.9[M+H] +

[0635] Step 4: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutanamido)propionamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-13-4)

[0636] 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyramido)propionamido)-2-((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl (1-((1-(((3R,5S)-1-((S)-2-((1R,2R))-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy (2-(2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-(methyl)amino)-3-methyl-1-oxobutan-2-yl)-(methyl)carbamate (800.2 mg, 57.7 μmol) was dissolved in DMF (5 mL), and diethylamine (126.6 mg) was added. The mixture was reacted at 25°C for 0.5 hour. After completion of the reaction, the solvent was drained, and a mixed solution of ethyl acetate / petroleum ether (60 mL, ethyl acetate / petroleum ether = 1 / 3) was added. The solution was stirred for 2 hours and filtered to give the title compound (604.5 mg, 0.52 mmol).

[0637] Its structural characterization data are as follows:

[0638] MS m / z(ESI):1164.7[M+H] +

[0639] Step 5: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3-methylbutyrylamino)propionamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-13-5)

[0640] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutyramido)propionamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxopropyl) (4-Heptyl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (100 mg, 85.9 μmol) was dissolved in DMF (3 mL), and DIPEA (35 mg) and 2,5-dioxopyrrolidin-1-yl 3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoate (54.7 mg, 103.1 μmol) were added. The mixture was reacted at 25°C for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 0.6) and freeze-dried to obtain the title compound (90.2 mg, 56.9 μmol).

[0641] Its structural characterization data are as follows:

[0642] MS m / z(ESI):1580.8[M+H] +

[0643] Step 6: Preparation of 4-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3-methylbutanamido)propionamido)-2-((methylamino)methyl)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-13-6)

[0644] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3-methylbutyrylamino)propionamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2- To the reaction mixture of 5-(2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-(methyl)amino)-3-methyl-1-oxobutan-2-yl)-(methyl)carbamate (90.2 mg, 56.9 μmol) was dissolved in DMF (2 mL), and tetrakistriphenylphosphine palladium (13.2 mg, 11.4 μmol) was added, along with 35 μL of formic acid and 70 μL of N-methylmorpholine. The mixture was stirred at room temperature under nitrogen for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 0.6) and freeze-dried to obtain the title compound (69.5 mg, 46.4 μmol).

[0645] Its structural characterization data are as follows:

[0646] MS m / z(ESI):1496.8[M+H] +

[0647] Step 7: 2,2',2"-(10-(2-((5-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3-methylbutanamido)propionamido)-2-((12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2 Preparation of triacetic acid (F-13)

[0648] 4-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3-methylbutanamido)propionamido)-2-((methylamino)methyl)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino To the mixture of 2-[(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (70.1 mg, 0.14 mmol) and 1-[(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)-1-[(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (70.1 mg, 0.14 mmol)] was added. After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (6.86 mg, 3.6 μmol).

[0649] Its structural characterization data are as follows:

[0650] MS m / z(ESI):1883.1[M+H] +

[0651] Its preparation method is as follows:

[0652] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0653] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0654] Example 5: 2,2',2"-(10-(2-((5-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenyl Preparation of triacetic acid (F-14)

[0655] Step 1: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (F-14-1)

[0656] Allyl (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (800 mg, 1.64 mmol) and (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanic acid (649.2 mg, 1.64 mmol) were dissolved in DCM (30 mL) and MeOH (6 mL). 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1.22 g, 4.92 mmol) was added and the mixture was reacted at 25°C for 16 hours. After completion of the reaction, the mixture was purified by flash column chromatography (silica gel, ethyl acetate / petroleum ether = 1) and concentrated again to obtain the title compound (995.5 mg, 1.15 mmol).

[0657] Its structural characterization data are as follows:

[0658] MS m / z(ESI):885.3[M+H2O] +

[0659] Step 2: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamic acid allyl ester (F-14-2)

[0660] Dissolve allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (995.5 mg, 1.15 mmol) in DMF (10 mL). Add pyridinium hydrofluoride (3.5 mL) dropwise in an ice bath. After addition, react at 25°C for 16 hours. After completion of the reaction, purify by flash column chromatography (silica gel, ethyl acetate / petroleum ether = 1) and reconcentrate to obtain the title compound (506.5 mg, 0.81 mmol).

[0661] Its structural characterization data are as follows:

[0662] MS m / z(ESI):647.3[M+H2O] +

[0663] Step 3: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)(methyl)carbamic acid allyl ester (F-14-3)

[0664] Dissolve allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (506.5 mg, 0.81 mmol) in DMF (3 mL), add DIPEA (209 mg) and bis(4-nitrophenyl) carbonate (295.5 mg, 0.972 mmol). After addition, react at 25°C for 16 hours. After completion of the reaction, purify by flash column chromatography (silica gel, ethyl acetate / petroleum ether = 1) and reconcentrate to obtain the title compound (515.2 mg, 0.65 mmol).

[0665] Its structural characterization data are as follows:

[0666] MS m / z(ESI):795.3[M+H] +

[0667] Step 4: Preparation of 4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)-2-((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-14-4)

[0668] Allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)(methyl)carbamate (515.2 mg, 0.65 mmol) and MMAE (CAS: 474645-27-7; 699.1 mg, 0.98 mmol) were dissolved in DMF (3 mL). DIPEA (251.6 mg) and 1-hydroxybenzotriazole (87.8 mg, 0.65 mmol) were added, and the mixture was reacted at 25°C for 2 hours. After completion of the reaction, the product was purified by flash column chromatography (C18, water / acetonitrile = 0.6) and freeze-dried to obtain the title compound (624.7 mg, 0.46 mmol).

[0669] Its structural characterization data are as follows:

[0670] MS m / z(ESI):1373.8[M+H] +

[0671] Step 5: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-amino-5-ureidopentanamido)benzyl(1-((1-(((3R,5S)-1)-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-14-5)

[0672] 4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)-2-((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl (1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl (methyl)amino)-3-methyl-1-oxobutan-2-yl)-3-(3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-(methyl)amino)-3-methyl-1-oxobutan-2-yl)-(methyl)carbamate (624.7 mg, 0.46 mmol) was dissolved in DMF (5 mL), and diethylamine (120 mg) was added. The mixture was reacted at 25°C for 0.5 hour. After the reaction was completed, the solvent was drained, and a mixed solution of ethyl acetate / petroleum ether (60 mL, ethyl acetate / petroleum ether = 1 / 3) was added. The solution was stirred for 2 hours and filtered to obtain the title compound (450.1 mg, 0.39 mmol).

[0673] Its structural characterization data are as follows:

[0674] MS m / z(ESI):1151.8[M+H] +

[0675] Step 6: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-14-6)

[0676] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-amino-5-ureidopentanamido)benzyl(1-((1-(((3R,5S)-1)-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine (164.8 mg, 0.47 mmol) were dissolved in DMF (5 mL), and DIPEA (151 mg) and HATU (223.1 mg) were added sequentially. The mixture was reacted at 25°C for 1 hour. The product was purified by flash column chromatography (C18, water / acetonitrile = 0.8) and freeze-dried to obtain the title compound (459.3 mg, 0.31 mmol).

[0677] Its structural characterization data are as follows:

[0678] MS m / z(ESI):1472.9[M+H] +

[0679] Step 7: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-14-7)

[0680] 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl (1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1 (459.3 mg, 0.31 mmol) was dissolved in DMF (5 mL) and diethylamine (90 mg) was added. The mixture was reacted at 25°C for 0.5 h. After the reaction was completed, the solvent was drained and a mixed solution of ethyl acetate / petroleum ether (50 mL, ethyl acetate / petroleum ether = 1 / 3) was added, stirred for 2 h, and filtered to obtain the title compound (348.8 mg, 0.28 mmol).

[0681] Its structural characterization data are as follows:

[0682] MS m / z(ESI):1250.8[M+H] +

[0683] Step 8: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3-methylbutyrylamino)-5-ureidopentanamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-14-8)

[0684] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutyramido)-5-ureidopentanamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl- 1-Oxohept-4-yl(methyl)amino)-3-methyl-1-oxobutan-2-yl(methyl)carbamate (100 mg, 80 μmol) was dissolved in DMF (3 mL). DIPEA (31 mg) and 2,5-dioxopyrrolidin-1-yl 3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoate (63.7 mg, 0.12 mmol) were added, and the mixture was reacted at 25°C for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 0.7) and freeze-dried to obtain the title compound (90.3 mg, 54.1 μmol).

[0685] Its structural characterization data are as follows:

[0686] MS m / z(ESI):1666.8[M+H] +

[0687] Step 9: Preparation of 4-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3-methylbutyrylamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy)-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-14-9)

[0688] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3-methylbutyrylamino)-5-ureidopentanamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy To the reaction mixture was dissolved (90.3 mg, 54.1 μmol) of (4-(2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-(2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-(2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-(2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)-(2-methyl-3 ...)carbamate (90.3 mg, 54.1 μmol) was dissolved in DMF (2 mL), and tetrakistriphenylphosphine palladium (12.5 mg, 10.8 μmol) was added, followed by the addition of 40 μL of formic acid and 80 μL of N-methylmorpholine. The mixture was stirred at room temperature under nitrogen for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water / acetonitrile =

[0689] Its structural characterization data are as follows:

[0690] MS m / z(ESI):1582.9[M+H] +

[0691] Step 10: 2,2',2"-(10-(2-((5-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane Preparation of triacetic acid (F-14)

[0692] 4-((S)-2-((S)-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3-methylbutyrylamino)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy)-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl To the mixture of 2-[(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (56.9 mg, 113.6 μmol) and 3-[(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)-1,4,7-triyl)-3-methyl-1-oxobutan-2-yl)-(methyl)carbamate (59.9 mg, 37.9 μmol) was dissolved in DMF (2 mL), and DIPEA (25 mg) was added. After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (14.6 mg, 7.4 μmol).

[0693] Its structural characterization data are as follows:

[0694] MS m / z(ESI):[M+H] + =1969.1

[0695] Its preparation method is as follows:

[0696] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0697] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0698] Example 6: 2,2',2"-(10-(2-((5-((9S,12S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-9-isopropyl-12-methyl-1,4,7,10-tetraoxo-2,5,8,11-tetraazatridecane-13-amide)-2-((12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R Preparation of (1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)benzyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (F-15)

[0699] Step 1: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(2-(2-aminoacetamido)acetamido)-3-methylbutanamido)propionamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane)-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-15-1)

[0700] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutyramido)propionamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptyl) (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine (44.1 mg, 125.4 μmol) were dissolved in DMF (3 mL). DIPEA (42 mg) and HATU (60.1 mg) were added sequentially. The mixture was reacted at 25°C for 1 hour. The product was purified by flash column chromatography (C18, water / acetonitrile = 0.8) and freeze-dried to give the title compound (125.2 mg, 93.7 μmol).

[0701] Its structural characterization data are as follows:

[0702] MS m / z(ESI):1501.1[M+H] +

[0703] Step 2: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(2-(2-aminoacetamido)acetamido)-3-methylbutanamido)propionamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane)-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-15-2)

[0704] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(2-(2-aminoacetamido)acetamido)-3-methylbutanamido)propionamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane)-2-yl)amino)-1-methoxy- (2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl) (methyl)amino)-3-methyl-1-oxobutan-2-yl) (methyl)carbamate (125.2 mg, 93.7 μmol) was dissolved in DMF (3 mL), and diethylamine (100 mg) was added. The mixture was reacted at 25°C for 0.5 hour. After completion of the reaction, the solvent was drained, and a mixed solution of ethyl acetate / petroleum ether (30 mL, ethyl acetate / petroleum ether = 1 / 3) was added and stirred for 2 hours. The mixture was filtered and dried to give the title compound (97.3 mg, 75.8 μmol).

[0705] Its structural characterization data are as follows:

[0706] MS m / z(ESI):1278.8[M+H] +

[0707] Step 3: 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((9S,12S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-9-isopropyl-12-methyl-1,4,7,10-tetraoxo-2,5,8,11-tetraazatridecane-13-amido)benzyl (1-((1-(((3R,5S)-1-((S)-2 Preparation of -((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-15-3)

[0708] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(2-(2-aminoacetamido)acetamido)-3-methylbutanamido)propionamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane)-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy- 5-Methyl-1-oxohept-4-yl(methyl)amino)-3-methyl-1-oxobutan-2-yl(methyl)amino)-3-methyl-1-oxobutan-2-yl(methyl)carbamate (48.3 mg, 37.8 μmol) was dissolved in DMF (2 mL), and DIPEA (35 mg) and 2,5-dioxopyrrolidin-1-yl 3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoate (72.6 mg, 0.15 mmol) were added. The mixture was reacted at 25°C for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 0.6) and freeze-dried to give the title compound (57.2 mg, 33.8 μmol).

[0709] Its structural characterization data are as follows:

[0710] MS m / z(ESI):1694.9[M+H] +

[0711] Step 4: 4-((9S,12S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-9-isopropyl-12-methyl-1,4,7,10-tetraoxo-2,5,8,11-tetraazatridecane-13-amido)-2-((methylamino)methyl)benzyl (1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1- Preparation of (hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)amino)-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (F-15-4)

[0712] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((9S,12S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-9-isopropyl-12-methyl-1,4,7,10-tetraoxo-2,5,8,11-tetraazatridecane-13-amido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1- To the reaction mixture (57.2 mg, 33.8 μmol) was dissolved in DMF (2 mL) and tetrakistriphenylphosphine palladium (7.8 mg, 6.8 μmol) was added: (1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl) (1-oxobutan-2-yl) (1-methyl)amino)-1-oxobutan-2-yl) (1-methyl)carbamate (57.2 mg, 33.8 μmol) was added: (1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl) (1-methyl)amino)-1-oxobutan-2-yl) (1-methyl)amino)-3-methyl-1-oxobutan-2-yl) (1-methyl)carbamate (57.2 mg, 33.8 μmol) was dissolved in DMF (2 mL) and tetrakistriphenylphosphine palladium (7.8 mg, 6.8 μmol) was added: (1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl) (1-methoxy-5-methyl-1-oxohept-4-yl) (1-methyl)amino)-1-oxobutan-2-yl) (1-methyl)carbamate (57.2 mg, 33.8 μmol) was dissolved in DMF (2 mL) and tetrakistriphenylphosphine palladium (7.8 mg, 6.8 μmol) was added: (1-methoxy-2-methyl-3-oxopropyl)

[0713] Its structural characterization data are as follows:

[0714] MS m / z(ESI):1610.8[M+H] +

[0715] Step 5: 2,2',2"-(10-(2-((5-((9S,12S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-9-isopropyl-12-methyl-1,4,7,10-tetraoxo-2,5,8,11-tetraazatridecane-13-amide)-2-((12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R Preparation of (1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)benzyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (F-15)

[0716] 4-((9S,12S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-9-isopropyl-12-methyl-1,4,7,10-tetraoxo-2,5,8,11-tetraazatridecane-13-amido)-2-((methylamino)methyl)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino) To the mixture of 1,4-dioxopyrrolidin-1-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (43.5 mg, 27.0 μmol) was dissolved in DMF (2 mL), and DIPEA (18 mg) was added, followed by 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (40.6 mg, 81.0 μmol). After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (12.61 mg, 6.3 μmol).

[0717] Its structural characterization data are as follows:

[0718] MS m / z(ESI):1997.1[M+H] +

[0719] Its preparation method is as follows:

[0720] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0721] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0722] Example 7: 2,2',2"-(10-(2-((5-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amide)-2-((12R)-11-((S)-sec-butyl)-12-(2 Preparation of ((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazacyclododecane-1,4,7-triyl)triacetic acid (F-16)

[0723] Step 1: 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amido)benzyl (1-((1-(((3R,5S)-1- Preparation of ((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-16-1)

[0724] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutyramido)propionamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxohept-4-yl A mixture 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, 28.1 mg, 38.7 μmol) and 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) was dissolved in DMF (2 mL). DIPEA (15 mg) and HATU (15.1 mg) were added sequentially. The mixture was reacted at 25°C for 1 hour. Purification by flash column chromatography (C18, water / acetonitrile = 0.6) and freeze-dried to give the title compound (33.8 mg, 18.1 μmol).

[0725] Its structural characterization data are as follows:

[0726] MS m / z(ESI):1872.1[M+H] +

[0727] Step 2: 4-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexadecane-26-amido)-2-((methylamino)methyl)benzyl (1-((1-(((3R, 5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-

[0728] Preparation of -2-yl)(methyl)carbamate (F-16-2)

[0729] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosanoyl-26-amido)benzyl (1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1 To the reaction mixture of 1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (33.8 mg, 18.1 μmol) was dissolved in DMF (2 mL), and tetrakistriphenylphosphine palladium (4.2 mg, 3.6 μmol) was added, along with 20 μL of formic acid and 40 μL of N-methylmorpholine. The mixture was stirred at room temperature under nitrogen for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 0.55) and freeze-dried to give the title compound (22.6 mg, 12.7 μmol).

[0730] Its structural characterization data are as follows:

[0731] MS m / z(ESI):1788.1[M+H] +

[0732] Step 3: 2,2',2"-(10-(2-((5-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amide)-2-((12R)-11-((S)-sec-butyl)-12-(2 Preparation of ((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazacyclododecane-1,4,7-triyl)triacetic acid (F-16)

[0733] 4-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexadecane-26-amido)-2-((methylamino)methyl)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)( To the solution of (1,4-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (19.1 mg, 38.1 μmol) was added 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (22.6 mg, 12.7 μmol) in DMF (2 mL). After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (5.17 mg, 2.4 μmol).

[0734] Its structural characterization data are as follows:

[0735] MS m / z(ESI):[M / 2+H] + =1087.1

[0736] Its preparation method is as follows:

[0737] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0738] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0739] Example 8: 2,2',2"-(10-(2-((5-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-1,20,23-trioxo-25-(3-ureidopropyl)-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amide)-2-((12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R Preparation of (1-((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (F-17)

[0740] Step 1: 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-1,20,23-trioxo-25-(3-ureidopropyl)-5,8,11,14,17-pentaoxa-2,21,24-triazahexadecane-26-amido)benzyl (1-((1-(((3R,5S)- Preparation of 1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-17-1)

[0741] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino))-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl Carbamate (100.0 mg, 80.0 μmol) and 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosane-20-oleic acid (87.2 mg, 120.0 μmol) were dissolved in DMF (3 mL). DIPEA (31 mg) and HATU (60.8 mg) were added sequentially. The mixture was reacted at 25°C for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 0.6) and freeze-dried to obtain the title compound (93.8 mg, 47.9 μmol).

[0742] Its structural characterization data are as follows:

[0743] MS m / z(ESI):1958.1[M+H] +

[0744] Step 2: 4-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-1,20,23-trioxo-25-(3-ureidopropyl)-5,8,11,14,17-pentaoxane-2,21,24-triazahexacosane-26-amido)-2-((methylamino)methyl)benzyl (1-((1-(((3R,5S))-1-( Preparation of methyl (S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-17-2)

[0745] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-1,20,23-trioxo-25-(3-ureidopropyl)-5,8,11,14,17-pentaoxa-2,21,24-triazahexadecane-26-amido)benzyl (1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)- To the reaction mixture (93.8 mg, 47.9 μmol) was dissolved (1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (97.8 mg, 47.9 μmol) in DMF (2 mL), and tetrakistriphenylphosphine palladium (11.1 mg, 9.6 μmol) was added, along with 40 μL of formic acid and 80 μL of N-methylmorpholine. The mixture was stirred at room temperature under nitrogen for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 0.5) and freeze-dried to give the title compound (62.8 mg, 33.5 μmol).

[0746] Its structural characterization data are as follows:

[0747] MS m / z(ESI):1874.1[M+H] +

[0748] Step 3: 2,2',2"-(10-(2-((5-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-1,20,23-trioxo-25-(3-ureidopropyl)-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amide)-2-((12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R Preparation of (1-((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (F-17)

[0749] 4-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-1,20,23-trioxo-25-(3-ureidopropyl)-5,8,11,14,17-pentaoxane-2,21,24-triazahexacosane-26-amido)-2-((methylamino)methyl)benzyl (1-((1-(((3R,5S))-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptyl- To the solution of methyl (4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-1-oxobutan-2-yl)(methyl)carbamate (62.8 mg, 33.5 μmol) in DMF (2 mL) were added DIPEA (22 mg) and 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (50.4 mg, 100.6 μmol). After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (20.47 mg, 7.4 μmol).

[0750] Its structural characterization data are as follows:

[0751] MS m / z(ESI):[M / 2+H] + =1130.1

[0752] Its preparation method is as follows:

[0753] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0754] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0755] Example 9: 2,2',2"-(10-(2-((5-((28S,31S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-28-isopropyl-31-methyl-1,20,23,26,29-pentaoxo-5,8,11,14,17-pentaoxa-2,21,24,27,30-pentaazadotriacontane-32-amide)-2-((12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R Preparation of ((1S,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (F-18)

[0756] Step 1: 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((28S,31S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-28-isopropyl-31-methyl-1,20,23,26,29-pentaoxo-5,8,11,14,17-pentaoxa-2,21,24,27,30-pentaazatriacontane-32-amido)benzyl (1-((1-(((3R, Preparation of (5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-18-1)

[0757] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(2-(2-aminoacetamido)acetamido)-3-methylbutanamido)propionamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane)-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino Carbamate (48.0 mg, 37.6 μmol) and 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosane-20-carboxylic acid (41.1 mg, 56.4 μmol) were dissolved in DMF (3 mL). DIPEA (20 mg) and HATU (21.4 mg) were added sequentially. The mixture was reacted at 25°C for 1 hour. The product was purified by flash column chromatography (C18, water / acetonitrile = 0.6) and freeze-dried to give the title compound (52.2 mg, 26.3 μmol).

[0758] Its structural characterization data are as follows:

[0759] MS m / z(ESI):1986.1[M+H] +

[0760] Step 2: 4-((28S,31S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-28-isopropyl-31-methyl-1,20,23,26,29-pentaoxo-5,8,11,14,17-pentaoxa-2,21,24,27,30-pentaazadotriacontane-32-amido)-2-((methylamino)methyl)benzyl (1-((1-(((3R,5S)-1- Preparation of ((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-18-2)

[0761] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((28S,31S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-28-isopropyl-31-methyl-1,20,23,26,29-pentaoxo-5,8,11,14,17-pentaoxa-2,21,24,27,30-pentaazadotriacontanoic acid-32-amido)benzyl (1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S To the reaction mixture (52.2 mg, 26.3 μmol) was dissolved (4-(2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (52.2 mg, 26.3 μmol) in DMF (2 mL), and tetrakistriphenylphosphine palladium (7.8 mg, 6.8 μmol) was added, along with 30 μL of formic acid and 60 μL of N-methylmorpholine. The mixture was stirred at room temperature under nitrogen for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 0.55) and freeze-dried to give the title compound (35.1 mg, 18.4 μmol).

[0762] Its structural characterization data are as follows:

[0763] MS m / z(ESI):1902.1[M+H] +

[0764] Step 3: 2,2',2"-(10-(2-((5-((28S,31S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-28-isopropyl-31-methyl-1,20,23,26,29-pentaoxo-5,8,11,14,17-pentaoxa-2,21,24,27,30-pentaazatriacontane-32-amide)-2-((12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R Preparation of ((1S,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (F-18)

[0765] 4-((28S,31S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-28-isopropyl-31-methyl-1,20,23,26,29-pentaoxo-5,8,11,14,17-pentaoxa-2,21,24,27,30-pentaazadotriacontane-32-amido)-2-((methylamino)methyl)benzyl (1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptyl) To the mixture of 1,4-dioxopyrrolidin-1-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (35.1 mg, 18.4 μmol) was dissolved in DMF (2 mL), and DIPEA (18 mg) was added, followed by 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (46.3 mg, 92.0 μmol). After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (23.17 mg, 10.1 μmol).

[0766] Its structural characterization data are as follows:

[0767] MS m / z(ESI):[M / 2+H] + =1144.1

[0768] Its preparation method is as follows:

[0769] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0770] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0771] Example 10: 2,2',2"-(10-((6S,9S,12S)-1-amino-12-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-6-((4-((12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxo Preparation of triacetic acid (F-19)

[0772] Step 1: 4-((9S,12S,15S)-9-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-1-(9H-fluoren-9-yl)-12-isopropyl-3,10,13-trioxo-15-(3-ureidopropyl)-2-oxa-4,11,14-triazahexadecane-16-amido)benzyl (1-((1-(((3R,5S)-1-((S)-2 Preparation of -((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-19-2)

[0773] N 6 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 2 2-(3,5-Bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoyl)-L-lysine (25 mg, 31.90 μmol) and VC-MMAE (CAS: 644981-35-1; 42.5 mg, 38.30 μmol) were dissolved in DMF (2 mL). DIPEA (16 mg) and HATU (19 mg) were added and the mixture was reacted at 25°C for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water / acetonitrile = 0.7) and freeze-dried to obtain the title compound (36.2 mg, 19.1 μmol).

[0774] Its structural characterization data are as follows:

[0775] MS m / z(ESI):1890.1[M+H] +

[0776] Step 2: Preparation of 4-((S)-2-((S)-2-((S)-6-amino-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)hexanoylamino)-3-methylbutanamido)-5-ureidopentanamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R))-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (F-19-3)

[0777] 4-((9S,12S,15S)-9-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-1-(9H-fluoren-9-yl)-12-isopropyl-3,10,13-trioxo-15-(3-ureidopropyl)-2-oxa-4,11,14-triazahexadecane-16-amido)benzyl (1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1- Carbamate (36.2 mg, 19.1 μmol) was dissolved in DMF (2 mL) and diethylamine (50 mg) was added. The mixture was reacted at 25°C for 0.5 h. After completion of the reaction, the product was purified by flash column chromatography (C18, water / acetonitrile = 0.5) and freeze-dried to give the title compound (25.5 mg, 15.3 μmol).

[0778] Its structural characterization data are as follows:

[0779] MS m / z(ESI):1667.9[M+H] +

[0780] Step 3: 2,2',2"-(10-((6S,9S,12S)-1-amino-12-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-6-((4-((12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropane Preparation of triacetic acid (F-19)

[0781] 4-((S)-2-((S)-2-((S)-6-amino-2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)hexanoylamino)-3-methylbutanamido)-5-ureidopentanamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R))-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan- To the solution of (2-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (23.1 mg, 45.9 μmol) was added 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (23.1 mg, 45.9 μmol). After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (12.09 mg, 5.9 μmol).

[0782] Its structural characterization data are as follows:

[0783] MS m / z(ESI):[M / 2+H] + =1027.1

[0784] Its preparation method is as follows:

[0785] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0786] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0787] 2. Antibody Preparation

[0788] 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.

[0789] 3. Conjugation of Compounds Containing Cellular Bioactive Molecules and Linkers to Antibodies

[0790] 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.

[0791] 1. Preparation of Trastuzumab-L-1 (DAR 2)

[0792] 0.31 mL of trastuzumab antibody (16.2 mg / mL) was adjusted to pH 7.40 with 1M Na₂HPO₄ solution. A 5-fold amount of the antibody dissolved in 1L-1 (18.15 μL, 10 mM) of dimethyl sulfoxide (DMSO) was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 18 hours. Afterwards, 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 (i.e., trastuzumab-L-1). The average loading (DAR) value determined by mass spectrometry was 2.28, of which DAR2 accounted for 60.28%.

[0793] 2. Preparation of Trastuzumab-L-4 (DAR 2)

[0794] 0.123 mL of trastuzumab antibody (16.2 mg / mL) was diluted with 200 μL of 20 mM PB (pH 7.60), then the pH was adjusted to 7.40 with 1 M Na₂HPO₄ solution. A 5-fold amount of L-4 dissolved in dimethyl sulfoxide (7.10 μL, 10 mM) was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 18 hours. Afterwards, 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 (i.e., trastuzumab-L-4). The DAR value determined by mass spectrometry was 2.35, of which DAR2 accounted for 57.97%.

[0795] 3. Preparation of Trastuzumab-F-15

[0796] 2.027 mL of trastuzumab antibody (14.8 mg / mL) was diluted with 101 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. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 113.6 μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. Six times the amount of the antibody was then added in sequence. F-15 (135.7 μL, 10 mM) dissolved in dimethyl sulfoxide (DMSO) was mixed thoroughly. The mixture was allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., trastuzumab-F-15). The DAR value determined by mass spectrometry was 4.12, of which DAR4 accounted for 88.43%.

[0797] 4. Preparation of Trastuzumab-F-16

[0798] 1.7 mL of trastuzumab antibody (14.8 mg / mL) was diluted with 85 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then the pH was adjusted to 7.60 with 1 M Na2HPO4 solution. A 10 mM TCEP (tris(2-carboxyethyl)phosphine, 94.7 μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. Six times the amount of the antibody was then added, followed by a solution of F-16 dissolved in dimethyl sulfoxide (110.5 μL, 10 mM), mixed thoroughly, and allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., trastuzumab-F-16). The DAR value determined by mass spectrometry was 3.6, of which DAR4 accounted for 72.01%.

[0799] 5. Preparation of Trastuzumab-F-18

[0800] 2.027 mL of trastuzumab antibody (14.8 mg / mL) was diluted with 101 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then the pH was adjusted to 7.60 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 113.6 μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. Six times the amount of the antibody was then added, followed by mixing thoroughly with a solution of F-18 dissolved in dimethyl sulfoxide (131.8 μL, 10 mM). The mixture was allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., trastuzumab-F-18). The DAR value determined by mass spectrometry was 4.08, of which DAR4 accounted for 92.32%.

[0801] IV. Detecting the inhibitory effect of compounds on tumor cell proliferation

[0802] 1. Efficacy testing of anti-human Her2 antibody-drug conjugates in the NCI-N87 model

[0803] 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, 3Around 30 minutes later, the patients were randomly divided into groups according to the size of the tumor, in the following order: vehicle control group (i.e., negative control, Vehicle group), Trastuzumab-F-16 1 mg / kg, Trastuzumab-F-15 1 mg / kg, Trastuzumab-F-18 1 mg / kg of the present invention, and control DS8201 1 mg / kg (Note: DS8201 is an ADC targeting human HER2 developed by Daiichi Sankyo, and the sample used in this experiment was prepared by Kelun Botai). Each group was injected by tail vein (iv), and the present invention was administered on Day 0 and Day 7, for a total of 2 times. The control DS8201 was administered on Day 0, Day 7, and Day 14, for a total of 3 times. After administration, the tumor diameter was measured with a vernier caliper twice a week, and the tumor volume was calculated according to 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.

[0804] The following formula was used to calculate the tumor growth inhibition rate (TGI) to evaluate the tumor inhibition efficacy of ADC:

[0805] 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%.

[0806] Where V T末 : Mean tumor volume of treatment group at the end of the experiment;

[0807] V T0 : Mean tumor volume at the start of drug administration in the treatment group;

[0808] V C末 : Mean tumor volume of negative control group at the end of the experiment;

[0809] V C0 : Mean tumor volume of the negative control group at the beginning of drug administration;

[0810] The following formula was used to calculate the relative tumor proliferation rate T / C (%), which was used to evaluate the tumor inhibition efficacy of ADC:

[0811] T / C(%)=(V T末 / V T0 ) / (V C末 / V C0 )*100%.

[0812] 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-F-16, 1 mg / kg trastuzumab-F-15, 1 mg / kg trastuzumab-F-18, and DS8201 groups were 177.42%, 176.60%, 176.53%, and 91.25%, respectively. On Day 27, there were no animal deaths or significant weight loss in any treatment group, and no significant drug toxicity was observed. The mice tolerated the ADC of this invention well during treatment. Specific results are shown in Table 11, Figures 1, 2, 3, and 4.

[0813] Table 11 Human gastric cancer cell NCI-N87 CDX model

[0814] 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 the formula Ab-[MLED] x The structure shown, 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 the structural fragment between the 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: M is selected from the following structural fragments:

3. The antibody-drug conjugate according to claim 1, wherein: M is selected from the following structural fragments:

4. The antibody-drug conjugate according to any one of claims 1 to 3, wherein: L is selected from a divalent structural fragment consisting of one or more of the following groups: C 1-6 alkylene, 6-10 membered arylene, 5-6 membered heteroarylene, -N(R')-, carbonyl, -O-, the residue of an amino acid or its analog (the amino acid is a natural amino acid or a non-natural amino acid, 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 or Lys (COCH2CH2(OCH2CH2) r OCH3)), a fragment of a short peptide composed of two or more amino acids (the short peptide is, for example, selected from 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 and Gly-Gly-Gly-Gly-Gly), Lys(R'), wherein R' and R" each independently represent 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(C 1-6 alkyl)-C(=O)-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) r -C 1-6 alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), or NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue), wherein r is selected from an integer of 1 to 20, such as 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; s is selected from an integer of 1-20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, preferably 5, 8 or 9; t is selected from an integer of 1-5; preferably, t is 1; Preferably, L is selected from a divalent structural fragment consisting of one or more of the following groups: s is an integer selected from 1 to 20; Preferably, L is selected from: s is an integer selected from 1-20.

5. The antibody-drug conjugate according to any one of claims 1 to 4, wherein: E is a single bond, -NH-CH2-, or is selected from the following structural fragments: Preferably, E is -NH-CH2- or More preferably, E is 6. The antibody-drug conjugate according to any one of claims 1 to 5, wherein: The cytotoxic drug is an anti-tubulin agent; Preferably, the microtubule protein inhibitor is an auristatin compound; Preferably, the auristatin compound 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; Preferably, the cytotoxic drug fragment D is selected from:

7. The antibody-drug conjugate according to any one of claims 1 to 6, wherein: -MLED is formed by any drug-linker selected from F'-1 to F'-12, F'-16 to F'-18, L'-6 to L'-11, F-1 to F-20 and L-1 to L-11, preferably selected from F-1 to F-20 and L-1 to L-11, for example, by substitution reaction (for example, removal of methylsulfonyl or pentafluorophenoxy structures thereon) or by addition reaction: wherein n is selected from an integer of 1-20, preferably n is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 5-15, 5-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 9 or 10;.

8. The antibody-drug conjugate according to any one of claims 1 to 7, 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: 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; The variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, or At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, or the variant has one or 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; 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.

9. The antibody-drug conjugate according to any one of claims 1 to 8, 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 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.

10. The antibody-drug conjugate according to claim 8 or 9, 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, 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; such as 1, 2, 3, 4 or 5 conservative substitutions of amino acids); Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO:35 and a light chain constant region (CL) as shown in SEQ ID NO:

36.

11. The antibody-drug conjugate according to any one of claims 1 to 10, 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.

12. The antibody-drug conjugate of any one of claims 1 to 11, wherein M is linked to a sulfhydryl (-SH) or amino (-NH2) group on Ab.

13. The antibody-drug conjugate according to any one of claims 1 to 12, wherein: The antibody or antigen-binding fragment thereof is selected from the antibody or antigen-binding fragment thereof according to claim 11; -MLED is as defined in claim 7; x is 1 to 10; preferably, x is 1 to 8, or x is 1 to 4.

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

15. A composition comprising one or more antibody drug conjugates according to any one of claims 1 to 14, wherein the DAR value (drug antibody conjugate ratio) of the composition is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7 ~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.

16. A pharmaceutical composition comprising the antibody drug conjugate according to any one of claims 1 to 14 or the composition according to claim 15, and one or more pharmaceutical excipients.

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

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

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

20. The antibody drug conjugate, composition or pharmaceutical composition of claim 19, wherein the cancer is selected from a solid tumor or a hematological malignancy; for example, selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) and urothelial carcinoma.

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

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

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